Minding the calcium store: Ryanodine receptor activation as a

Pharmacology & Therapeutics 125 (2010) 260–285

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Pharmacology & Therapeutics

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Minding the calcium store: Ryanodine receptor activation as a convergentmechanism of PCB toxicity

Isaac N. Pessah ⁎, Gennady Cherednichenko, Pamela J. LeinDepartment of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, CA 95616, USA

⁎ Corresponding author. Tel.: 530 752 6696.E-mail address: [email protected] (I.N. Pessah).

0163-7258/$ – see front matter © 2009 Elsevier Inc. Aldoi:10.1016/j.pharmthera.2009.10.009

a b s t r a c t
a r t i c l e i n f o
Keywords:
Ryanodine receptor (RyR) Calcium-induced calcium releaseCalcium regulationPolychlorinated biphenylsTriclosanBastadinsPolybrominated diphenylethersDevelopmental neurotoxicityActivity dependent plasticity
Chronic low-level polychlorinated biphenyl (PCB) exposures remain a significant public health concern sinceresults from epidemiological studies indicate that PCB burden is associated with immune systemdysfunction, cardiovascular disease, and impairment of the developing nervous system. Of these variousadverse health effects, developmental neurotoxicity has emerged as a particularly vulnerable endpoint inPCB toxicity. Arguably the most pervasive biological effects of PCBs could be mediated by their ability to alterthe spatial and temporal fidelity of Ca2+ signals through one or more receptor-mediated processes. Thisreview will focus on our current knowledge of the structure and function of ryanodine receptors (RyRs) inmuscle and nerve cells and how PCBs and related non-coplanar structures alter these functions. Themolecular and cellular mechanisms by which non-coplanar PCBs and related structures alter local and globalCa2+ signaling properties and the possible short and long-term consequences of these perturbations onneurodevelopment and neurodegeneration are reviewed.

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© 2009 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
2. Ryanodine receptor macromolecular complexes:
significance to polychlorinated biphenyl-mediated Ca2+ dysregulation . . . . . . . . . . . . . . . . . . . 263
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
1. Introduction

1.1. Polychlorinated biphenyls: Occurrence and concerns to public health

Polychlorinated biphenyls (PCBs) are synthetic chlorinated aro-matic hydrocarbons that are non-flammable, chemically stable andhave high boiling points. In the United States, PCBs were synthesizedand marketed primarily as Aroclor® mixtures whose degree ofchlorination was identified by a four-digit designation (e.g., 1248,1254, 1260, etc.), with the first two digits identifying the mixture asPCBs and the last two digits identifying the percent of chlorine usedduring synthesis. A higher degree of PCB chlorination increasesmelting point and lipophilicity, whereas lower chlorination increasesvapor pressure and water solubility. Similar PCB mixtures were

synthesized worldwide and identified under several trade names suchas Clophen® and Kanechlor®. PCB mixtures, especially those of inter-mediate chlorination, such as Aroclor 1248 and Aroclor 1254, werewidely used in several industries for their insulation and heat dis-sipating properties. PCBs were also broadly incorporated into a varietyof common products such as pesticide extenders, plastics, varnishes,adhesives, carbonless copy paper, newsprint, fluorescent light ballastsand caulking compounds (Ross, 2004).

By 1977, when PCBs were banned, more than 600,000 tons weremanufactured in the United States, and global production is estimatedat over 1.5 million tons (Breivik et al., 2002). Because of their exten-sive industrial use and chemical stability, PCBs have accumulated inthe environment and biota. PCBs have been identified in approxi-mately one third of the sites listed on the National Priorities List (NPL)and Superfund Sites (Anonymous, 2007). The average PCB levels inthe environment and human blood have steadily declined since 1977.However, geographic “hotspots” of relatively high PCB contaminationpersist due to improper disposal, and mobilization of PCBs from

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261I.N. Pessah et al. / Pharmacology & Therapeutics 125 (2010) 260–285

historical end uses in and around urban environments (legacy PCBs).Specific examples of PCB hotspots in the United States that contributeto higher human exposures include the San Francisco Bay watershed(Davis et al., 2007), the Hudson River watershed (Schneider et al.,2007; Asher et al., 2007), Chicago air (Sun et al., 2006; Hu et al., 2008;Zhao et al., 2009), and regions of Lake Erie near urban centers (Sunet al., 2007; Robinson et al., 2008). Thus, chronic low-level PCBexposures remain a significant public health concern since resultsfrom epidemiological studies indicate that PCB burden is associatedwith immune system dysfunction (Heilmann et al., 2006; Selgrade,2007; Park et al., 2008a,b), cardiovascular disease (Hennig et al., 2005;Humblet et al., 2008; Dziennis et al., 2008; Everett et al., 2008; Helyaret al., 2009), and impairment of the developing nervous system(Jacobson et al., 1992; Chen et al., 1992; Koopman-Esseboom et al.,1996; Schantz et al., 2003; Grandjean & Landrigan, 2006; Roegge &Schantz, 2006; Rogan & Ragan, 2007; Stewart et al., 2008). Of thesevarious adverse health effects, developmental neurotoxicity hasemerged as a particularly vulnerable endpoint in PCB toxicity.Whether neurological, immunological and cardiovascular impair-ments are interrelated by one or more convergent mechanisms, orarise independently from biologically distinct mechanisms continuesto be debated. Furthermore, which PCB structures confer specifichealth risks to the general public or to a susceptible population,remains unclear.

1.2. Non-dioxin-like polychlorinated biphenylstructures—convergent mechanisms mediated by ryanodine receptors

Of the 209 possible PCB congeners that were synthesized ascommercial mixtures, most of the scientific and regulatory attentionhas beendirected toward the so-called dioxin-like PCBs that lack at leasttwo chlorines in the ortho-positions. The phenyl rings of dioxin-likePCBs, for example PCB 77 (3,3′,4,4′-tetrachlorobiphenyl) and PCB 126(3,3′,4,4′,5-pentachlorobiphenyl), assume a coplanar orientation thatmimics the planar structure of dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD), the archetypical agonist for the arylhydrocarbonhydroxylase receptor (AhR) (Fig. 1). Coplanarity and lipophilicity are

Fig. 1. Coplanar structure of dioxin and two examples of dioxin-like PCBs. Non-dioxin-like Pthereby promoting non-coplanar geometry, as typified by PCB 95 and PCB 153. 3-D projectiLib. Med.).

arguably the two most important physicochemical parameters foroptimizing tight binding to AhR, although the position of para andmetasubstituents influences apparent binding affinity. A growing number ofenvironmental chemicals are known to activate or inhibit AhR, therebyregulating its translocation to the nucleus where it dimerizes with AhRnuclear translocator (ARNT) (Denison & Nagy, 2003). The AhR–ARNTcomplex binds to the DNA core sequence 5′-GCGTG-3′ in the promoterregion of dioxin-responsive genes to regulate their expression. Pro-longed activation of AhRand its responsive genes has been implicated indiverse toxicological sequelae associated with chronic, low-level expo-sures to TCDD, polycyclic aromatic hydrocarbons (PAHs), and coplanarPCBs (Carpenter, 2006; Mitchell & Elferink, 2009). Thus, the risk tohuman, fish and wildlife associated with PCB exposures is assessed byassigning an equivalence factor (TEF) that reflects the AhR activity ofany individual PCB congener relative to TCDD, which is arbitrarilyassigned a TEF of 1.0.

Several limitations of the TEF concept have been identified (Vanden Berg et al., 2006). Arguably the most important limitation forpredicting PCB toxicity based solely on an AhR-based TEF is the factthat PCBs having one or more chlorines in the ortho-positions arenon-coplanar structures with very low or no activity towards the AhRyet they exhibit significant toxicological activity. In vitro studies haveidentified PCB 95 (Fig. 1) among the most biologically active non-coplanar structures and its occurrence in human and environmentalsamples has been recently scrutinized using improved analyticalmethods. PCB 95 has been detected in human tissues (Covaci et al.,2002; Chu et al., 2003a; DeCaprio et al., 2005; Jursa et al., 2006), and inenvironmental samples including indoor and outdoor air, top soil,tidal marsh sediments, grass, diets, and human feces (Robson &Harrad, 2004; Harrad et al., 2006; Hwang et al., 2006; Zhao et al.,2009; Wong et al., 2009). Recent studies indicate that non-coplanarPCBs currently predominate in biological and environmental samples.For example, PCB 153 (Fig. 1) has been identified as a major con-tributor to total PCB burden in humans (Longnecker et al., 2003;Moonet al., 2009; Agudo et al., 2009; Axelrad et al., 2009).

The ortho-rich PCBs and metabolites of both ortho-rich and ortho-poor PCBs have a number of actions independent of the AhR that have

CBs have N2 chlorine substitutions in the ortho-position that introduce steric hindranceons were calculated using the Molecular Dynamics Tool of ChemIDplus Advanced (Nat.

262 I.N. Pessah et al. / Pharmacology & Therapeutics 125 (2010) 260–285

been termed “non-dioxin-like”. Mono-ortho-substituted PCBs mayhave dioxin-like and non-dioxin-like activities. However mono-orthocongeners commonly detected in tissues, such as PCB 118 (2,3,4,4′,5-pentachlorobiphenyl) and PCB 156 (2,3,3′,4,4′,5-hexachlorobiphenyl),have extremely low TEF values (≪0.0001), and their apparent AhRactivity could be largely attributed to coplanar contaminants (Peters,et al. 2006). Several biological activities have been experimentallydemonstrated with non-dioxin-like PCBs, and these were recentlyreviewed (Mariussen & Fonnum, 2006; Fonnum et al., 2006).Endocrine effects include weak estrogenicity (Safe, 2004), enhancedinsulin (Fischer et al., 1999) and arachidonic acid secretion (Baeet al., 1999a), and disruption of the hypothalamo–pituitary–thyroidaxis. Two possibly convergent mechanisms actively being investi-gated include (1) disruption of thyroid hormone metabolism andsignaling (Zoeller et al., 2000; Zoeller, 2005; Knerr & Schrenk,2006), and (2) perturbations in cellular Ca2+ signaling (Tilson andKodavanti, 1998; Pessah, 2001). Arguably the most pervasivebiological effects of PCBs could be mediated by their ability toalter the spatial and temporal fidelity of Ca2+ signals through one ormore receptor-mediated processes. The pivotal roles of Ca2+ signalsin regulating movement, metabolism, growth, proliferation, genetranscription and protein translation in virtually all cell types is wellestablished. Kodavanti et al. first reported that exposure to non-coplanar PCBs elevate cytoplasmic Ca2+ in cultured cerebellargranule neurons (Kodavanti et al., 1993), and several mechanismswere proposed including disruption of membrane properties(Kodavanti et al., 1996). A selective receptor-targeted mechanismwas also proposed based on the stringent structure–activityrelationship of PCBs for enhancing the activity of ryanodinereceptors (RyRs), a family of intracellular Ca2+ channels (Wong &Pessah, 1996). For example, PCB 95 and PCB 153 at concentrations≤10 µM lack detectable AhR activity, yet significantly enhance theactivity of type 1 (RyR1) and type 2 (RyR2) isoforms at concentra-

Fig. 2. Relative concentration of 28 non-coplanar PCBs needed to double [3H]ryanodine bindin Fox River fish, marsh sediments, and human serum (red bars). PCBs in parentheses are c

tions ≤1 µM (Wong & Pessah, 1996; Pessah et al., 2006). Fig. 2demonstrates the relative activity of 28non-coplanar PCBs toward RyR1and their relative contribution to total PCB burden reported in Fox Riverfish (52%) (Kostyniak et al., 2005), San Francisco Bay tidal marshsediments (∼50%) (Hwang et al., 2006), and human serum (∼45%)(DeCaprio et al., 2005). Becausenot all thePCBsdetected in these studieshave been tested for RyR activity, these estimates of the occurrence ofRyR-active PCBs are conservative. Therefore, the PCB congeners found inhighest abundance in environmental samples and tissues collected fromhumans and animals are capable of directly and potently interactingwith a major family of intracellular Ca2+ channels. RyRs are broadlyexpressed in most cell types where they participate in shapingtemporally and spatially defined Ca2+ signals that are essential forregulating several aspects of cellular signaling that regulate growth,movement, metabolism, secretion and plasticity.

Significant to the neurotoxic potential of PCBs, RyR channelactivity regulates a variety of physiological and pathophysiologicalprocesses in the central (Berridge, 2006; Dai et al., 2009), and periph-eral nervous systems (Jackson & Thayer, 2006; Buchholz et al., 2007;Behringer et al., 2009). Decrements in neonatal reflexes, cognitivefunction, motor activity, tremors, changes in autonomic functioning,and hearing impairments are consistent findings with developmentalPCB exposures in studies of humans and animals, and are primarilyattributed to adverse effects on the developing CNS (Stewart et al.,2000; Mariussen & Fonnum, 2006; Roegge & Schantz, 2006; Kenetet al., 2007; Darras, 2008; Fitzgerald et al., 2008). The possibility thatPCBs might directly influence peripheral neurons and their effectorsincluding skeletal, cardiac, and smoothmuscle, and cochlear hair cells,all of which express functionally essential RyRs, has not receivednearly as much investigation as their influence on the developingendocrine and central nervous systems.

In addition to TH, studies on the endocrine disrupting effects ofPCBs and related organohalogens have also focused on estrogen,

ing to ryanodine receptor type 1 (RyR1; black bars) and their corresponding occurrenceo-eluting congeners.


insulin, and their respective signaling pathways (Fonnum et al., 2006;Zoeller, 2007; Carpenter, 2008). Considering that RyRs have beenshown to regulate several aspects of these same endocrine functions(Islam, 2002; Dillmann, 2002; Muchekehu & Harvey, 2008), acommon convergent mechanism may contribute to pathologicalendocrine signaling and abnormal responses in target organs thatdepend on RyR activity for mediating appropriate Ca2+ signals.

