R

Rm

TSN

a

ARRAA

KCPIP

1

rdtaroaas((dpisRpce

Cf

0d

Cell Calcium 45 (2009) 527–534

Contents lists available at ScienceDirect

Cell Calcium

journa l homepage: www.e lsev ier .com/ locate /ceca

eview

egulation of Ca2+ entry by inositol lipids in mammalian cells by multipleechanisms

amas Balla ∗

ection on Molecular Signal Transduction, Program for Developmental Neuroscience, Eunice Kennedy Shriver National Institute of Child Health and Human Development,ational Institutes of Health, Bethesda, MD 20892, United States

r t i c l e i n f o

rticle history:eceived 2 February 2009eceived in revised form 18 March 2009ccepted 20 March 2009

a b s t r a c t

Increased phosphoinositide turnover was first identified as an early signal transduction event initiatedby cell surface receptors that were linked to calcium signaling. Subsequently, the generation of inositol1,4,5-trisphosphate by phosphoinositide-specific phospholipase C enzymes was defined as the major link

2+

vailable online 22 April 2009

eywords:alciumhosphoinositideon channel

between inositide turnover and the cytosolic Ca rise in response to external stimulation. However, in thelast decades, phosphoinositides have been emerging as major regulatory lipids involved in virtually everymembrane-associated signaling process. Phosphoinositides regulate both the activity and the traffickingof almost all ion channels and transporters contributing to the maintenance of the ionic gradients thatare essential for the proper functioning of all eukaryotic cells. Here we summarize the various meansby which phosphoinositides affect ion channel functions with special emphasis on Ca2+ signaling and

t gov

hospholipase C outline the principles tha

. Introduction

The requirement of calcium ions for contractility of the heart wasecognized by Sydney Ringer in 1883 marking the beginning of theevelopment of our understanding of the role of Ca2+ in muscle con-raction (see [1] for historical details). Calcium since became knowns one of the most universal intracellular signaling molecules thategulates virtually every aspect of a cell’s life and death. These notnly include rapid processes such as contraction and secretion butlso long-term responses such as regulation of metabolic enzymesnd ultimately gene expression. To act as an effective intracellularignal cytosolic Ca2+ concentration ([Ca2+]i) must be kept at a low∼100 nM) resting level, but also needs to rapidly rise to high levelsup to 10–100 �M) and quickly return to baseline. Therefore, theelicate control of cytoplasmic Ca2+ concentration has been a highriority during evolution. The source of Ca2+ for the [Ca2+]i increase,

n most cases, is the extracellular fluid but cells can also use Ca2+

tored in organelles, a mechanism highly evolved in skeletal muscle.

apid release of Ca2+ from intracellular stores [mostly the endo-lasmic reticulum (ER)] is a general mechanism to rapidly elevateytosolic Ca2+, but increased influx of Ca2+ is usually necessary tolicit a full biological response.

∗ Correspondence address: National Institutes of Health, Bldg 49, Rm 6A35, 49onvent Drive, Bethesda, MD 20892-4510, United States. Tel.: +1 301 496 2136;

ax: +1 301 480 8010.E-mail address: ballat@mail.nih.gov.

143-4160/$ – see front matter. Published by Elsevier Ltd.oi:10.1016/j.ceca.2009.03.013

ern the highly compartmentalized roles of these regulatory lipids.Published by Elsevier Ltd.

The mechanism of Ca2+ signal generation in so-called non-excitable tissues has become a center of interest when a group ofhormones and neurotransmitters acting on cell surface receptorswas found to activate cells without production of cAMP, the thenrecently discovered “second messenger” (see [2]). These stimulitermed “calcium-mobilizing agonists” were often linked with cGMPproduction and increased turnover of phosphatidylinositol (PtdIns)and both Ca2+ release and influx responses [3]. For a period, it wasbelieved that the source of the internal Ca2+ release was the mito-chondria, an organelle known for its ability to take up and releasesignificant amounts of Ca2+ [4]. Two major discoveries have finallyprovided with an explanation of how the Ca2+ signal was generated.First, it was recognized that PtdIns(4,5)P2 breakdown by PLC in theplasma membrane (PM) is the first step in the increased turnover ofPtdIns after hormonal stimulation [5,6], and second, it was demon-strated that one of the products of this reaction, Ins(1,4,5)P3 wascapable of releasing Ca2+ from non-mitochondrial internal Ca2+

stores [7]. With the finding of the Ins(1,4,5)P3 receptors (InsP3Rs) inthe ER [8] and identifying them as Ca2+ release channels [9], the linkbetween PtdIns turnover and Ca2+ release has been established.