This review will focus on our current knowledge of the structureand function of RyRs in muscle and nerve cells and how PCBs andrelated non-coplanar structures alter these functions. Our currentknowledge of how RyRs assemble into arrays and clusters, how theygenerate local releases of Ca2+ termed sparks, and trigger global Ca2+

waves is most advanced in studies of skeletal, cardiac and smoothmuscle. The topic is reviewed here first to provide context to themolecular mechanisms by which PCBs and related structuresinfluence RyR structure and function. The molecular and cellularmechanisms by which non-coplanar PCBs and related structures alterlocal and global Ca2+ signaling properties and the possible short andlong-term consequences of these perturbations on neurodevelopmentand neurodegeneration will then be discussed.

2. Ryanodine receptor macromolecular complexes:significance to polychlorinated biphenyl-mediated Ca2+ dysregulation

As might be predicted by their size and critical contribution tomuscle function, multiple factors contribute to the precise regulationof RyR channel activity. In skeletal and cardiac muscle at least 20protein–protein interactions have been described for the two majorisoforms RyR1 (type 1 RyR) and RyR2 (type 2 RyR), respectively(Fig. 3), and these interactions influence important aspects of Ca2+

channel function that are either essential for excitation–contraction(EC) coupling or fine-tune the spatial and temporal properties of localand global Ca2+ signals in the myocyte. Many of these interactions,when disrupted, have been shown to contribute to RyR-mediatedsusceptibility to muscle damage and to the progression of several

Fig. 3. Several proteins interact with RyRs to regulate their function as high conductanceinteracts with ion channels in the plasma membrane, cytoplasmic signaling proteins,transmembrane assembly that anchors RyRs to the ER/SR interacts with proteins that fine-tunhave been left out of the schematic.

muscle disorders. Similar functional and/or physical coupling of RyRsto context-specific proteins have been identified in smooth muscle,neurons and other cell types.

The composition of RyRmacromolecular complexes is therefore animportant consideration when interpreting the seemingly diverse invitro and in vivo actions attributed to non-coplanar PCB congenersand Aroclor mixtures in a wide variety of tissues and cell types. PCBshave been shown to enhance Ca2+ release from sarcoplasmicreticulum/endoplasmic reticulum (SR/ER) and mitochondrial stores,and to promote Ca2+ entry, but whether these effects stem fromconvergent receptor-mediated mechanisms or multiple “nonspecific”influences onmembrane integrity has been debated (Pessah, &Wong,2001; Inglefield et al., 2001). PCBs also alter the activities of RyR“accessory” proteins including the multifunctional protein kinases,PKA (Inglefield et al., 2002; Llansola et al., 2009) and PKC (Kodavantiet al., 1994), calmodulin (Olivero & Ganey, 2001; Benninghoff &Thomas, 2005), and FKBP12 (FK506 binding protein 12 kDa), themajor T-cell immunophilin (Wong & Pessah, 1997; Wong et al.,2001b; Gafni et al., 2004). Additionally, PCB toxicity in excitable andnon excitable cells appears to be mediated at least in part by oxidativestress and involves biotransformation via quinone and hydroxylatedmetabolites, and altered activities of key antioxidant defense enzymessuch as glutathione transferases, NADH/NAD(P)H oxidoreductases,and possibly selenoproteins (Howard et al., 2003; Murugesan et al.,2007; Duntas, 2008; Wei et al., 2009; Liu et al., 2009a). Many ofthese proteins have been directly implicated in redox regulation ofRyRs.

2.1. Organization, function anddysfunctionof ryanodine receptor complexes

Ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphatereceptors (IP3Rs) are a family of Ca2+ channels that are broadlyexpressed throughout the central and peripheral nervous systems.The distribution, structure, and function of RyRs are best understoodin striated muscle where expression of RyR1 and RyR2 isoforms are

Ca2+ channels in striated muscle. The large cytoplasmic assembly (“junctional foot”)and cytoplasmic enzymes that regulate phosphorylation and redox sensing. Thee communication with the Ca2+ stores within the SR/ER lumen. For clarity, interactions


essential for engaging the process of EC coupling in skeletal andcardiac muscle, respectively. In mice, targeted deletion of RyR1 resultsin a birth-lethal phenotype (Takeshima et al., 1994; Buck et al., 1997),whereas RyR2 null mice do not survive past embryonic day 9(Takeshima et al., 1998). These phenotypes are consistent with thefailure to engage EC coupling in these tissues at times critical for theanimal's survival. A third isoform, RyR3, is not essential for ECcoupling although it is transiently expressed in skeletal muscle duringembryonic development and is down regulated in most, but not all,fibers postnatally (Tarroni et al., 1997; Conti et al., 2005; Legrandet al., 2008). Targeted deletion of RyR3 does not impair musclefunction or survival, but does seem to impact neurobehavioral func-tion (Section 2.5.2).

2.2. Ryanodine receptors in striated muscle

In striated muscle, RyR channels are anchored to specializedregions of the SR of the developing myotube and adult fiber, termedjunctional regions, where the SR and transverse tubule membranesapproach within 10–15 nm of each other (Fig. 4). In the context ofthese junctions, RyRs organize into tetrameric arrays or clusters thatspan the junctional SR-T tubule space (Franzini-Armstrong et al.,2005; Di Biase & Franzini-Armstrong, 2005). Each tetramer has four-fold symmetry and constitutes a functional channel of ∼2.2 MDa thatregulates releases of Ca2+ stored within the lumen of the SR into thecytoplasm.

Coordinated gating (activation and inactivation) of multiple RyRchannels generates spatially limited and temporally defined release ofCa2+ sparks (Gonzalez et al., 2000; Chun et al., 2003; Gyorke et al.,2007; Cheng & Lederer, 2008). Thus sparks represent quantal releasesof Ca2+ from the lumen of junctional SR to a restricted area of thecytoplasm. Although spontaneous and evoked Ca2+ sparks arecommonly observed in cardiomyocytes, smooth muscle myocytes,and invertebrate skeletal muscle, they are rarely detected in intactadult mammalian skeletal muscle under physiological conditions. Thelack of sparks in mammalian skeletal muscle is likely because the L-type Ca2+ channel CaV1.1 confers direct negative regulation of RyR1(Lee et al., 2004; Zhou et al., 2006). However, Ca2+ sparks can bereadily detected in mammalian skeletal muscle under pathophysio-logical conditions that generate reactive oxygen species (ROS)(Martins et al., 2008). This is not unexpected since both RyR1 andRyR2 are exquisitely sensitive to sulfhydryl modification by com-pounds that generate ROS (Abramson et al., 1988; Favero et al., 1995),redox cyclers, and cysteine arylators (Abramson et al., 1988; Pessahet al., 1990; Feng et al., 1999; Pessah et al., 2001; Pessah et al., 2002;

Fig. 4. (A) Electron micrograph of the T-tubule/SR junction of negatively stained skeletal mlarge cytoplasmic domain of a row of RyR1s that span the junctional space between theorientation of four CaV1.1 (i.e., α1s) L-type Ca2+ channel subunits (brown) and RyR1 (gree

Gao et al., 2005). Highly reactive (hyper-reactive) cysteine residueswithin the RyR sequence and possibly accessory proteins have beenidentified, and these confer redox sensing properties to the Ca2+

channel complex (Liu & Pessah, 1994; Liu et al., 1994; Voss et al.,2004; Phimister et al., 2007; Jurynec et al., 2008). Shifts in RyR activityin response to localized changes of glutathione (GSH/GSSG), nitricoxide, oxygen, and ROS appear to constitute a fundamental physio-logical and pathophysiological mechanism that adjusts local andglobal cellular Ca2+ signals to the changing local redox environment.The mechanisms appear to involve glutathionylation, nitrosylation,electron delocalization, and oxidation of hyper-reactive cysteineswithin RyR complexes (Zable et al., 1997; Xia et al., 2000; Feng &Pessah, 2002; Aracena et al., 2003; Aracena-Parks et al., 2006; Sunet al., 2008; Terentyev et al., 2008). PCBs appear to mediate toxicity, atleast in part, by mechanisms involving oxidative stress (Howard et al.,2003; Lyng & Seegal, 2008; Glauert et al., 2008; Liu et al., 2009a). Thus,in addition to direct binding of PCBs to RyRs, PCB metabolites thatpossess redox active moieties, such as quinones and semiquinones(Machala et al., 2004; Spencer et al., 2009), would be expected toconfer additional activities towardmodifying RyRs and their signalingevents. In this regard, site-selective oxidation of RyRs was alreadydemonstrated for naphthoquinones, benzoquinones, and benzo[a]pyrene 7,8-dione (Feng et al., 1999; Pessah et al., 2001; Gao et al.,2005).

As spark frequency and spatial spread of Ca2+ increases withphysiological (e.g. plasma membrane depolarization) or pharmaco-logical (e.g., caffeine) stimuli, Ca2+waves are initiated that are capableof propagating throughout the cell in which they occur (Cheng &Lederer, 2008). Thus activation of RyRs is a means of eliciting con-trolled release of SR Ca2+ stores and is amajor source of both local andglobal Ca2+ signals that are essential for regulating contractility ofstriated and smooth muscle.

2.2.1. Could non-coplanar polychlorinatedbiphenyls alter ryanodine receptor function in striated muscle?

Skeletal and cardiac muscle represent major organs for the initialdisposition of PCBs following exposure (Matthews & Anderson, 1975;Matthews & Tuey, 1980; Brandt et al., 1981; Birnbaum, 1983). Muscletissues are therefore considered critical compartments when formu-lating physiologically based pharmacokinetic models that faithfullyreproduce tissue:blood partitioning (Parham et al., 1997). A recentstudy of Galapogos sea lions identified the congener profile of PCBsfound in muscle biopsies (Alava et al., 2009). Results from this studyindicate that the 10 most active congeners identified based on [3H]ryanodine-binding analysis (Fig. 2) represented nearly 25% of the total

yotubes. Arrows indicate the position of densely staining “junctional feet” that are thetwo membranes (adapted from (Protasi et al., 1998)). (B) 3-D model of the relativen) based on cyroEM reconstruction studies (adapted from (Wolf et al., 2003).


PCB burden found inmuscle (Alava et al., 2009). In rodents exposed toPCB 136, the low-dose group displayed significantly higher PCB levelsin cardiac and skeletal muscles compared to other tissues, such asadipose tissue and the liver (Kania-Korwel et al., 2008a,b).

Surprisingly, if and how non-coplanar PCBs modify the propertiesof striated muscle EC coupling has only recently been examined. Thephysical interactions between CaV1.1 and RyR1 that trigger skeletalmuscle EC coupling provide a relevant experimental model for testingwhether non-coplanar PCBs can impair the fidelity of this otherwisetight form of conformational coupling between two Ca2+ channels.The amplitude of Ca2+ transients in mouse embryonic myotubeselicited by low frequency (0.1 Hz) electrical pulses was significantlyenhanced within 2.5 min after initiating perfusion of 5 µM PCB 95 inthe external medium compared to the solvent control, and PCB 95prevented recovery of the Ca2+ signal to its original baseline (Fig. 5Aand B) (Cherednichenko & Pessah, in preparation). When higherfrequency electrical pulse trains were evoked (2.5 and 5 Hz), PCB 95not only amplified Ca2+ transient amplitudes but also caused ectopicCa2+ transients immediately after electrical stimuli were suspended(arrows, Fig. 5C), and these were not observed in the absence of PCB.How non-coplanar PCBs and related structures influence the couplingbetween RyRs and plasmalemmal Ca2+ channels (Fig. 4), and theirdirect impacts on skeletal and cardiac muscle function clearly needsmore attention.

2.2.2. Ryanodine receptor associations with plasma membrane proteinsOver the last 15 years major advances have been made in

understanding how RyR structure relates to function and identifyingkey accessory proteins that regulate activation and inhibition of Ca2+

release from ER/SR stores. Most of our understanding comes fromstudies of RyR1 in skeletal muscle and RyR2 in cardiac muscle wherethey assemble inmacromolecular complexes termed Ca2+ release units(CRUs). The central component of the CRU is the RyR homotetramer,

Fig. 5. Skeletal myotubes acutely exposed to 5 µM PCB 95 exhibited significantly higherCa2+ transient amplitudes evoked by low frequency (0.1 Hz) electrical pulse trains(A&B; pb0.05) and a failure to recover their original baseline (i.e., resting Ca2+ level)compared to the corresponding control period when solvent (DMSO) alone wasperfused (Ctrl). (C) Responses to 10 s electrical pulse trains of 2.5 or 5 Hz resulted insignificantly higher transient amplitudes compared to the corresponding control (Ctrl)period. Ectopic Ca2+ transients (red arrows) are frequently observed soon afterelectrical stimuli ceased in the PCB-exposed myotubes and were not observed incontrol. Adapted from (Cherednichenko, in preparation).

with a small C-terminal transmembrane assembly anchored within themembrane and a massive cytoplasmic (“junctional foot”) assemblyspanning the T-tubule SR junction (Franzini-Armstrong et al., 2005; DiBiase & Franzini-Armstrong, 2005) (Fig. 4). Although the composition ofkey proteins comprising the CRUs of skeletal and cardiac muscles arestrikingly similar (Fig. 3), skeletal muscle does not require Ca2+ entrythrough the L-type voltage-gated Ca2+ channel (CaV1.1) to triggeractivation of RyR1 and SR Ca2+ release. Rather, CaV1.1 and RyR1physically interact such that four CaV1.1 subunits in the T-tubulemembrane orient over every secondRyR1anchored to the junctional SR.This structural arrangement engages a form of bidirectional signaling inwhichT-tubule depolarization is sensedby the S4 segmentof CaV1.1 andtransmitted to RyR1 through conformational shifts of the “criticaldomain” (residues 720–765 of CaV1.1) residing within the cytoplasmicloop between repeats II and III (Grabner et al., 1999). RyR1 transmits aretrograde signal to not only enhance L-type Ca2+ entry current (Nakaiet al., 1996; Fleig et al., 1996; Sheridan et al., 2006), but also affectprecise alignment of CaV1.1 and associated subunits into tetradic arrayswithin the T-tubule membrane (Protasi et al., 1998). In myotubes andsome adult fibers, retrograde signaling from RyR1 to CaV1.1 appears tobe essential for preventing decrement and ultimate failure of ECcoupling during prolonged high frequency (tetanic) electrical pulsetrains, through a process termed excitation-coupled Ca2+ entry (ECCE)(Cherednichenko et al., 2004a).

Initial results using pharmacological tools indicated that CaV1.1was the voltage sensor that triggers ECCE, and that the L-type Ca2+

channel was unlikely to constitute the Ca2+ conduction pathway forECCE. More recent evidence indicates that the L-type channel is amajor contributor to ECCE (Bannister et al., 2009). Pharmacologicalagents that influence conformational states of RyR1 influenceelectrophysiological properties of the L-type Ca2+ current and ECCEin similar ways. For example, experimental conditions that permit thealkaloid ryanodine to lock RyR1 channels in a persistently closed (non-conducting) conformation (Zimanyi et al., 1992) also causes signifi-cant reduction in the inter-tetrad distances of the CaV1.1 complex(Paolini et al., 2004) and influences the activation–deactivationkinetics of ECCE (Cherednichenko et al., 2004a; Bannister et al.,2009). Importantly, missense mutations that affect RyR1 function candramatically alter the properties of both orthograde and retrogradesignaling, modifying channel functions on both side of the junction(Hurne et al., 2005; Yang et al., 2007a; Cherednichenko et al., 2008;Andronache et al., 2009; Bannister et al., 2009).

In contrast to skeletal muscle, conformational coupling between L-type Ca2+ channels (CaV1.2) and RyR2 is not sufficient to trigger ECcoupling in the absence of Ca2+ entry across the T-tubular membranein cardiomyocytes. Rather tight functional coupling between L-typeCa2+ entry mediated by CaV1.2 channels within the T-tubulemembrane and RyR2s occurs across narrow 12 nm junctions throughthe process of Ca2+-induced Ca2+ release (CICR) (Bers, 2008).Depolarization of the T-tubule membrane triggers CaV1.2-mediatedlocalized Ca2+ influx (termed sparklets) that in turn triggers RyR2-mediated sparks immediately across the junctional space. Dependingon the intensity of stimuli arriving at the T-tubule membrane andother local “environmental” factors that influence RyR2 activity (e.g.,phosphorylation state), summation of sparks can produce local andpropagated Ca2+waves through the process of CICR (Cheng & Lederer,2008).

In addition to interactions with voltage-gated channels (CaV1.1 andCaV1.2) as described above, RyRs have been shown to directly orindirectly interactwith and regulate the functionof store-operatedCa2+

channels (SOCCs) in some, but not all muscle tissues examined. Forexample, the transient receptor protein channels TrpC3 (Kiselyov et al.,2000; Lee et al., 2006a; Woo et al., 2009), TrpM4 (Morita et al., 2007),and TrpV4 (Earley et al., 2005) were shown to interact with RyRcomplexes. However, Trp–RyR interactions may not be the majormechanismresponsible for store-operatedCa2+entry (SOCE) in skeletal


muscle. Knockdown of STIM and Orai, the essential components of theCa2+-release activated Ca2+ current (ICRAC), greatly suppresses SOCE inskeletalmuscle cellswithout impairing ECCE (Lyfenko&Dirksen, 2008),and SOCE is not dependent on RyR1 expression (Cherednichenko et al.,2004a; Lyfenko&Dirksen, 2008). However, STIMwas recently shown togate TrpC channels by electrostatic interaction (Zeng et al., 2008).

Homer is a family of proteins shown to regulate signal transduc-tion, synaptogenesis, and receptor trafficking in neurons (Xiao et al.,2000; Szumlinski et al., 2006). Both short and long forms of Homerinteract with RyR1 and RyR2 and regulate channel function in anadditive biphasic manner that is highly dependent on their relativeconcentration, but is independent of multimerization via the coiled-coil domains found in Homer long forms (Feng et al., 2002; Wardet al., 2004; Feng et al., 2008; Pouliquin et al., 2009). As demonstratedearlier in neurons where Homer links IP3R responses to mGluR1signaling, Homer may also play a scaffolding function in striatedmuscle linking RyR1 and RyR2 to CaV1.1 or CaV1.2, respectively, toregulate the gain of EC coupling (Huang et al., 2007; Pouliquin et al.,2009). In skeletal muscle, Homer appears to also link TrpC channels tothe cytoskeletal matrix and function to regulatemechanotransduction(Stiber et al., 2008).

Three important consequences of co-localization and functionalcoupling of RyRs with Ca2+ channels residing in the surfacemembrane include: (1) it permits local signaling microdomains; (2)it confers a mechanism for reciprocal regulation between channels inthe plasma membrane and RyRs anchored within the SR/ERmembrane; and (3) it provides direct feedback about the state ofCa2+ filling within the SR/ER lumen. Thus chemical agents thatinteract with RyR and alter its conformation and function are likely toinfluence not only the properties of Ca2+ release from stores, but alsoCa2+ entry through SOCE and ECCE mechanisms.

2.2.3. Ryanodine receptor associations with cytoplasmic proteinsTwo T-cell immunophilins, FK506 binding protein 12 kDa (FKBP12)

and its isoform FKBP12.6 (also referred to as calstabins 1 and 2) cantightly bind to RyR1 (Jayaraman et al., 1992) and RyR2 (Timerman et al.,1996). Although up to four FKBPs can bind per functional RyR channel(one per subunit), the ratio of FKBP isoform/RyR is likely to varyaccording to tissue and with changing physiological or pathophysio-logical states (Zalk et al., 2007; Lehnart, 2007). Cryo-electron micros-copy (EM) reconstruction of frozen hydrated RyR1 tetramers revealedthat FKBP12binds adjacent to cytoplasmic domain 9 of the clamp regioncomplex (Wagenknecht et al., 1997; Samso et al., 2006; Serysheva et al.,2008) (Fig. 6A, top view of cytoplasmic “foot” domain). Mutagenesisstudieswith RyR1 suggest essential contributions of Val-2461-Pro-2462(corresponding RyR2 residues Ile-2427-Pro-2428) in the bindinginteraction (Gaburjakova et al., 2001), but the N-terminal residues305-1937 of RyR2 may also contribute to binding FKBP12.6 (Masumiya

Fig. 6. (A) 3-D structure of RyR1 in the closed state at 10 Å resolution showing the location otriggered Ca2+ release from skeletal junctional SR is inhibited by pre-incubating with rapam

et al., 2003). Since the first report that FKBP12 stabilizes the functionalbehavior of RyR1 (Brillantes et al., 1994), disruption of FKBP12/12.6 RyRcomplexes has been associatedwith heritable and acquired disorders ofcardiac (Zalk et al., 2007; Gyorke& Carnes, 2008) and skeletal (Bellingeret al., 2008; Bellinger et al., 2009) muscle.

PCB-triggered Ca2+ release from junctional SR membrane vesiclesisolated from skeletal muscle can be selectively negated by pretreat-ment with either the immunosuppressive drug FK506 or rapamycin(Fig. 6B)without inhibiting responses to other RyR1 channel activatorssuch as caffeine (Wong & Pessah, 1997). FK506 and rapamycin tightlybind to the greasy binding pocket of FKBP12 promoting dissociation ofthe FKBP12/RyR1 complex and possibly preventing rebinding. Rapa-mycin and FK506 interferewith the actions of PCB 95 (and other activePCBs) in the same concentration range that promotes the dissociationof the FKBP12/RyR1 complex, suggesting that PCBs interact with abinding site formed at the FKBP12/RyR1 complex interface to enhancechannel open probability (Fig. 6A). However, an allosteric mechanismhas not been ruled out.

Nevertheless, themolecular mechanism bywhich PCB 95 affects theRyR1/FKBP12 ismediatedbydirect stabilizationof the channel in the fullopen state (Fig. 7). Samso andcoworkers (2009) recentlyutilizedPCB95to better understand the basis for RyR1 conformational transitionsbetween closed and open states. Single-channel biophysical character-ization of the two states in bilayer lipid membranes (Fig. 7, left andmiddle panels) and cryoelectron microscopy of frozen single-particlesin their hydrated state (Fig. 7 right panel) were performed on identicalsamples and conditions to permit direct correspondence betweenbiophysical state and structural conformation of the channel. PCB 95appears to invert the thermodynamic stability of the RyR1/FKBP12channel complex producing extremely long-lived openings inter-spersed with extremely short-lived transitions to the closed state,although the unitary current is indistinguishable from the native openstate. By contrast, the presence of very lowCa2+on the cytoplasmic side(pCa2+b108 set in the presence of the Ca2+ chelator EGTA) after fusionof an actively gating channel completely stabilized the fully closed stateof the channel. The corresponding three-dimensional structures at∼10 Å resolution provided information about the structure surroundingthe ion pathway indicating the presence of two right-handed bundlesemerging from the putative ion gate (the cytoplasmic “inner branches”and the transmembrane “inner helices”) (Samso et al., 2009). The PCB95 modified state causes a precise relocation of the inner helices andinner branches resulting in an ∼4 Å increase in diameter of the ion gate(Fig. 7, right panel). Six of the identifiable transmembrane segments ofRyR1 have similar organization to those of the mammalian Kv1.2potassium channel. Upon gating to the PCB 95-induced open state, thedistal cytoplasmic domains move towards the transmembrane domainwhile the central cytoplasmic domains move away from it, and alsoaway from the four-fold axis (Samso et al., 2009).

f the FKBP12 docking site near domain 9 (adapted from (Samso et al., 2009). (B) PCB 95ycin that disrupts the FKBP12/RyR1 complex (adapted from (Wong & Pessah, 1997).

Fig. 7. PCB 95 directly stabilizes the fully open (conducting) conformation of the RyR1/FKBP12 channel complex reconstituted in the bilayer lipid membrane preparation, whereasEGTA fully closes the channel (left and middle panels). Right panels show the corresponding structural shifts calculated from cryoEM reconstruction of single hydrated particlesshowing themain constrictions along the ion pathway in the closed and open states. The cytosolic constriction (cc) relaxes and the inner branches (ib) becomemore separated in theopen state. Adapted from (Samso et al., 2009).


Similar FKBP12/RyR1-dependent Ca2+ release anddirect activationof RyR1 channels have been demonstratedwith bastadin-5 (Fig. 8) andbastadin-10, two members of a family of over 20 bromotyrosine-derived macrolactams that have been isolated from the Verongidmarine sponges Ianthella sp. (Mack et al., 1994; Chen et al., 1999). LikePCB 95, bastadin-10 (B10) dramatically stabilizes the open conforma-tion of the RyR1 channel, possibly by reducing the free energyassociated with closed to open channel transitions. The stability of thechannel open state induced by B10 sensitized the channel to activationby Ca2+ to such an extent that it essentially obviated regulation byphysiological concentrations of Ca2+ and relieved inhibition byphysiological Mg2+. These actions of B10 were produced only on thecytoplasmic face of the channel, were selectively eliminated bypretreatment of channels with FK506 or rapamycin, and werereconstituted by exogenous addition of human recombinant FKBP12.Bastadin-10 dramatically enhances spark frequency and duration inadult frog skeletal muscle fibers (Gonzalez et al., 2000), whereasbastadin-5 was shown to influence both resting Ca2+ and caffeine-evoked transients in cultured myotubes (Pessah et al., 1997; Yanget al., 2007b). The reduced pharmacophore that confers RyR activityresideswithin the ‘eastern’ and ‘western’non-coplanar bromocatecholether moieties that resemble hydroxylated brominated dipheny-lethers defined by the dashed boxes in Fig. 8 (Masuno et al., 2006).

The widely used antibacterial triclosan, a non-coplanar chloroca-techol ether (Fig. 8), was shown to activate RyR1 and mobilize Ca2+

from SR stores of intact primary skeletal myotubes (Ahn et al., 2008).Both non-coplanar PCBs and bastadins also enhance RyR2 activityalthough the requirement for FKBP12.6 has not been reported.

Calmodulin (CaM) and S100A1 are two widely expressed Ca2+

binding proteins that interact with RyR1 and RyR2 isoforms in a Ca2+-

dependent manner. Both proteins appear to compete with a commonconserved site within the clamp domains corresponding to residues3614-3643 (Wright et al., 2008). Although ApoCaM can enhance RyRactivity, it is thought that Ca2+-CaM provides the major physiologicalregulatory role, inhibiting RyR channel activity as local Ca2+ concen-tration rises (Meissner, 2002). By contrast, Ca2+-S100A1 stimulates RyRchannel activity. Competition between Ca2+-CaM and Ca2+-S100A1 onRyRs has been proposed to confer tight but dynamic regulation of ECcoupling gain with temporal changes in local Ca2+ concentration inskeletal and cardiacmuscle (Wright et al., 2008). PCB 95 and bastadin-5and 10 were shown to dramatically shift the Ca2+ dependence of RyR1channel activation and inhibition independently of exogenously addedCaM or SA1001A (Wong & Pessah, 1996; Chen et al., 1999). Whetherthese compounds shift the dynamic regulation mediated through CaMand S100A1 remains to be investigated.