Finding the mechanism responsible for the subsequent Ca2+

influx has proven to pose a greater challenge. In 1986, James Putneypostulated that during stimulation of calcium mobilizing receptors,

depletion of the ER Ca2+ stores was sufficient to activate Ca2+ influxwithout the need for any of the messengers formed by PLC action[10]. This mechanism has become known as store-operated Ca2+

entry (SOCE) and was believed not to depend on phosphoinosi-tides other than indirectly through Ins(1,4,5)P3, a regulator of Ca2+

5 ium 45

rrtcrbfmocectitPfbreTEae(egat

bicbppqrcoweinaPotnitita

2

2

tirpi

28 T. Balla / Cell Calc

elease from the ER. The nature of the channel responsible for SOCEemained elusive and for several years TRPC channels had beenhe most favored candidates [11]. TRP channels are non-specification channels first identified in Drosophila eye as the proteinsesponsible for a characteristic light-induced change in the mem-rane potential (transient receptor potential) in electric recordingsrom the eye [12]. After cloning of several similar channels from

ammalian sources [13], research on TRP have dominated the fieldf SOCE [11]. However, the ion selectivity and I/V profile of TRPhannels in electrical recordings did not match those of ICRAC, thelectrophysiological correlate of SOCE previously identified in mastells and T-cells [14,15], both of which display massive SOCE, ques-ioning whether TRP channels were responsible for the Ca2+ influxn these cells. The other unsolved question was the means by whichhe decreased luminal ER Ca2+ ([Ca2+]ER) is communicated to theM to activate Ca2+ entry. The most accepted model termed “con-ormational coupling” assumed some sort of molecular proximityetween the ER and the PM, where ER-resident proteins couldegulate PM ion channels by direct interaction [16], although thexistence of a diffusible messenger has been also considered [17].he final answers to these questions were found recently when theR proteins, STIM1 and 2, were discovered as the ER Ca2+ sensorsnd the Orai1/CRACM proteins as essential component of SOCE andxpression of these two proteins reconstituted both ICRAC and SOCEsee [18–20]). However, it should be noted that SOCE may not bexclusively attributed to the Orai channels, as recent evidence sug-ests that STIM1 can also communicate to TRPC proteins [21,22]nd that elimination of either Orai1 or TRPC channels can decreasehe native SOC pathway in some cells [23].

Although the link between SOCE and phosphoinositides haseen firmly established (via Ins(1,4,5)P3 production) several stud-

es suggested that a variety of other ion channels and transportersan also be regulated by PLC-coupled receptors and ultimatelyy membrane phosphoinositides (see [24,25]). Therefore, thehosphoinositide-regulation of ion channel and membrane trans-ort activities has emerged as a research topic parallel to theuestions on SOCE and became an important new aspect of neu-oscience and cell biology [26,27]. A third thread of researchonverging on this subject matter originated from the questionsf how newly synthesized channels are delivered to the PM andhether the channels were active within the internal membranes

n route to their final destination in the PM (see [28]). Even moremportantly, the removal and insertion of ion channels using inter-alization and recycling machineries of the cells, was recognized asway of rapidly regulating the number of channels available in theM. These processes linked ion channels to the general questionsf cell biology namely membrane assembly and movements withinhe cells. In this review we will try to highlight some examples of theumerous distinct ways of regulation of ion channels by phospho-

nositides. Because of the extensive literature in each of these topics,his review will not provide detailed in depth discussion availablen several comprehensive reviews, but will attempt to emphasizehe important overlaps between these otherwise disparate researchreas that all relate to some aspect of Ca2+ signaling.