The Ca2+ binding protein sorcin and presenilin 2 (PS2) areubiquitously expressed in various tissues including neurons andcardiomyocytes and each has been demonstrated to influence RyRfunction. Sorcin interactswith the C-terminal endoproteolytic fragmentof PS2 in a Ca2+ dependent manner, but not with full-length PS2 (Pack-Chung et al., 2000), but the interaction may not be essential formodulationofRyR. Inheart tissues, PS2was shown tophysically interactwith RyR2. Papillary muscle PS2 knockout mice display enhanced peakamplitudes of Ca2+ transients and peak tension compared to wild type(Takeda et al., 2005). Sorcin inhibits Ca2+ channel activity and atten-uates spark activity and was proposed to contribute a physiologicalmeans for terminating Ca2+ induced Ca2+ release in cardiac muscle(Farrell et al., 2003; Stern & Cheng, 2004; Farrell et al., 2004).

At least three Ser residues within the junctional foot assembly ofRyR1 (Ser 2844) and RyR2 (Ser 2808, Ser 2814, Ser 2030), can be

Fig. 8. Bastadin 5 showing “eastern” and “western” dibromocatechol ethers (red boxes) that are the putative pharmacophores for RyR1. The structure of the antibacterial triclosan isshown in the lower right.


phosphorylated by PKA (Wehrens et al., 2006; Aydin et al., 2008), Ca2+-CaM kinase II (Currie et al., 2004; Huke & Bers, 2008) and possibly PKC(Takasago et al., 1991). Evidence that the scaffolding protein mAKAPtethers PKA in close proximity to protein phosphatase A1 and A2 (PPA1and PPA2) within the RyR2 complex, possibly through a leucine/isoleucine zipper (LIZ) motif, suggests tight regulation of RyR2phosphorylation in response to changes in cellular cAMP, such asthose that normally occur with β-adrenergic stimulation (Marx et al.,2001; Marks et al., 2002). However, RyR phosphorylation is complex inboth striated muscle and neurons, and appears to be dynamicallyregulated with shifting physiological and pathophysiological states(Zalk et al., 2007; Dai et al., 2009).

Altered patterns of RyR phosphorylation are associated withchanges in RyR nitrosylation, glutathionylation, and depletion ofFKBP12/12.6 from its binding site located within the clamp region.Importantly these changes have been strongly implicated in theetiology of several heritable and acquired disorders of striatedmuscle,including malignant hyperthermia susceptibility (MHS) and centralcore disease (CCD) (Durham et al., 2008), Duchenne musculardystrophy (Bellinger et al., 2009), catecholaminergic polymorphicventricular tachycardia (CPVT), arrhythmogenic right ventriculardysplasia type 2 (ARVD2), and ischemic heart failure (Blayney & Lai,2009). Over 120 missense or deletion mutations within RyR1 havebeen associated with MHS, central core disease (CCD) and/ormultiminicore disease (MmD) in skeletal muscle (Robinson et al.,2006). Nearly 25 mutations have been identified within RyR2 thatcontribute to CPVT (Gyorke, 2009; Liu et al., 2009b). Significantincreases in RyR Ser phosphorylation and Cys nitrosylation have beenassociatedwith an increased Ca2+ leak through RyR channels carryinga few of these mutations and these can markedly reduce the Ca2+

content of the ER/SR lumen. RyR mutations also alter the fidelity ofECCE (Yang et al., 2007a; Cherednichenko et al., 2008) perhaps furthercompounding SR Ca2+ depletion. Whether hyper-phosphorylation,nitrosylation, and/or glutathionylation of RyRs with mutations arecommon convergence points of CRU dysfunction and progression ofthese diverse disorders is currently being intensely investigated. Ofrelevance to the toxicity of PCBs and related chemicals that alter thefunction of RyRs, it should be noted that RyR mutations may remainphenotypically silent or subclinical until triggered by one or moreenvironmental exposures or stressors. Recently, changes in thephosphorylation state (at Ser 2808 and Ser 2814) were associated

with functional impairments in cardiac RyR2 channels in thestreptozotocin-induced model of type 1 diabetes (Shao et al., 2009).

Several enzymes and proteins involved in regulating cellular redoxstatus have been also demonstrated to directly regulate RyR channelactivity. The mu isoform of glutathione-s-transferase (GST mu) wasshown to promote inactivation of RyR1 and RyR2, whereas its distantrelative CLIC-2 that functions as a chloride channel appears toselectively attenuate the activity of RyR2 in the presence of a GSH:GSSG redox buffer (Abdellatif et al., 2007; Jalilian et al., 2008; Menget al., 2009). Selenoprotein N (SepN) is physically associated withRyR1 of skeletal muscle and it appears to be essential for conferringregulation of RyR1 channels by GSH:GSSG redox potential (Jurynecet al., 2008). Importantly the absence of SepN in skeletal muscleappears to contribute to a subset of congenital myopathies and alteredredox regulation of RyR1 has been implicated in their etiology.

NAD(P)H oxidases are major sources of superoxide generation inmyocytes, especially during arrhythmia and after acute myocardialinfarction. For example tachycardia augments the association of theNAD(P)H oxidase cytosolic subunit p47phox to the SR fraction,without modifying the content of the membrane integral subunitgp91phox (Sanchez et al., 2005). The enzymatic oxidation of NADH istightly linked with negative regulation of RyR2 channel activity incardiacmyocytes. Inhibition of NADH oxidase activity with nanomolarrotenone or pyribaben relieves this negative regulation and increasesRyR2 channel activity, spark frequency, and Ca2+ oscillations incardiomyocytes (Cherednichenko et al., 2004b).

RyRs are emerging as a central integrator of not only physiologicalsignals, but also of pathophysiological responses to oxidative stress thatmay arise from mutations in the RyR proteins themselves, theiraccessory proteins, metabolic imbalances, or insults from xenobioticchemicals such as PCBs. Whether RyR-active PCBs and related non-coplanar structures influence the phosphorylation, nitrosylation, and/orglutathionylation state of the receptor complex has not been explored.

2.2.4. Ryanodine receptor associations withintegral and luminal sarcoplasmic reticulum proteins

Within the junctional SR sacks of cardiac and skeletal muscle, RyRsform a protein complex with triadin and junctin (which are anchoredwithin the membrane), and calsequestrin that resides within the SRlumen (Liu & Pessah, 1994; Guo et al., 1996). Ultrastructural andbiochemical evidence supports a model in which triadin and junctin


interact directlywith RyRs andmay act to scaffold calsequestrin (a lowaffinity SR Ca2+ binding protein) near the RyR lumen to control theavailability of Ca2+ near RyRs thereby providing luminal control(feedback) to the CRU about the local filling state of the Ca2+ store(Beard et al., 2009). Ablation of either triadin or calsequestrinexpression in the heart results in ventricular arrhythmias (Chopraet al., 2007, 2009). Both PCBs and bastadins were shown to influencethe balance of regulated RyR1 channels (i.e., those sensitive toryanodine) and their ryanodine-insensitive “leak” states in isolatedSR and BC3H1 cells, possibly by converting low conductance leakstates into high conductance channel states (Wong & Pessah, 1997;Pessah et al., 1997). More recently, bastadin 5's ability to convertryanodine-insensitive Ca2+ leak to ryanodine-sensitive channels wasshown to lower resting free Ca2+ in intact myotubes expressing wildtype RyR1 and those expressingMH susceptibility mutations, but onlyin the presence of RyR1 channel blockers (ryanodine or FLA365) (Yanget al., 2007b). In this regard, myotubes with MH mutations havesignificantly higher resting intracellular Ca2+ levels than thoseexpressing wild type RyR1, and the reductions afforded by bastadin5 in the presence of ryanodine are significantly greater (Fig. 9).

Junctophilins are integral ER/SR proteins that form non-covalentinteractions with membrane lipids through their MORN (membraneoccupation recognition nexus) motifs, and are primarily responsible forstabilizing close apposition of the junctional regions SR/ER and theplasmalemma in bothmuscle cells and neurons (Takeshima et al., 2000;Kakizawa et al., 2008). Junctophilins are therefore essential for creatinga specialized milieu that permits the large junctional foot assembly ofRyRs to physically and functionally interact with proteins in the plasmamembrane. The importance of proper assembly of junctional assemblyhas been recently underscored in mice with targeted deletions ormutations in junctophilins. Deletion of junctophilin isoform 1 (JP1), themajor form expressed in skeletal muscle, results in weak EC couplingand contractile failure that is lethal shortly after birth (Ito et al., 2001).The lack of JP1 may also negate critical aspects of channel regulationbecause JP1 and RyR1 interact in a conformationally sensitive mannerthat involves hyper-reactive Cys residues (Phimister et al., 2007).Deletion of junctophilin isoform 2 (JP2), the major form in cardiacmuscle functionally uncouples CaV1.2 and RyR2 and is embryonic lethaldue to contractile failure, whereas 5 missense mutations are associatedwith hypertrophic cardiomyopathy in humans (Matsushita et al., 2007;Landstrom et al., 2007).

Mice lacking neural junctophilins (JP3 and JP4) exhibit impairedexploratory behavior in the open-field task, and impaired performancein the Y-maze and multiple-trial passive avoidance tests indicatingimpaired short- and long-term memory (Moriguchi et al., 2006).Ablation of JP3 and JP4 uncouples functional interactions amongNMDA receptors, ryanodine receptors, and small-conductance Ca2+-activatedK+ channels activation. These results reveal that activation ofsmall-conductance Ca2+-activatedK+ channels,which is necessary forafterhyperpolarization in hippocampal neurons, requires Ca2+ release

Fig. 9. Bastadin 5 in the presence of a ryanodine concentration that blocks RyR1channels reduces resting Ca2+ to a greater extent in cells expressing missensemutations that confer MH susceptibility to humans than in cell expressing wild typeRyR1 (Wt). ⁎pb0.05 adapted from (Yang et al., 2007a, b).

through RyRs, and is triggered by NMDA receptor-mediated Ca2+

influx. Junctophilins stabilize close apposition of “junctional” postsyn-aptic membranes that permit crosstalk between RyRs and theirsignaling partners in the plasmamembrane. These observations revealthe essential role of RyRs in mediating changes in synaptic plasticity(Kakizawa et al., 2008). These newly discoveredmechanismsmight bedirectly relevant to how PCBs mediate neurotoxicity.

2.3. Structure–activity of polychlorinated biphenyls toward RyR1 and RyR2

Non-coplanar PCBs were shown to potently and selectivelysensitize both RyR1 and RyR2 channel activities in studies with SRmembranes isolated from mammalian skeletal and cardiac muscle,respectively, using radioligand binding studies with [3H]ryanodine([3H]Ry) and macroscopic Ca2+ flux measurements across isolated SRmembrane vesicles (Wong & Pessah, 1996). [3H]Ry binds to RyR1withhigh selectivity and specificity only to an activated conformation ofRyRs, thereby providing a rapid and quantitativemethod for screeningchemicals that enhance or inhibit channel activity (Pessah et al., 1985;Pessah et al., 1987). Fig. 10 (left panel) highlights two importantaspects of the PCB structure–activity relationship towards RyR1: (1)chlorine substitutions at the ortho-positions which restrict thebiphenyl rings to non-coplanarity; and (2) the contribution of para-substituents which can reduce or eliminate activity. For example PCB126, one of the most potent PCB congeners toward the arylhydro-carbon hydroxylase (Ah) receptor, lacks activity toward RyR1 at itssolubility limits. In general, PCBs lacking at least one ortho-substitutionare inactive toward RyR1 and RyR2, regardless of the degree ofchlorination. A similar structure–activity profile applies for activationof RyR2 channels isolated from cardiacmuscle (Wong& Pessah, 1996),

ER preparations isolated from adult cerebellum, hippocampus orcortex contain all three RyR isoforms, although RyR1 and RyR2predominate (Wong et al., 1997a). Of the congeners assayed on ERpreparations from brain, non-coplanar PCB 95 exhibited the highestpotency toward activating high-affinity [3H]Ry binding, whereasmono-ortho PCB 66 (2,3,4,4′-tetrachlorobiphenyl) and PCB 126 wereinactive. Ca2+ transport measurements made with cortical ER vesiclesrevealed that PCB 95 discriminates between inositol 1,4,5-trispho-sphate- and ryanodine-sensitive stores, and PCB 95 induced Ca2+ wasdose-dependent and completely inhibited by ryanodine receptorblockers (Wong et al., 1997a).

A more detailed structure–activity analysis was completed withRyR1. Fig. 2 shows a ranking of the 28 most active congeners of 35tested based on the concentration required to double [3H]Ry bindingactivity to RyR1 (Pessah et al., 2006). Many of these are found inenvironmental and human samples and collectively they cancomprise up to 50% of the total PCB burden. PCB 95 and PCB 136(2,2′,3,3′,6,6′-hexachlorobiphenyl) share asymmetric chlorine sub-stitutions on the phenyl rings (2,3,6 and 2′,3′,6′ for PCB 136 vs. 2, 5and 2′,5′ for PCB95) and both are racemic mixtures of twoatropisomers (see below). The 2′,5′-Cl configuration of PCB 95 isequivalent to a 3′6′-Cl configuration of PCB 136 (i.e. 2,3,6,2′,5′ vs.2,3,6,3′,6′) assuming a calculated dihedral angle of ∼90° based on thecrystal solution of PCB 84 (Lehmler et al., 2005b). The 2,3,6-Clconfiguration on one ring with ortho, meta on the other is optimal forrecognizing a binding site within the RyR1 complex and for sensitizingchannel activation. Para-chloro substitution lowers the maximumefficacy towards RyR1 regardless of the presence of one or moreortho-substitutions. Comparing the relative activities of PCB 30 andPCB 75 (2,4,6,4′-tetrachlorobiphenyl), the additional para-Cl-substi-tution completely eliminates activity towards RyR1. Thus completelack of activity observed here with PCB 75 is likely due to the di-para-chloro substitution. In general, PCB structures possessing 4,4′-Clexhibit lower activity towards RyR1, regardless of the presence of oneor more ortho-substitutions (Pessah et al., 2006). Hydroxylated PCBsare appearing in human tissues and there is currently great interest in

Fig. 10. Dose–response relationship of selected PCBs towards enhancing the binding of [3H]Ry to RyR1 showing the importance of ortho-substitutions (non-coplanarity (left panel)and substitutions at the para position.