. Phosphoinositide regulation of calcium signaling

.1. Regulation of the SOCE pathway by phosphoinositides

With the recent discovery of the STIM and Orai/CRACM proteins,

he basic mechanism by which Ca2+ depletion of the ER leads to Ca2+

nflux has been largely uncovered [18–20]. As detailed in severalecent reviews [19,29,30], the ER-resident single transmembranerotein STIM1 undergoes oligomerization upon Ca2+ unbinding

n its Ca2+ sensing luminal EF-hand domain. This oligomerization

(2009) 527–534

induces an interaction between the ER-bound STIM1 protein andthe Orai/CRACM channels located in the PM, leading to the openingof the latter and stabilization of ER–PM contact sites [31,32]. STIM1contains a polybasic sequence at its very C-terminus and its simi-larity to polybasic sequences found in other proteins that interactwith negatively charged membrane phospholipids raised the pos-sibility that STIM1–PM interactions are also facilitated by anionicphospholipids [31]. However, STIM1 constructs lacking the polyba-sic domain still can respond to Ca2+ depletion with oligomerizationand activate Orai1-mediated Ca2+ influx indicating, that polybasicdomains are not essential for SOCE [22,33,34].

Nevertheless, the pivotal role of the luminal ER [Ca2+] in the con-trol of SOCE together with the central importance of Ins(1,4,5)P3in the regulation of Ca2+ release from the ER, tightly links SOCEto Ins(1,4,5)P3 formation (Fig. 1A). Any intervention that reducesthe level of Ins(1,4,5)P3 (such as inhibition of PLC, or depletion ofits substrate, PtdIns(4,5)P2), immediately reduces SOCE providedthat the ER Ca2+ uptake mechanism is functional. This was demon-strated recently when rapid elimination of the PM PtdIns(4,5)P2led to a quick reversal of SOCE activation by a Ca2+-mobilizing ago-nist [35]. Similarly, several reports have shown that inhibition ofthe phosphatidylinositol 4-kinase(s) (PI4Ks) that supply the PMwith PtdIns4P [and hence PtdIns(4,5)P2] leads to a depletion ofPtdIns(4,5)P2 and a diminishing Ins(1,4,5)P3 generation in cellsstimulated by Ca2+-mobilizing agonists (e.g. [36,37]) ultimatelyresulting in an inhibition of SOCE. These results, however, did nottell whether SOCE itself requires phosphoinositides. This questioncan only be studied if SOCE is activated without Ins(1,4,5)P3, namelyby inhibition of the SERCA Ca2+ pump [by thapsigargin or thereversible inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone, TBHQ]. Afew studies have investigated this question. Broad et al. [38] havereported that high concentration of the PI3K inhibitors wortman-nin (Wm) and LY294002 inhibited SOCE and endogenous ICRAC inrat basophilic leukemia cells. They showed that this inhibition wasnot caused by the lack of InsP3 or DAG and it correlated betterwith changes in PtdIns4P than in PtdIns(4,5)P2 levels [38]. Theseauthors also showed that the PLC inhibitor, U73122 also inhibitedthe SOCE and ICRAC but only if added before its activation and thedrug failed to close SOCE once it got activated. Rosado et al. also ana-lyzed the effects of LY294002 on SOCE in platelets and concludedthat the inhibitory effect was not due to depolarization [39]. Theinterpretation of the experiments is complicated by the fact thatWm at concentrations used in these studies (but not LY294002)also inhibits MLCK, and MLCK inhibitors have been shown to inhibitSOCE [40]. This question was recently reexamined in our groupusing overexpressed STIM1 and Orai1 proteins. We found thatthe movements of STIM1 were slightly impaired in cells acutelydepleted in PtdIns(4,5)P2 but this had no impact on SOCE. However,PtdIns4P depletion either by using PI3K inhibitors at concentra-tions that inhibit type-III PI4Ks, or by PLC activation by agonistshad a significant inhibitory effect on SOCE. These inhibitory effectscorrelated with changes in PtdIns4P rather than PtdIns(4,5)P2, andwere not related to STIM1 movements (Korzeniowski et al., sub-mitted). These studies raise the possibility that Orai1 activationrequires PtdIns4P generation. These findings are even more intrigu-ing as the PI4K that is responsible for the generation of the PMpool of PtdIn4P is mostly ER localized in mammalian cells [41]. Thismakes the ER–PM contact sites stabilized by STIM1–Orai1 interac-tion a special cellular compartment potentially important for lipidtransfer between the two membranes.