Fig. 11. Correlation between the PCB concentration needed to double [3H]Ry binding toRyR1 and the initial rate of PCB-induced Ca2+ efflux from SR vesicles (data for eachcongener were normalized to respective parameters obtained with PCB 95). BZnumbers are given, Adapted from (Pessah et al., 2006).


determining whether these metabolites are more biologically activethan the corresponding parent structures (Fernandez et al., 2008; Parket al., 2008a,b; Park et al., 2009; Liu et al., 2009a). The 4-OH derivativeof PCB 30 (4′OH-PCB 30) was found to be significantly more activetoward RyR1 than the parent PCB 30 (2,4,6-Cl) (Fig. 10, right panel).Thus a para-OH group on the phenyl ring that carries no otherdeactivating substitution confers potency and efficacy towardsactivating RyR1. Two possible mechanisms could be responsible.First, the acidic property of the lone phenyl-OH substituent is likely tocontribute hydrogen-bonding potential to stabilize interactions withthe receptor site. Alternatively, if the hydroxyl group is partially orwholly ionized, then electrostatic interactions would be expected tostabilize the PCB-RyR1 complex. In support of this interpretation, thedi-OHderivative of PCB30, 3′,4′-di-OH,2,4,6-PCB,was found to possesslower potency but similar efficacy to PCB 30. The presence of twoadjacent –OH moieties would be expected to promote intramolecularhydrogen bonding and could preclude stabilizing interactions withRyR1 that impact the apparent affinity and efficacy for channelactivation (Pessah et al., 2006). There is an excellent correlation(r2=0.87) between the initial rate of PCB-induced Ca2+ efflux and theconcentration needed to increase [3H]Ry binding by two fold, a mea-sure of potency (Fig. 11), and PCB-induced Ca2+ release from ER/SRmembrane without inhibiting the SR/ER Ca2+-ATPase (SERCA pump).Moreover, the release can be completely inhibited by prior block ofRyR channels with ryanodine or ruthenium red, suggesting that aselective receptor-mediated mechanism is responsible.

2.3.1. Enatioselectivity of chiralpolychlorinated biphenyls toward ryanodine receptors

Nineteen of the possible 209 polychlorinated biphenyl (PCB)structures exist as pairs of atropisomers (also referred to as enantio-mers) that are sufficiently stable to permit separation by gas or liquidchromatography using chiral column matrices (Haglund, 1996). Stableenantiomeric PCB structures have unsymmetrical chlorine substitutionsin their respective phenyl rings and possess ≥3 chlorine atoms in theortho-positions that restrict the degree of rotation about the biphenylbond. Evidence of enantioselective enrichment of PCB atropisomers inenvironmental samples (Pessah, 2001; Wong et al., 2001a, 2004;Robson & Harrad, 2004; Asher et al., 2007; Jamshidi et al., 2007; Wonget al., 2007), food (Bordajandi et al., 2005; Bordajandi et al., 2008;Bordajandi & Gonzalez, 2008), as well as biological tissues from animals(Chu et al., 2003b; Kania-Korwel et al., 2007, 2008a,b) and humans(Chu et al., 2003a; Harrad et al., 2006) are being widely reported.Recently (−) PCB136was shown todirectly sensitize activation of RyR1and RyR2, whereas (+) PCB 136 did not when assayed at either 25 or37 °C (Pessah et al., 2009) (Fig. 12). (−) PCB 136 rapidly mobilized SR

Ca2+ stores by activating RyR1 in the presence of low (resting) levels ofcytoplasmic Ca2+, and (+) PCB 136 failed to competitively inhibit theactions of (-) PCB 136. Themechanism bywhich (−) PCB 136 promotesRyR activity is to coordinately stabilize the open, while destabilizing theclosed states of the RyR1 channel.

The enantiospecificity of (−) PCB 136 indicates that the spatialconfiguration of the chlorine substitutions about the biphenyl is signifi-cantly more important than the overall physicochemical properties ofthe PCB for optimizing interactions with RyRs and implies a highlyordered binding interaction between active PCBs and their site(s) ofinteraction within RyR complexes. It is interesting to note that the fourmost active PCBs toward RyR thus far tested are chiral (Figs. 2 and 11)although thedegreeof enantiospecificity toward sensitizingRyRactivitymay vary (Lehmler et al., 2005a; Pessah et al., 2009).

2.4. Ryanodine receptors in smooth muscle

All three RyR isoforms are expressed in smooth muscle myocytes(McGeown, 2004). The patterns of RyR isoform expression and theircontribution to smoothmuscle contractility differ among specific organsin which they are found including vascular, urinary bladder, ureter,airway andthegastrointestinal track (McGeown,2004; Cheng&Lederer,

Fig. 12. (A) PCB 136 is chiral because the asymmetric distribution of chlorines prevents interconversion of its (+) and (-) atropisomers. Upon separation, (-) PCB 136was found to beactive towards enhancing the activity of RyR1 (B) and RyR2 (C), whereas (+) PCB 136 was not active. Adapted from (Pessah et al., 2009).


2008). RyRs also localize to regions of SR that are in close proximity(≤100 nm) to the plasma membrane of a variety of smooth musclemyocytes where they are responsible for generating spontaneous anddepolarization evoked CICR (Lesh et al., 1998; Gollasch et al., 1998). Thepatterns of expression of RyR isoforms are highly dependent on the typeof smooth muscle and multiple isoforms can be expressed within asmooth muscle cell (Chalmers et al., 2007). For example, recent RT-PCRresults have indicated that RyR2 and RyR3 are expressed in culturedmyocytes from rat mesenteric artery (Berra-Romani et al., 2008) or vasdeferens (Ohno et al., 2009), although the ratio of the two isoforms canchange with proliferation. In contrast, uterine smooth muscle expressestwo splice variants of RyR3 that are differentially expressed innonpregnant and pregnant myometrium, although their functionalsignificance has been recently questioned (Noble et al., 2009). It appearsthat expressionof a functional long formofRyR3 is responsive to caffeineand cADP ribose, whereas expression of a non-functional short form caninhibit the function of the long form when they are concomitantlyexpressed (Dabertrandet al., 2007). At the endof pregnancy, the relativeexpression of RyR3 long form appears to increase suggesting physiolog-ical regulation of RyR3 alternative splicing may be important inregulating uterine contractility at the end of pregnancy.

In smooth muscle that undergoes action potential driven phasiccontraction (e.g., smooth muscle of the uterer), the periodicity of Ca2+

sparks appear to be functionally coupled to pacemaker ionic currentsthat, collectively, set the rhythmof the firing of action potentials (Wanget al., 2002; Burdyga &Wray, 2005;Wray et al., 2005). By contrast, RyRsexpressed in arterial and urinary bladder smooth muscles are co-localized with and tightly coupled to large conductance Ca2+-activatedpotassium channels (BKCa) thatmediate spontaneous outward currents(STOCs) (Nelson et al., 1995; Jaggar et al., 1998; Perez et al., 1999;Herrera & Nelson, 2002). Voltage-activated Ca2+ entry into arterialsmoothmuscle is primarily responsible for enhancing tone.However, animportant consequence of the co-localization and tight functionalcoupling betweenRyRs andBKCa is that activation of sub-plasmalemmalRyR-mediated Ca2+-induced Ca2+ release efficiently enhancesSTOCs thereby hyperpolarizing the surface membrane and shutting offvoltage-dependent Ca2+ entry (Nelson et al., 1995). Therefore,activation of RyRs plays a pivotal physiological role in arterial smoothmuscle relaxation and the abnormal RyR-BKCa coupling has been shownto cause hypertension and left ventricular hypertrophy (Pluger et al.,2000; Brenner et al., 2000; Amberg et al., 2003).

2.4.1. Cellular toxicity of polychlorinated biphenyls to smooth muscleExposure of uterine smooth muscle cells isolated from gestation

day 10–pregnant rats to non-coplanar PCB 4 (2,2′-dichlorobiphenyl)was shown to inhibit contractility and synchronization (Chung & LochCaruso, 2005). These effects on contractility were initially attributed

to MAPK-mediated phosphorylation of connexin 43 that resulted ininhibition of myometrial gap junctions. However, additional studiesrevealed that antioxidants could reverse the inhibitory influence ofPCB on contraction and synchronicity without restoring gap junctionfunction (Chung & Caruso, 2006), suggesting other mechanisms areinvolved. Paradoxically, complex PCB mixtures significantly stimulat-ed uterine contraction frequency with the least chlorinated mixture,Aroclor 1242, being most potent. The actions of micromolar Aroclor1242 on uterine smooth muscle contractility seem at least in partmediated by enhanced Ca2+ entry through a nifedipine-sensitivepathway (Bae et al., 1999b; Wrobel et al., 2005). Aroclor 1260 did notexhibit significant effects on rat uterine strips in vitro (Bae et al., 2001;Tsuneta et al., 2008). However subsequent to microbial metabolism, apartially dechlorinated mixture dominated by ortho-substituted PCBswith ≤4 chlorines substituents increased uterine contraction fre-quency over 7-fold. Exposure of bovine myometrial cells to A1248initially increased the spontaneous force of contractions but withlonger exposures (≥24 h) was inhibitory (Wrobel et al., 2005). Non-coplanar PCB-153 (2,2′,4,4′,5,5′-hexachlorobiphenyl) increased thespontaneous and oxytocin-evoked frequency of myometrial contrac-tions, and these effects were greater in cells isolated before than afterovulation. On the other hand, PCB 153 delayed or inhibited oxytocin-stimulated rise in intracellular Ca2+ concentration without alteringcell viability (Wrobel et al., 2005; Wrobel & Kotwica, 2005). Ortho-substituted PCBs and their metabolites were found to reduceproliferation of myometrial cells originating from pregnant womenexposed in vitro (Bredhult et al., 2007).

The mechanisms responsible for the seemingly paradoxical effectsof PCBs on uterine smooth muscle contractility (decreased contractil-ity produced by PCB 4 vs. enhanced contractility induced by an ortho-rich mixtures and PCB153) are currently not understood. Collectivelythese results suggest that non-coplanar PCBs, especially lightlychlorinated congeners, are most active toward altering myometrialcontractility through a mechanism involving altered Ca2+ signaling.

2.5. Polychlorinated biphenyl developmental neurotoxicity

2.5.1. Ryanodine receptors in the nervous systemNeurons have an extensive ER membrane system that extends

deep into the soma to envelope the nucleus and out into proximalregions of the dendrites and axon. Specialized regions of the ER closelyappose the plasma membrane and are present in more distal aspectsof the neuron such as growth cones, axon terminals and dendriticspines. As in non-neuronal cells, Ca2+ release from neuronal ER storescan be evoked by stimulation of RyRs or IP3Rs, and both receptor typescan couple to the activation of neurotransmitter-gated receptorsand voltage-gated Ca2+ channels on the plasma membrane. This


organization enables the ER to function not only as a buffer and sourceof Ca2+ in axonal and somatodendritic compartments but to alsodiscriminate between different types of neuronal activity and inte-grate Ca2+-dependent signaling between the plasma membrane,cytosol and nucleus (Berridge, 2006; Bardo et al., 2006). Ca2+ spark-like events arising from both RyRs and IP3Rs in nerve growth factor-differentiated PC12 cells and cultured hippocampal neurons exhibitdifferent kinetic properties than their counterparts found in cardiacmuscle, with greater spatial width and significantly longer duration(Koizumi et al., 1999a,b). Long lasting local Ca2+ signals spreadingseveral microns, called “syntillas” have been measured withinpresynaptic terminals of basket cells of the cerebellum (Collin et al.,2005), and in peptidergic terminals of murine hypothalamic neuronsand neuroendocrine (chromaffin) cells (De Crescenzo et al., 2004;ZhuGe et al., 2006; De Crescenzo et al., 2006). Syntillas observed inhypothalamic neuronal terminals are triggered by membrane depo-larization and are restricted near the inner leaflet of the plasmamembrane. RyR1s anchored to the ER in very close proximity toplasmalemmal L-type voltage-dependent Ca2+ channels engage in aform of voltage-induced Ca2+ release (VICaR) that is similar to muscleEC coupling (De Crescenzo et al., 2006).

High-affinity [3H]ryanodine-binding sites are expressed in ratbrain microsomal fractions from cerebral cortex, cerebellum, hippo-campus and brainstem (Zimanyi & Pessah, 1991) and form caffeinesensitive Ca2+ channels (McPherson et al., 1991). All three RyRisoforms are expressed in the central nervous system (CNS) but aredifferentially distributed between specific brain regions, cell types andsubcellular localizations, reflecting their participation in specializedfunctions. In situ hybridization studies of mouse brain (Mori et al.,2000) indicate that during embryogenesis, RyR1 is predominantlyexpressed in the rostral cortical plate whereas RyR3 is prominent inthe caudal cortical plate and hippocampus. However, from postnatalday 7 into adulthood, RyR2 expression is upregulated and RyR3 ex-pression downregulated so that RyR1 and RyR2 are more highly andbroadly expressed than RyR3. In the postnatal brain, RyR1 expressionis most prominent in the dentate gyrus and Purkinje cell layer but thisisoform has also been detected in the caudate/putamen nuclei, olfac-tory tubercle, olfactory bulb, and cortex. RyR2 is the major isoformexpressed in many brain regions and RyR2 transcripts have beenobserved in the hippocampus, cerebellum, medial habitual nuclei,amygdala, cortex and more anterior brain regions including thegranular cell layer of the olfactory bulb. In the cerebellum, RyR2mRNAspecifically localized to the granular cell layer. RyR3, which accountsfor about 2% of the total RyR protein in the brain (Murayama & Ogawa,1996), shows distinctive patterns of expression in several structures ofmouse brain. RyR3-specific antibodies appeared to preferentially stainthe granular layer in the cerebellum even though the signal intensitywas less than RyR2. Immunocytochemical studies have also detectedRyR3 in the hippocampus and sporadic patterns of RyR3 staining havebeen reported in the thalamus and the caudate/putamen nuclei.

Detailed in situ hybridization (Furuichi et al., 1994; Giannini et al.,1995; Mori et al., 2000) and immunocytochemical (Hertle & Yeckel,2007) studies of the distribution of RyR isoforms in the hippocampus ofrodent brains indicate that RyR1 is enriched in the stratum oriens,stratum pyramidale and stratum radiatum of CA1 and CA3 subfields ofhippocampus. The densest RyR1 immunolabeling localized to thesomata of pyramidal cells within the stratum pyramidale and portionsof their apical dendrites in the stratumradiatum(Hertle&Yeckel, 2007).By contrast, RyR1 has relatively lower expression in dentate gyrus. RyR2is primarily expressed in the cells of thedentate gyrus and in the stratumlucidum of CA3 with weaker staining in pyramidal cells in stratumpyramidale andwithin stratum radiatum of CA1 (Lai et al., 1992; Hertle& Yeckel, 2007). RyR2 is localized in axons to a greater degree than indendrites, and is most densely distributed in the hippocampal mossyfiber pathway and in axon bundles traversing the cortical laminae(Seymour-Laurent & Barish, 1995; Hertle & Yeckel, 2007). RyR3

immunoreactivity was detected in all hippocampal subfields but wasstronger in CA1 than CA3. Intense RyR3 immunoreactivitywas detectedalong the dentate gyrus/hilus border and within the hilus.

RyR expression has also been documented in non-neuronal cells ofthe CNS. Diffuse staining of the hippocampal neuropil indicates thatRyR3 is expressed in astrocytic processes (Matyash et al., 2002), andseparate studies of cultured glial cells derived from rodent brainconfirmed RyR expression in not only astrocytes (Matyash et al., 2002;Straub & Nelson, 2007) but also oligodendrocytes (Haak et al., 2001;Weerth et al., 2007). These studies further indicated that astrocytes(Matyash et al., 2002) and oligodendrocyte progenitors (Haak et al.,2001) express RyR3 but not RyR1 or RyR2. In oligodendrocyteprogenitor, RyR3 and IP3R type 2 were shown to have a differentialdistribution within their processes that might explain differences inlocal and global Ca2+ signals mediated by these two channel types,and their functional interactions appear to determine the spatial andtemporal characteristics of Ca2+ signaling in these cells. In contrast,human microglia cultured from both fetal and adult brain samplesexpress mRNA for RyR1 and RyR2, whereas RyR3 mRNA can bedetected only in fetal microglial cells. RyR expression has also beenexamined in peripheral nervous system (PNS) neurons. Transcriptsencoding RyR2 and RyR3 but not RyR1 have been detected insympathetic ganglia isolated from adult rats and ganglionic levels ofRyR3 but not RyR2 mRNA decline with advanced age (Vanterpoolet al., 2006). Immunohistochemical studies of dorsal root ganglia(DRG) revealed immunoreactivity for RyR3 but not RyR1 or RyR2(Lokuta et al., 2002; Ouyang et al., 2005) in these sensory neurons.

2.5.2. Ryanodine receptor-mediated mechanisms in neurodevelopmentGiven that RyRs are expressed in both presynaptic and postsyn-

aptic sites in virtually all brain areas (Ogawa & Murayama, 1995;Nozaki et al., 1999; Hidalgo, 2005) as well as in CNS glial cells and PNSneurons, it is perhaps not unexpected that experimental evidenceindicates that RyRs contribute to fundamentally important aspects ofneuronal excitability and use-dependent synaptic plasticity (Berridge,1998; Korkotian & Segal, 1999; Matus, 2000; Kennedy, 2000; Segal,2001). In axon terminals, RyRs mediate spontaneous, evoked andfacilitated neurotransmission (Liu et al., 2005; Collin et al., 2005;Dunn & Syed, 2006; De Crescenzo et al., 2006; Shimizu et al., 2008)and regulate the mobilization and recycling of synaptic vesicles(Shakiryanova et al., 2007; Levitan, 2008). RyRs localized to thesomatodendritic domain influence the intracellular encoding ofneural activity and are implicated in modulating both neurochemical(Berridge, 2006; Bardo et al., 2006) and structural (Segal et al., 2000)aspects of synaptic plasticity. Diverse neurochemical changes associ-atedwith dendritic synaptic plasticity have been shown to rely on RyRfunction, including: 1) activity-dependent postsynaptic translation(Jourdi et al., 2009) and secretion (Kolarow et al., 2007) ofneurotrophins, 2) modulation of Gq-coupled receptor function bystress peptides and hormones (Riegel & Williams, 2008); 3) activity-dependent enhancement of glutamate responses and the associatedincrease of GluR1 within spines mediated by synaptopodin, an actin-binding protein that co-localizes with RyRswithin the spine apparatusof hippocampal neurons (Vlachos et al., 2009); and 4) sequentialactivation of CaM kinases, CREB and transcription of genes encodingCa2+-regulated proteins triggered by repetitive or prolonged depo-larization of hippocampal neurons (Deisseroth et al., 1998). RyRssimilarly function in peripheral neurons to regulate the release of(Cong et al., 2004; Ouyang et al., 2005; Huang et al., 2008) andresponse to (Brain et al., 2001; Locknar et al., 2004) neurotransmittersand neuropeptides, and their function underlies the exocytotic releaseof glutamate from astrocytes (reviewed in Reyes & Parpura, 2009).Changes in postsynaptic efficacy are also associated with morpholog-ical changes in dendritic spines. Pulse application of caffeine, a RyRagonist, has been reported to cause a rapid and significant increase inthe size of existing dendritic spines in mature cultures of hippocampal


neurons and this effect is blocked by antagonizing concentrationsof ryanodine (Korkotian & Segal, 1999), supporting the involvementof RyR in mediating activity-dependent changes in dendritic spinemorphology.

Consistent with the demonstrated role of RyRs in neurotransmis-sion and synaptic plasticity at the cellular level, ligands that directlymodulate RyR, such as ryanodine, alter functional aspects of neuro-plasticity including long-term potentiation (LTP) (Wang et al., 1996)and long-term depression (LTD) (Wang et al., 1997) in the hippo-campus. FK506 and rapamycin, which deregulate RyR2 by dissociatingthe RyR2/FKBP12/12.6 complexes also inhibit LTD (Li et al., 1998).Thus, RyRs appear to play a critical role in use-dependent plasticitythat underlies the early stages of associativememory. Earlier evidencesuggested that RyR2 through its interaction with calexcitin might alsoalter Ca2+ signaling over a longer time frame, implying a critical rolefor RyRs in the consolidation phase of associative memory (Alkonet al., 1998). Most intriguing is a work showing a tight correlationbetween acquisition of spatial learning and selective upregulation ofRyR2 in the dentate gyrus and CA3 (Cavallaro et al., 1997), implicatingRyR2 in storage phases of associative memory (Alkon et al., 1998).Additional studies demonstrate that mice with targeted deletion ofRyR3 have deficits in contextual fear conditioning but improvedspatial learning in the Morris water maze task (Futatsugi et al., 1999;Kouzu et al., 2000). Interestingly, comprehensive behavioral pheno-typing of RyR3 knockout mice revealed decreased social interaction,hyperactivity and mildly impaired prepulse inhibition, whereas nomeasurable impairments in motor function and working andreference memory tests were detected (Matsuo et al., 2009).

A recent discovery is that RyRs also function as a key element ofthe output pathway from the molecular circadian clock in neurons ofthe suprachiasmatic nucleus (SCN). Electrophysiological and calciummobilization experiments indicate that RyR activation by administra-tion of caffeine or 100 nM ryanodine increased the firing frequency ofSCN neurons, whereas inhibition of RyRs by dantrolene or 80 μMryanodinedecreasedfiring rate, suggesting that RyRs are involved in thecircadian rhythmicity of SCN neurons (Aguilar-Roblero et al., 2007).Subsequent behavioral studies demonstrated that RyR activationinduced a significant shortening of the endogenous period, whereasRyR inhibitiondisrupted circadian rhythmicity. Inboth experiments, theperiod of rhythmicity returned to basal levels upon cessation ofpharmacological treatment (Mercado et al., 2009). In light of thesestudies, it is interesting to note that it had been reported some 25 yearsearlier that PCBs interferedwith rhythmic pituitary–adrenal function inrats (Dunn et al., 1983).

Dynamic changes in intracellular Ca2+ concentrations play a crucialrole in not only neuronal excitability and synaptic plasticity but also incell proliferation and differentiation, cell movement, and cell death(Cline, 2001; Spitzer et al., 2004; Moody & Bosma, 2005; Zheng & Poo,2007). Thus, it might be predicted that RyRs function to regulatediverse aspects of neurodevelopment, and emerging evidence sup-ports that prediction. Early reports that RyRs are upregulated duringNGF-induced differentiation of adrenal chromaffin cells (Jimenez &Hernandez-Cruz, 2001) suggested the possibility that RyRs mediatethe CICR necessary for neurogenesis and neuronal differentiation.In support of this hypothesis, it was recently shown that activation ofL-type Ca2+ channels, GABAA receptors or RyRs promoted neuronaldifferentiation, while inhibition of these channels/receptors had theopposite effect on mouse embryonic stem (ES) cells (Yu et al., 2008).Moreover, the activity of intracellular Ca2+ signaling, expression of theneuronal transcription factor NeuroD and the rate of neurogenesis weresignificantly inhibited in neuronal cells derived from embryonic stemcells obtained from RyR2 deficient mice relative to wild type controlseven in the presence of L-type Ca2+ channel and GABAA receptoractivation. Apoptosis is another neurodevelopmental process that isessential to normal brain development (Martin, 2001; Dikranian et al.,2001), occurring in proliferative zones and in postmitotic cells in both

the fetal and postnatal brain (White & Barone, 2001). The spatiotem-poral pattern of apoptosis in the developing CNS is tightly regulated anddisruption of either the timing or themagnitude of apoptosis in a givenbrain region can alter cell number and thus connectivity, causingdeficitsin higher order function even in the absence of obvious pathology(Sastry & Rao, 2000; Barone et al., 2000; Martin, 2001). Increased Ca2+

andROS are significant triggers of apoptosis, and RyRactivation a criticalcomponent of apoptotic signaling pathways (Berridge et al., 2000;Robertson et al., 2001; Carmody & Cotter, 2001; Ermak & Davies, 2002;Ravagnan et al., 2002).

RyRs have also been implicated in regulating morphogeneticprocesses in the developing nervous system. Specifically, RyR3 hasbeen shown to be necessary for astrocyte migration (Matyash et al.,2002), and for axonal growth cone responses tonitric oxide (Welshhans& Rehder, 2007) or activation of cell adhesion molecules (Ooashi et al.,2005). With respect to the latter, in the presence of RyR3-mediatedCICR, growth cones exhibited attractive turning, but in the absence ofRyR3-mediated CICR, Ca2+ signaling elicited growth cone repulsion.The authors suggest on the basis of these observations that the source ofCa2+ influx, rather than its amplitude or the baseline Ca2+ level, is theprimary determinant of the direction of axonal growth cone turning.Based on pharmacological inhibition studies, RyRs have also beenimplicated in regulating Ca2+-dependent neurite outgrowth (Arie et al.,2009) in DRG neurons, a cell type that extends only axons, as well asactivity-dependent dendritic growth and retraction in retinal ganglianeurons (Lohmann et al., 2002; Lohmann et al., 2005). A generalizationemerging from studies of activity-dependent dendritic growth is thatCa2+ exerts bimodal effects on dendritic structure. Thus, increasedintracellular Ca2+ has been linked to both dendritic growth and todendritic retraction (reviewed in Redmond et al., 2002; Segal et al.,2000; Lohmann&Wong, 2005). Twopossibilities havebeenproposed toexplain why Ca2+ signaling stimulates dendritic growth in some caseswhile inhibiting dendritic plasticity in others. First, Segal et al. (2000)suggest that moderate and/or transient increases in intracellular Ca2+

cause growth of dendritic branches and spines, whereas large Ca2+

increases cause destabilization and retraction of these dendriticstructures (Fig. 13). Consistent with this possibility, relatively sustainedincreases in intracellular Ca2+ vs. transient changes in Ca2+ influxactivate different Ca2+-dependent signaling pathways with distincteffects on dendrites (Wilson et al., 2000; Redmond et al., 2002). Thesecond possibility is that dendritic responses to Ca2+ differ withneuronal maturation such that early in development, increased Ca2+

promotes dendritic growth, while later in development, increased Ca2+

functions to stabilize dendritic structure (Lohmann & Wong, 2005).While pharmacological manipulation of RyRs has been shown toinfluence activity-dependent dendritic morphogenesis, the specificisoforms associated with any specific effect have yet to be determined.