2.2. Regulation of Ca2+ transport by the plasma membranephosphoinositides

The first reports on a direct role of membrane PtdIns(4,5)P2 ona calcium transport system was the finding of a stimulatory effect

T. Balla / Cell Calcium 45

Fig. 1. Different forms of regulation of ion channels relevant to Ca2+ signaling. (A)In the canonical phosphoinositide signaling pathway, phospholipase C (PLC) acti-vation from cell surface receptors leads to the generation of Ins(1,4,5)P3, which, inturn, releases Ca2+ from the endoplasmic reticulum (ER) Ca2+ stores via Ins(1,4,5)P3

receptor-channels. The decreased ER luminal Ca2+ is sensed by the ER residentSTIM proteins that respond with oligomerization and interaction with PM (PM)Orai/CRACM channels at ER–PM junctional sites activating Ca2+ influx. This formof Ca2+ entry is indirectly linked to Ins(1,4,5)P3 production, but direct regulation ofthe Orai channels and possibly STIM interaction with the PM by phosphoinositideshas also been proposed. (B) Direct regulation of ion channel (and transporter) activ-ities by PM phosphoinositides. Here the lipid directly interacts with some molecularcomponent of the channel usually by electrostatic interaction involving positivelycharged regions of the channel facing the cytoplasm. Phosphoinositide levels canchange as a result of PLC activation, or due to a shift in the balance of 5-phosphataseand PIP 5-kinase ctivities. Channels with the highest affinity to inositides may notrespond to decreasing inositide levels if they tightly hold on to their bound lipid,therefore, the same lipid change can evoke a variety of activity changes depend-ing on the lipid affinities of the channels. Potassium channels are also known tobe sensitive to phosphoinositide changes and because they have significant impacton the membrane potential, they can indirectly affect Ca2+ entry by changing theelectrochemical driving force of Ca2+. This is especially important for voltage-gatedCa2+ channels or for the inwardly rectifying ICRAC channels. (C) Ion channels are alsomoved around within the cells as a result of membrane trafficking. The synthesisand assembly of channels usually occures in the ER and the channels are glycosy-lated in the Golgi from where they are heading their final destination, the PM. Itis now believed that most channels are not active within the internal membranes,although there are examples suggesting otherwise. PtdIns(4,5)P2 is present in thePM and channels and transporters that require this lipid for activity gain function-ality only upon insertion in the PM. In the opposite process, active channels can beremoved from the PM by various interanlization routes, some clathrin-dependent,some – independent. Internalized channles are moved into recycling compartmentsfrom where they can recycle back to the membrane or head to degradation. All ofthese trafficking events are regulated by phosphoinositides.

(2009) 527–534 529

of acidic phospholipids on the PM Ca2+ pump that was similar tothose elicited by calmodulin or limited proteolysis [42]. Similarly,purified sarcoplasmic reticulum Ca2+ ATPase was shown to containtightly associated phosphoinositides suggesting a possible regula-tory interaction between the Ca2+ pump and these phospholipids[43]. However, fewer studies addressed whether the lipid regulationshown in isolated membranes or purified proteins does play a rolein an intact cell system. Our studies using Wm at concentrationsthat inhibit type-III PI 4-kinases in combination with agonist-induced PLC activation showed that cells depleted in PtdIns(4,5)P2still can reduce their [Ca2+]i very effectively with Ca2+ extrusion[44]. On the other hand, LY294002 was shown to inhibit Ca2+ extru-sion in platelets at concentrations that also inhibit PI 4-kinases andperhaps other enzymes [45].

Although the effect of anionic phospholipids on the Na+/Ca2+

exchange activity in reconstituted systems have been described ear-lier [46], Donald Hilgemann was the first to demonstrate that theeffects of ATP on the Na+/Ca2+ exchanger (NCX1) and on KATP potas-sium channels in giant excised patches of the heart were mediatedby the synthesis of anionic phospholipids, mainly PtdIns(4,5)P2[47]. However, the changes in phosphoinositides in the heart are notparticularly robust after hormonal influences and it is still puzzlingwhether Na+/Ca2+ exchange activities are controlled by changingPtdIns(4,5)P2 levels or the high PtdIns(4,5)P2 of the PM is requiredas a permissive environment restricting the transport activity ofthe NCX1 protein to the PM [48]. It is noteworthy, though, thatCa2+-calmodulin has a strong stimulatory effect on ATP-inducedPtdIns(4,5)P2 (but not PtdIns4P) synthesis in crude cardiac mem-branes [49] and osmotically induced shrinkage increases bothPtdIns4P and PtdIns(4,5)P2 in several cell types [49]. However,whether PtdIns(4,5)P2 levels change during contraction and relax-ation in the heart is yet to be determined.