In addition to regulating neurodevelopment and physiologicalprocesses in the central and peripheral nervous system, RyRs are alsoimplicated in Ca2+ dysregulation associated with cell toxicity, agingand neurodegeneration (reviewed in Thibault et al., 2007). The modelthat has been proposed is that aging and/or pathological changesoccur in both L-type Ca2+ channels and RyRs and these interact toabnormally amplify Ca2+ transients. In turn, the increased transientsresult in dysregulation of multiple Ca2+-dependent processesultimately accelerating functional decline. Diverse pathogenic stimuli,including HIV-1 Tat (Norman et al., 2008), amyloid-beta and prionpeptides (Ferreiro et al., 2008) as well as mutations associated withneurodegeneration such as the presenilin (PS2) mutation (Lee et al.,2006a,b) activate apoptotic or excitotoxic pathways via RyR-depen-dent mechanisms that increase intracellular Ca2+ levels in neurons.Consistent with this model, two recent reports confirm a role forinteractions between presenilins and RyRs in regulating release ofcalcium from both presynaptic and postsynaptic ER (Zhang et al.,2009; Chakroborty et al., 2009). This interaction is perturbed in 3xTg-ADmice such that RyR-evoked Ca2+ release in CA1 pyramidal neurons

Fig. 13. Proposed relationship between intracellular Ca2+ concentration and dendriticgrowth. Adapted from (Segal et al., 2000).


is markedly increased resulting in altered synaptic homeostasis ofthese neurons relative to wild type mice (Chakroborty et al., 2009).Interestingly, the RyR2 isoform was found to be selectively increasedmore than fivefold in the hippocampus of 3xTg-AD mice relative tocontrols and the authors propose this as themechanism to explain thedeviant, yet functional calcium signaling evident in presymptomatic3xTg-AD mice long before the onset of AD histopathology. RyR-dependent Ca2+ release from presynaptic internal stores is also re-quired for ethanol to increase spontaneous γ-aminobutyric acidrelease onto cerebellum Purkinje neurons (Kelm et al., 2007), and theimbalance between excitatory and inhibitory circuits that underliesNMDA receptor-mediated epileptiform persistent activity is blockedby inhibition of the ryanodine receptor (Gao & Goldman-Rakic, 2006).These observations support the hypothesis that factors that alter RyRexpression and/or function have the potential to interfere withnormal neurodevelopment and neuronal function.

2.5.3. Experimental evidence of ryanodinereceptor-mediated mechanisms of polychlorinated biphenyl neurotoxicity

Of the various adverse effects associated with PCBs, developmentalneurotoxicity has emerged as a particularly vulnerable endpoint(NIEHS, 1999; Schantz et al., 2003; Carpenter, 2006). Population-based studies have consistently demonstrated that PCBs negativelyimpact neuropsychological function in exposed children (Schantz et al.,2003; Carpenter, 2006; Korrick & Sagiv, 2008). PCB exposure has beenpositively correlated with decreased IQ scores, impaired learning andmemory, lower reading comprehension, attentional deficits andpsychomotor dysfunction (Chen et al., 1992; Jacobson et al., 1992;Koopman-Esseboomet al., 1996; Schantz et al., 2003; Roegge & Schantz,2006;Grandjean&Landrigan, 2006; Rogan&Ragan, 2007; Stewart et al.,2008). Comparable cognitive and psychomotor behavioral deficits areobserved in primate and rodent models following developmental PCBexposures (Schantz et al., 1989; Tilson et al., 1990; Schantz et al., 1995;Schantz, 1996; Rice, 1998; Tilson & Kodavanti, 1998). More recently, ithas been postulated that exposure of the developing brain to PCBs mayalso influence the susceptibility of the adult brain to acute stressors andneurodegeneration (Lein et al., in press). This hypothesis is derived inpart fromamortality study of 17,000workers occupationally exposed toPCBs that revealed an increased incidence of PD in female subjects(Steenland et al., 2006) and findings of a positive correlation betweenPCB exposure and depression and impaired memory and learningamong adults 49-86 years of age living inMichigan and exposed to PCBsvia consumptionof Great Lakesfish (Schantz et al., 2001) and adults 55–74 years of age livingalong regionsof theHudsonRiver inNewYork thathave been heavily contaminated with PCBs (Fitzgerald et al., 2008). Inthe latter study, the PCB body burdens of affected individuals weresimilar to those of the general population, suggesting persistent neuro-

psychological effects from prior exposures. Whether early-life expo-sures to PCBs increase susceptibility to neurodegenerative processesthat contribute to dementia and motor deficits is difficult to determinefrom these studies because the critical exposure period could not beidentified. However, several recent studies using experimental animalmodels support this possibility. The first set of studies utilized a well-established model of focal cerebral ischemia, middle cerebral arteryocclusion (MCAO) in rats, to demonstrate that exposure to Aroclor 1254in the maternal diet throughout gestation and lactation confers neuro-protection against focal ischemic stress in theadult brain (Dziennis et al.,2008). Congener-specific analyses of tissues harvested from adultanimals immediately following MCAO indicated no difference in PCBlevels between control and PCB-exposed brains, suggesting that PCBeffects on stroke outcome may reflect PCB interactions with develop-mental processes. In the second set of studies, developmental PCBexposure was shown to influence seizure susceptibility in the weanlingand young adult rat. Seizure susceptibility was assessed by quantifyingthe threshold for seizures induced by flurothyl (bis-2,2,2-triflurothylether), a convulsive drug used to investigate seizure susceptibility inrodents (Ferland & Applegate, 1998; Szot et al., 2001) and the responseto pentylenetetrazole (PTZ), which kindles seizures within 15–20injections of initially subconvulsive doses in rats (Corda et al., 1991).Exposure to PCB 95 in thematernal diet from gestational day 5 throughweaning on postnatal day 21 significantly decreased seizure thresholdsin animals challenged with flurothyl on postnatal day 35 and causedfaster kindling with PTZ on postnatal days 60–83 (Lein et al., in press).Considered collectively, these studies support the possibility thatexposure of the developing brain to PCBs may elicit persistent changesin the brain that influence the susceptibility of the adult brain to sub-sequent stressors.

How developmental PCB exposure causes persistent neuropsycho-logical impairment in either children or adults has yet to be resolved.Several lines of evidence suggestmechanisms independent of the AhR.First, congener-specific analyses of brain tissues from human subjects(Corrigan et al., 1998) and experimental animals (Dziennis et al., 2008;Yang et al., 2009) exhibiting neuropsychological impairment associ-ated with exposure to complex PCB mixtures indicate enrichment ofnon-coplanar ortho-rich congeners. Second, studies utilizing purifiedcongeners have consistently demonstrated developmental neurotox-icity associated with non-coplanar ortho-rich congeners (Tilson &Kodavanti, 1998; Schantz et al., 2003; Kodavanti, 2005; Fonnum et al.,2006;Mariussen& Fonnum, 2006). For example, perinatal exposure toPCB 95 has been shown to persistently alter cognitive and psychomo-tor activity in rodent models (Schantz et al., 1997) as well as LTP inhippocampal slice cultures (Wong et al., 1997b),and to alter the ratioof excitatory to inhibitory neurotransmission in the developingauditory cortex (Kenet et al., 2007) and hippocampal slice cultures(Kim et al., 2009a).

Themechanisms underlying the neurotoxic effects of non-coplanarPCBs are only partially understood. Biological activities associatedwithortho-rich and hydroxyl metabolites of ortho-poor PCBs include: 1)endocrine disruption, specifically weak estrogenicity (Safe, 2004),enhanced insulin (Fischer et al., 1999) and arachidonic acid secretion(Bae et al., 1999a), and disruption of the hypothalamic–pituitary–thyroid axis (Zoeller et al., 2000; Zoeller, 2005; Knerr & Schrenk,2006); 2) reduced levels of dopamine and other biogenic amines in thebrain and in cultured neurons (Seegal, 1996; Mariussen & Fonnum,2006); and 3) increased levels of reactive oxygen species (ROS) andintracellular Ca2+ in neurons (Kodavanti, 2005; Mariussen & Fonnum,2006). There is evidence that non-coplanar PCBsmay induce increasesin intracellular Ca2+ by several mechanisms, including influx of extra-cellular Ca2+ through L-type voltage-sensitive Ca2+ channels or theNMDA receptor (Mundy et al., 1999; Inglefield & Shafer, 2000) or viarelease of intracellular Ca2+ stores subsequent to activation of IP3Rs(Inglefield et al., 2001) or RyR (Wong et al., 1997a); however, RyRactivation is the most sensitive of these mechanisms (Wong & Pessah,

Fig. 14. Developmental A1254 exposure interferes with normal dendritic growth andexperience-dependent dendritic plasticity. Dendritic morphology was analyzed amongP31 rats trained in the Morris water maze (Maze) and among littermates identicallyhoused and exposed but not trained (Non-Maze). As seen in representative cameralucida drawings of the basilar dendritic arbor of cortical neurons, developmentalexposure to A1254 at 1 or 6 mg/kg/d in the dam's diet throughout gestation andlactation significantly increased dendritic arborization relative to vehicle controls(0 mg/kg/d A1254). Maze training significantly increased dendritic complexity amonganimals in the control group but this experience-dependent plasticity was blockedamong animals in the A1254 treatment groups with amore pronounced effect observedin the lower treatment group. Data are presented as the mean±SEM (N=17-21neurons per group). The percent changes in dendritic length were calculated using dataobtained from 17-21 neurons per treatment group. The percent change in dendriticlength as a function of maze training was calculated as the difference in dendritic lengthof neurons in maze-trained animals vs. non-maze-trained animals divided by thedendritic length of neurons in maze-trained animals multiplied by 100. ⁎pb0.05;⁎⁎pb0.01; ⁎⁎⁎pb0.001. From (D. Yang et al., 2009).


1996; Wong et al., 1997a,b). It is interesting to note that thesemechanisms may not be mutually exclusive since RyR activationis known to interact with IP3R and with both L-type voltage-sensitiveCa2+ channels and theNMDA receptor (see Section 2.5.4.), and lowµMconcentrations of non-coplanar PCBs have been shown to enhancesignificantly the sensitivity of primary cultured neurons to NMDA- andAMPA-elicited Ca2+ signals (Gafni et al., 2004), revealing a functionallink between PCB amplification of RyR signaling and sensitivity toexcitatory amino acids.

The causal relationship between these biological activities of non-coplanar PCBs and the neuropsychological deficits associated withPCB developmental neurotoxicity remains a pressing question in thefield. It has been postulated that these biological activities contributeto the developmental neurotoxicity associated with non-coplanarPCBs by interferingwith the patterning of neuronal connectivity in thedeveloping brain (Seegal, 1996; Gilbert, 2000). Critical determinantsof neuronal connectivity include neuronal apoptosis (Sastry & Rao,2000; Barone et al., 2000; Martin, 2001) and dendritic morphogenesis(Matus, 2000; Kennedy, 2000). Altered patterns of neuronal apoptosisnot only impact neuronal connectivity in the developing brain, butalso influence the susceptibility of the adult brain to subsequentenvironmental insults or aging (Langston et al., 1999; Barlow et al.,2007). The shape of the dendritic arbor determines the total synapticinput a neuron can receive (Purpura, 1967; Purves, 1975, 1988), andinfluences the types and distribution of these inputs (Miller & Jacobs,1984; Sejnowski, 1997; Schuman, 1997). Altered patterns of dendriticgrowth and plasticity are associated with impaired neuropsycholog-ical function in experimental models (Berger-Sweeney & Hohmann,1997) and are thought to contribute to diverse disorders ofneurodevelopmental origin (Zoghbi, 2003; Pardo & Eberhart, 2007;Connors et al., 2008) as well as neurodegenerative diseases (de Ruiter& Uylings, 1987; Jagadha & Becker, 1989; Flood & Coleman, 1990).Each of the biological activities associated with non-coplanar PCBshave been shown to influence neuronal apoptosis and to contribute tothe dynamic control of dendritic growth; however, to date, experi-mental evidence linking these activities to PCB-induced alterationsin neuronal connectivity has been reported only for RyR-dependentmechanisms.

PCBs have been shown to induce caspase-dependent apoptosis inprimary cultures of hippocampal neurons (Howard et al., 2003).Neuronal apoptosis was induced by A1254 and non-coplanar PCB 47but not by coplanar PCB 77. Aroclor 1254 contains predominantlyortho-rich PCBs with significant activity at the RyR (Wong & Pessah,1996;Wong et al., 1997a), and SAR studies identified PCB 47 as a RyR-active congener and the coplanar PCB 77 as a congener with negligibleactivity at the RyR (Pessah et al., 2006). Further evidence that RyRactivation mediated the pro-apoptotic activity of PCBs includes theinhibition of PCB-induced apoptosis by FLA 365, a selective RyRantagonist (Chiesi et al., 1988;Mack et al., 1992) but not by antagonistspreviously shown to block PCB-mediated Ca2+ flux through L-typevoltage-sensitive channels, NMDA receptors, and IP3Rs in culturedneurons.