The reversal of the inactivation (termed run-down) ofthe Na+/Ca2+ exchanger or the KATP potassium channel inexcised macropatches by PtdIns(4,5)P2 and ATP (via synthesisof PtdIns(4,5)P2) [47] has established an experimental paradigmthat was followed by a large number of studies revealing thePtdIns(4,5)P2 requirement of several ion channels [24,25]. Theseincluded potassium channels of a great variety (see below) as wellas calcium conductive channels, such as the voltage-gated P/Q [50]and N-type [25,51] Ca2+ channels and several members of the TRPfamily of non-selective cation channels [52]. Recently this list wasextended to some of the ligand-gated channels such as the NMDAreceptors [53] and the ATP-gated P2X4 [54]. In almost all of thesestudies the question was raised whether PtdIns(4,5)P2 is merelya requirement for the proper functioning of the channels or itis actually regulated by PtdIns(4,5)P2 changes that occur duringactivation of PLC-coupled receptor mechanisms. This question hasbeen unequivocally answered in some cases, such as the M-current(mediated by the KCNQ potassium channels) [55,56] or the cold andmenthol sensitive TRPM8 channels [57,58]. The activity of the KCNQchannels follows very tightly the changes in PM PtdIns(4,5)P2 somuch so that it can be used as a PtdIns(4,5)P2 sensor [55]. However,there are other examples where the role of PtdIns(4,5)P2 is morecontroversial, being reported as inhibitory as well as a stimulatoryfactor. For example, an apparently opposing effects of PtdIns(4,5)P2are thought to depend on the levels of the lipid in the case ofP/Q channels. It was proposed that a high affinity lipid bindingsite stabilizes the channel, whereas binding of PtdIns(4,5)P2 to aputative lower affinity site would switch the channel into a “reluc-tant” mode [51]. In another example, TRPV1 channels were shownto be sensitized by PLC-coupled agonists apparently via reduction

of PtdIns(4,5)P2 [59] but the same lipid was found to be requiredfor the recovery of TRPV1 channels from desensitization [60]. Theseseemingly contradictory findings were resolved when it was shownthat PtdIns(4,5)P2 depletion contributes to the desensitization of

5 ium 45

tsic

ccatPtmioiePnPnoaoph

2p

ftveasctbar

t[pChbl(a[bta

red[ctlogn

30 T. Balla / Cell Calc

he TRPV1 channels at high capsaicin concentration, but at low cap-aicin doses PtdIns(4,5)P2 inhibited the same channels [61]. Thenositol lipid regulation of TRPM8 and TRPV1 channels are dis-ussed in detail in this issue by Tibor Rohacs.

The mechanism of direct inositol lipid regulation of these Ca2+

onductive channels remains elusive (Fig. 1B). Almost all of thesehannels contain clusters of basic residues in their membrane-djacent regions facing the cytosol or within their C-terminalails. In some TRP channels there are sequences identified as “halfH domains” that can form intermolecular PH domains poten-ially binding phosphoinositides [62], but lipid regulation was also

apped to the TRP domains of their cytoplasmic tails [57,63]. Annteresting and common feature of the PtdIns(4,5)P2 regulationf many potassium and TRP channels is that the lipid alters thenteraction of the channels with other specific regulators. In sev-ral instances this regulator is calmodulin, which competes withtdIns(4,5)P2 such as in TRPC6, TRPV1, voltage-gated Ca2+ chan-els and KCNQ potassium channels [64]. Other examples includetdIns(4,5)P2 modulation of the interaction of Kir3 potassium chan-els with ��-subunits [65], or the PtdIns(4,5)P2-mediated decreasef the apparent ATP affinity of KATP channels [66]. The direct inter-ction of phosphoinositides with ion channels whether regulatoryr permissive is a fascinating research area and adds to the com-lexity by which phosphoinositide changes can influence the ionicomeostasis of the cells.