Recent evidence suggests that PCBs may also interfere withneuronal connectivity in vivo. Developmental exposure to A1254was observed to enhance basal levels of dendritic growth but blockexperience-dependent dendritic growth in the cortex and cerebellumof weanling rats (Lein et al., 2007; Yang et al., 2009) (Fig. 14), anddevelopmental exposure to PCB 95was reported to disrupt the balanceof neuronal inhibition to excitation in the developing rat auditorycortex (Kenet et al., 2007). Several lines of evidence suggest that PCBsensitization of RyRs contributes to the effects of these compounds onneuronal connectivity. First, these changes in neuronal connectivityare associated with exposure to non-coplanar PCB congeners withhigh affinity for the RyR. Second, PCB-induced changes in dendriticgrowth and plasticity are coincident with increased [3H]ryanodine-binding sites and RyR expression in the brains of untrained animals

and inhibition of training-induced RyR upregulation. Moreover, thedose–response relationship for PCB effects on dendritic growth andplasticity was similar to that of PCB effects on RyR expression butnot to that of PCB effects on thyroid hormone levels or sex-steroid-dependent developmental endpoints (Yang et al., 2009). IncreasedRyR expression in brain tissues has also been associated with PCB-induced changes in gene expression (Royland et al., 2008; Royland &Kodavanti, 2008) and locomotor activity (Roegge et al., 2006). In vitrostudies confirmed a link between PCB sensitization of RyR and effectson dendritic arborization. PCB 95, a congener with potent RyR activity,but not PCB 66, a congener with negligible RyR activity, promoteddendritic growth in primary cortical neuron cultures and this effectwas blocked by pharmacological antagonism of RyR activity (D. Yanget al., 2009). The downstreammechanisms bywhich PCB sensitizationof the RyR influences dendritic arborization have yet to be established,but it is postulated these involve modulation of Ca2+-dependentsignaling pathways linked to activity-dependent dendritic growth andplasticity. Activation of a CaMK-CREB-Wnt signaling pathway hasrecently been demonstrated to link neuronal activity to transcriptionof gene products that regulate dendritic growth (Wayman et al., 2006)(Fig. 15); and activity-dependent translation is mediated by theserine-threonine protein kinase mammalian target of rapamycin(mTOR) (Takei et al., 2004; Kumar et al., 2005; Jaworski et al., 2005;Gong et al., 2006). RyR activation has been shown to cause sequentialactivation of CaM kinases, CREB and transcription of genes encodingCa2+-regulated proteins (Deisseroth et al., 1998); translationmechan-isms of activity-dependent growth are Ca2+-dependent; and PCBeffects on RyR-mediated Ca2+ signaling require interactions withFKBP12 (Wong & Pessah, 1997; Wong et al., 2001a,b; Gafni et al.,2004), which regulates mTOR activity (reviewed in Chen & Fang(2002)).

These data linking a direct molecular effect of PCBs (RyR activation)to disruption of specific neurodevelopmental events (neuronal

Fig. 15. Activity increases intracellular Ca2+ via NMDA receptor activation and calcium-induced calcium release, which alters dendritic growth via transcriptional or translationalmechanisms. The former involves CaMK I activation and enhanced CREB-dependent Wnt transcription; Wnt binds the Frizzled receptor to activate downstream effector moleculesβ-catenin, JNK and Rac. The latter involves Ca2+-dependent activation of mTOR, which relieves repression of initiation factor-4E by 4EBP1. mTOR is regulated by FKBP12, which istargeted by non-coplanar PCBs.


apoptosis anddendritic growth andplasticity) provide thefirst evidenceof a receptor-based mechanism for PCB developmental neurotoxicity.This not only provides a powerfulmeans for predictingwhich of the 209possible congeners within the PCB family present the greatest risks toneurodevelopment, but also supports the development of mechanism-based tools for screeningother chemical classes of environmental healthconcern, such as PBDEs. Human polymorphisms in RYR genes are linkedto environmentally triggered disorders including malignant hyperther-mia (MH) (Gronert et al., 2004), cardiac arrhythmias (Wehrens et al.,2005), and sudden death (Laitinen et al., 2004), suggesting the testablehypothesis that individuals with mutation(s) in one or more CRUproteins exhibit increased susceptibility to developmental neurotoxicityresulting from low-level environmental exposures to non-coplanarPCBs. In support of this hypothesis, we recently showed that non-coplanar PCB 95 is significantly more potent and efficacious in dis-rupting cation regulation of MH mutation R615C-RyR1 compared towild type RyR1 (Ta & Pessah, 2007). Considered together, theseobservations identify non-coplanar PCBs with high RyR activity ascandidate environmental risk factors in neurodevelopmental disordersand provide insight regarding potential gene-environment interactionsthat influence susceptibility to environmentally triggered neurodeve-lopmental deficits.

2.5.4. Ryanodine receptors as a point ofconvergence in polychlorinated biphenyl neurotoxicity

While emerging evidence clearly identifies RyRs as criticalmolecular targets in PCB developmental neurotoxicity, other biolog-ical activities have been ascribed to non-coplanar PCBs, includingincreased intracellular levels of ROS (Mariussen & Fonnum, 2006;Fonnum et al., 2006), disruption of thyroid hormone signaling(Zoeller, 2007; Crofton, 2008) and decreased levels of dopamine(Mariussen & Fonnum, 2006). Are these biological activities causallyrelated to PCB developmental neurotoxicity, and if so, do theyrepresent divergent or convergent mechanisms of PCB developmentalneurotoxicity?

In the case of PCB disruption of thyroid hormone signaling, animalstudies have shown that reductions in circulating TH levels with

developmental exposure to PCBs are associated with low frequencyhearing loss and damage to cochlear hair cells, especially outer haircells (Goldey et al., 1995; Lasky et al., 2002). Since TH is necessary fornormal cochlear development (Uziel, 1986), the loss of hair cells hasbeen considered the indirect consequence of TH deficiency duringcritical periods of development. However, the profile of cochleardamage following PCB exposure is not entirely consistent withmodelsof hypothyroidism, and TH replacement in PCB-exposed rats onlypartially reversed hearing deficits (Goldey & Crofton, 1998; Croftonet al., 2000; Crofton & Zoeller, 2005; Sharlin et al., 2006). Additionalevidence that an alternative, TH independent, mechanism contributesto PCB ototoxicity was recently presented (Powers et al., 2009).Interestingly, these effects on cochlear development are producedprimarily by non-coplanar PCBs (Kostyniak et al., 2005; Powers et al.,2006), and all three RyR isoforms are differentially expressedthroughout the organ of Corti, including inner and outer hair cells,and within spiral ganglion neurons (Morton-Jones et al., 2006; Grantet al., 2006). RyR1 is the major isoform expressed in the outer haircells where it is co-localized with nicotinic cholinergic receptors at“synaptic cisterns” resembling triadic junctions that are essential forengaging excitation–contraction coupling in skeletal muscle (Liou-dyno et al., 2004). RyR1 channels tightly couple Ca2+ release from ERstores in response to cholinergic input to the outer hair cell therebyregulating Ca2+-activated potassium currents that are necessary forlong-term survival of olivocochlear fibers and synapses (Murthy et al.,2009). RyRs expressed in inner hair cells functionally couple to Ca2+-activated potassium channels (Beurg et al., 2005), whereas in spiralganglion neurons, RyRs are functionally coupled to somatic AMPA-typeglutamate receptors (Morton-Jones et al., 2008). These observationssuggest the possibility that the cochlea represents a direct target of RyR-active PCBs, and that RyR-dependentmechanismswork in parallel or inseries with TH-dependent mechanisms to cause ototoxicity.

Emerging evidence from diverse areas of research raises the in-triguing possibility that RyR sensitization contributes to other knownbiological activities of PCBs. For example, it has been demonstrated thatPCBs activate the RyR causing release of Ca2+ from the ER (Wong &Pessah, 1997), which in turn can increase production of ROS (Ravagnan

Fig. 16. Mechanisms by which non-coplanar PCBs might induce apoptotic DNAfragmentation. Specific non-coplanar PCBs may directly activate the ryanodine receptor(RyR) causing release of Ca2+ from endoplasmic reticular (ER) stores. Increasedcytoplasmic Ca2+ activates caspases resulting in apoptosis. Increased cytoplasmic Ca2+

may also cause increased mitochondrial Ca2+ influx, which increases generation ofreactive oxygen species (ROS) thereby promoting caspase-dependent apoptosis. Alterna-tively, or in addition, PCBs may generate ROS directly, which then increase cytoplasmiclevels of Ca2+ via activation of RyRs. Blocking the L-type voltage-sensitive Ca2+ channelwith verapamil or the NMDA receptor with APV does not have any effect on PCB-inducedDNA fragmentation, suggesting that, in this model system, extracellular calcium is notinvolved in the apoptotic signaling pathway.


et al., 2002; Ermak & Davies, 2002) (Fig. 16). This might be a reciprocalinteraction in that ROS can directly modulate the channel activity of theRyR (Feng et al., 2000; Pessah, 2001). An interesting speculation is thatRyR-dependent mechanisms also contribute to the decreased levels ofcirculating thyroid hormone associated with PCB exposure. The thyroidgland is a major target organ of the sympathetic nervous system, andsympathetic neurons express RyRs (Vanterpool et al., 2006) and neuro-transmitter release from sympathetic neurons is regulated by RyRactivity (Cong et al., 2004). Conversely, since thyroid hormone regulatesRyR expression in at least the heart (Jiang et al., 2000; Dillmann, 2002;Hudecova et al., 2004), perhaps PCB effects on RYR expression aremediated in part by changes in thyroid hormone signaling.

Emerging evidence regarding a role for RyRs in regulatingdopamine homeostasis suggests the possibility that PCB sensitizationof RyRs contributes to the effects of PCBs on dopamine. Several mech-anisms are currently postulated to contribute to dopamine reductions

Fig. 17. 3-D projections of Bisphenol A, triclosan, and 2,2,′,4,4′-tetrabromodiphenyletheChemIDplus Advanced (Nat. Lib. Med.).

seen following PCB exposure, including inhibition of tyrosinehydroxylase and L-aromatic acid decarboxylase (Angus & Contreras,1996; Angus et al., 1997), two of the enzymes involved in the synthesisof dopamine, decreased striatal levels of the dopamine transport(DAT) (Caudle et al., 2006) and selective activation of oxidative stress-related pathways in dopaminergic neurons (Lee & Opanashuk, 2004).Ryanodine induces dopamine release from striatal dopaminergicneurons and this effect is significantly attenuated in striatal slicesisolated from RyR3 null mice (Wan et al., 1999). More recently, it hasbeen demonstrated that pharmacological manipulations of RyRactivity alter somatodendritic dopamine release (Patel et al., 2009)aswell as action potential- andNMDA receptor-evoked Ca2+ signaling(Cui et al., 2007; Harnett et al., 2009) in midbrain dopaminergicneurons, and that internal Ca2+stores are necessary for the abnormalrelease of dopamine via reverse transport through the dopaminetransporter caused by amphetamine and methamphetamine (Goodwinet al., 2009). Collectively these observations provide biological plausi-bility to the intriguing speculation that RyR sensitization may be aconvergent mechanism of PCB developmental neurotoxicity.

2.6. Convergent mechanism fornon-coplanar POPs: toward an alternative equivalence factor

The observation that RyRs play a critical role in diverse tissue typesand in numerous cellular processes raises an interesting challenge inlight of emerging data identifying RyRs as a direct molecular target inPCB neurodevelopmental toxicity. What factor(s) determine thespecificity of PCB toxicity? Why do PCBs seem to preferentially targetthe developing nervous system? Certainly the timing of exposure willinfluence the biological outcomes of PCB exposures, as will pharma-cokinetic parameters such as dosage, the metabolites produced, anddistribution of PCBs within the body. But other factors that could beequally important include expression patterns of RyRs and the com-plement of accessory proteins that comprise the calcium release unitas well as the antioxidant capacity of the cell. Alternatively, perhapsthe developing nervous system is not the only preferential target, andwe have simply missed toxic effects of PCBs on peripheral targetorgans (muscle, cochlea, pancreatic beta cells, etc) because littleattention has been paid to these endpoints. Given the role of the RyRin a number of peripheral tissues where PCBs have been shown tohave effects, at least in vitro, understanding how PCBs impact theseperipheral targets and their implications for human and animal healthand risk assessment seem warranted.

Another interesting hypothesis that emerges from consideration ofthe structure–activity relationship of PCB interactions with the RyR iswhether the RyR functions as a target for other toxicants that possessnon-coplanar structures. Obvious candidates are the polybrominateddiphenyl ethers (PBDEs) (Fig. 17). Recently Dingemans and coworkers

r (BDE-47). 3-D projections were calculated with the Molecular Dynamics Tool of


reported that the 6-hydroxylmetabolite of BDE-47 (20-120 µM) rapidlyinfluences intracellular Ca2+ homeostasis in PC12 cells, which can be atleast partially accounted for by release from the ER store (Dingemanset al., 2008). Preliminary evidence that certain PBDEcongeners and theirmetabolites directly alter RyR function has emerged (Kim et al., 2009b).Careful consideration of the 3-dimensional structure of these andrelated environmental contaminants of concern to human health mayreveal other RyR ligands. One such candidate, triclosan (Fig. 17), hasbeen shown in pilot studies to alter Ca2+ signals in a RyR-dependentmanner (Ahn et al., 2008). Another candidate, bisphenol A (Fig. 17),remains untested.Whether the effects of non-coplanar compounds thatare capable of altering subtle aspects of RyR function are additiveremains to be established. However, based on results obtained withnon-dioxin-like PCBs, the potential for toxicological significance is clear.The SAR data available for PCB interactions with RyRs, and theidentification of a RyR-based mechanism of neurodevelopmentaltoxicity, suggest the utility of developing an alternative TEF strategyfor non-dioxin-like PCBs based on their relative RyR activity. Thisstrategy has great appeal for assessing the risk of neurodevelopmentaltoxicity associated with exposure to mixtures of PCBs and has theflexibility to adjust to emerging data about exposures to existing andnew non-coplanar compounds and their metabolites based on RyRactivity.

Acknowledgments

We thank Drs. Kyung Ho Kim, Dongren Yang and Diptiman Bose forproviding editorial corrections. The authors gratefully acknowledge theartwork of Ms. Maureen Avakian presented in Fig. 12. Supported byNational Institutes of Health 1R01 ES014901 and 1P01 ES11269, theU.S. Environmental Protection Agency (U.S. EPA) through theScience to Achieve Results (STAR) program (Grant R829388 and833292), Autism Speaks/Cure Autism Now Environmental InnovatorAward, the UC Davis M.I.N.D. Institute, and the Superfund BasicResearch Program (P42 ES04699).

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Minding the calcium store: Ryanodine receptor activation as a