.3. Regulation of the removal or insertion of channels in thelasma membrane by phosphoinositides

Delivery of ion channels and transporters from a reserve poolound in subplasmalemmal vesicles could be a mechanism by whichhe number of channels can be rapidly increased in the PM. Con-ersely, channels can be rapidly moved from the membrane byndocytosis to decrease their number in the membrane (Fig. 1C). Inddition, the constitutive trafficking of Ca2+ channel subunits to theite of their assembly is important to supply the PM with functionalhannels. Regulation of membrane trafficking and fusion events,herefore, can indirectly influence ion fluxes through the mem-rane. Since phosphoinositides play important roles in both exo-nd endocytosis [67], they provide an additional means to channelegulation.

PI3K-dependent trafficking of voltage-gated Ca2+ channels tohe PM has been recently reported in myoblasts and COS-7 cells68]. This is mediated by PtdIns(3,4,5)P3 induced Akt activation andhosphorylation on a Ser residue of the �2a subunit of the N-typea2+ channels [68]. An IGF1-induced PI3K-dependent increase ofigh voltage-activated Ca2+ channels in cerebellar neurons has alsoeen described [69]. Another well-documented example of regu-

ated channel insertion into the PM is the epithelial sodium channelENaC) (even though this is not directly related to Ca2+ signaling),channel that also is regulated by PtdIns(4,5)P2 once in the PM

70]. ENaC channels rapidly incorporate into the apical membraney a Rho and PIP 5-kinase-dependent mechanism [71], whereasheir removal from the membrane and degradation is inhibited byPI3K-mediated mechanism [72].

There are other documented examples of the phosphoinositide-egulation of trafficking of TRP channels [28]. A curious stimulatoryffect of PLC�1 expression on the activity of TRPC3 channels (whichid not require the PLC activity of the protein) has been described73]. This was attributed to the enhanced surface expression of thehannels aided by an intermolecular PH domain formation between

he 1/2 PH-domains of PLC�1 and the other half within the intracel-ular tails of TRPC channels and the presumed inositol lipid bindingf the hybrid PH domain [62]. It has been shown recently thatrowth factor stimulation enhances the insertion of TRPC5 chan-els from vesicular pools by a PI3K and Rac1 mediated mechanism

(2009) 527–534

that also involves PIP5KI� [74]. Similarly, EGF-stimulated insertionof TRPC4 channels into the PM was reported. Channels of otherTRP subfamilies, including TRPV2 and TRPV1 were also shown totranslocate to the PM following NGF or IGF-1 stimulation [75,76].One of the most prominent examples of channel regulation by inser-tion into and removal from the PM is found in Drosophila eye, wherethe TRP-like, or TRPL channel was shown to undergo a light-inducedinternalization into a storage compartment from which the channelgets reinserted to the photoreceptor membrane during recovery inthe dark [77,78].

Several studies have shown the removal of ion channels fromthe PM as a way of reducing channel activity. The Kir1.1 K+

channels (also known as ROMK) in the kidney are down regu-lated by clathrin-dependent endocytosis during dietary potassiumrestriction by a mechanism involving the endocytic adaptor pro-tein, ARH and the “NPXY” internalization motif present in thesechannels [79]. Overexpression of PI 4-kinase and PIP 5-kinase sig-nificantly decreased rather than increased the current densitiesof NXC1, and a targeted expression of PI4KII� in the heart oftransgenic mice resulted in a large decrease of NXC1 current den-sity without a change in expression levels and an increased rateof clathrin-mediated endocytosis [80]. These data strongly sug-gested that the effects of phosphoinositides on the endocytosis andrecycling of these channels are dominant during long-term exper-iments.

These examples demonstrate that the acute regulation ofchannel activity in the PM by phosphoinositide changes is com-plemented with phosphoinositide control of the trafficking anddistribution of these channels between the various membranes,adding a new level of complexity to the control of Ca2+ influx intothe cell.

2.4. Regulation of Ca2+ influx by phosphoinositides indirectly bythe changing membrane potential

The amount of Ca2+ entering a cell through various Ca2+ conduc-tive channels is primarily determined by the open and closed stateof the channel pores. Activation and inactivation of voltage-gatedCa2+ channels are clearly regulated by the membrane potential, buteven channels that are not gated by voltage are sensitive to themembrane potential as the amount of Ca2+ flowing through themdepends on the electrochemical gradient of Ca2+. This is especiallytrue for the ICRAC channels that show a pronounced inward rec-tification and hence this form of Ca2+ entry is very sensitive todepolarization [81]. It is because of this feature of Ca2+ entry whyphosphoinositide-regulation of the various potassium channels isrelevant to Ca2+ signaling. Potassium channels of a great varietyshow a wide range of functions. Inwardly rectifying K+ (Kir) chan-nels regulate the pattern of firing of action potentials, contributeto metabolic regulation (via control of insulin secretion) or deter-mine potassium homeostasis (ROMK). A large family of two-poreK+ channels serve as “background channels” determining the basalmembrane potential in many neurons and neuroendocrine tissues[82], while members of the KCNQ family of K+ channels underliethe M-current (KCNQ-2, -3, -5) found in neurons as well as the I(Ks)current (KCNQ1) of cardiac tissues [83]. As outlined above, all theseK+ channels show a dependence and/or regulation by PM phospho-inositides that have been discussed in many recent reviews and willnot be further detailed here [84]. However, it is important to keep inmind that phosphoinositide changes can have profound influence

on both the basal membrane potential or on the shape of actionpotentials via their influence on these K+ channels, which, in turn,affects Ca2+ signaling (Fig. 1B). Such an indirect effect should not bemistaken with the direct regulation of Ca2+ conductive channels byphosphoinositides.

um 45 (2009) 527–534 531

3p

mcsTEptwwalpcPTtlibiw

ttCaI(itgH(ucPantbtttt

diitiTpnPbnfctieg

Fig. 2. Principles governing the compartmentalized regulation of ion channels (orother effectors) by phosphoinositides. (A) PtdIns(4,5)P2 in the plasma membraneexists in free form but is also bound to several proteins regulated by this lipid (effec-tors, Eff1-4). These could be channels, transporters, actin binding proteins, clathrinadaptors, enzymes and several other proteins. The enzymes that produce and con-vert these lipids [including PIP 5-kinases (PIPkin), phosphoinositide 5-phosphatases(5-ptase), Class I PI 3-kinases (PI3K) and phospholipase C (PLC) enzymes] act on theunbound fraction (in pink) which is in a dynamic equilibrium with the protein boundfractions (Eff1-4). Changes in the level of free PtdIns(4,5)P2 are reflected differentlyin the pools bound to the various effectors depending on their rates of dissociation.This represents a functional compartmentalization that does not necessarily meana phisical segregation but may result in different metabolic turnover rates. (B) Theo-retical example of the regulation of an effector (ion channel) by a phosphoinositide

T. Balla / Cell Calci

. Organization of localized changes and microdomains inhosphoinositide regulation

The extent to which phosphoinositide regulation is compart-entalized is being increasingly recognized. The PM has long been

onsidered as the relevant site of polyphosphoinositide synthe-is and PLC-mediated hydrolysis from a Ca2+ signaling standpoint.he only question regarding compartmentalization was how theR-synthesized PtdIns reaches the PM to serve as precursor forolyphosphoinositide synthesis leading to the identification ofhe PI transfer proteins [85,86]. This view has suddenly changedhen the yeast PI 3-kinase, Vps34p and its lipid product, PtdIns3Pere identified as key regulators of vacuolar sorting in yeast [87]

nd endocytic trafficking in mammalian cells [88]. This was fol-owed by the surprising localization of several PI 4-kinase andhosphoinositide phosphatase isoforms in the Golgi and endo-ytic compartments [89–91] challenging our understanding of howtdIns4P is made and reaches the PM in mammalian cells [92].herefore, when thinking of compartmentalization, most of us refero the unique phosphoinositides composition of various intracellu-ar membranes. While this view is certainly valid and importantn defining the inositide signature of internal membranes, it isecoming evident that further compartmentalization of phospho-

nositides takes place even within the PM, Golgi or any membraneithin the cell.

Some of these compartments can be physically unique, such ashe apparent enrichment of phosphoinositides in detergent resis-ant membrane domains often referred to as “rafts” [93]. Pike andasey [94] suggested that agonist-sensitive phosphoinositides aressociated with caveolin-rich rafts in A431 cells. Indeed, the typeI PI 4-kinase was found in and purified from detergent resistantthough non-caveolar) membrane fractions [95]. Similarly, severalmportant signaling molecules related to phosphoinositide syn-hesis and hydrolysis are present in “rafts” [96,97] leading to theeneral notion that phosphoinositides are mostly “raft” associated.owever, “raft” is a term that is not linked to any specific membrane

although most believe it is part of the PM) and thorough attemptssing the recently available phosphoinositide visualization toolsould not confirm that PtdIns(4,5)P2 are enriched in “rafts” in theM [98]. Moreover, the majority of the PI4KII� enzyme, mentionedbove, has been localized to the TGN and endosomal membraneetwork as opposed to the PM [89–91], although, undoubtedlyhis activity is present in the PM (the best example being the redlood cell membrane). Nevertheless, the possibility still remainshat some phsophoinositide regulation is linked to “rafts” and inhis context it is relevant that among the Ca2+ conductive channels,he TRPC1 channels are enriched in caveoli [99] and are responsibleo the cyclodextrin sensitivity of SOCE in some cell types [100].

Regardless of whether inositides are segregated in physicallyefinable compartments, strong evidence suggests that phospho-

nositides are functionally compartmentalized. This is a result of thenteraction of phosphoinositides, such as PtdIns(4,5)P2 with mul-iple effector proteins (Fig. 2A). These protein–phosphoinositidenteractions display a variety of affinities and dissociation rates.his implies that during activation of a PLC or phosphoinositidehosphatase enzyme, the decreasing free PtdIns(4,5)P2 level willot be uniformly followed by a similar decreases in the amount oftdIns(4,5)P2 bound to various interacting molecules. Proteins thatind PtdIns(4,5)P2 with high affinity and slow dissociation rate mayot “sense” immediately the decreasing lipid levels while those

rom which the lipid rapidly dissociates will “read” the free lipid

hanges very closely. This affinity-driven functional compartmen-alization was shown to play a role for example in the differentialnositide sensitivities of two-pore K+ channels [101]. Since the lat-ral diffusion of free phosphoinositides is fast, it is not possible toenerate a sharp lipid gradient in any particular membrane without

dedicated to the protein. Here the associated kinase or phosphatase ensures that thephosphoinositide is not diffusing away from the effector and does not contribute tothe larger “shared” pool of the inositol lipid in question.

association of the lipids with proteins with slower lateral mobility.The dissociation rate of the lipid from the binding protein (the effec-tor) together with the lateral mobility of the latter will determinehow far the lipid can diffuse on the “back” of an effector molecule.This has been elegantly analyzed recently using pleckstrin homol-ogy (PH) domains with various affinities to PtdIns(4,5)P2 [102]. Thismechanism, however, alone is probably not sufficient to generatefunctional inositide gradients. What is also needed is the highlylocalized production (or elimination) of the lipids in the close vicin-ity of the effector molecule. This model assumes that the inositollipid kinases (and/or phosphatase) are in close association with theeffector molecule (for this review, an ion channel) that is to be reg-ulated by the lipid. This arrangement has several advantages: thelocalized lipid change can control the channel without any “spilling”effect on neighboring proteins, and the lipid binding specificity ofthe channel is less critical for the regulation. The specificity is pro-vided by the kinase and phosphatase that is tightly associated withthe signaling complex. Although this signaling organization repre-sents the highest degree of specificity and is most likely widely usedin biology, it is the most difficult to demonstrate experimentally.Such highly localized lipid changes dedicated to specific effectormolecules will probably evade detection by the widely used GFP-

PH domain lipid binding tools unless the probe originates from thevery effector molecule itself. There are already few examples ofthe use of ion channels as reporters of phosphoinositide changes[55]. This organization explains why inositide kinases or phos-

5 ium 45

patpolchtawOiilrtcctsr[

toegacmpp

4

igrhrhcrptrwcot(ttatTthwTten

32 T. Balla / Cell Calc

hatases with apparently identical localization and biochemicalctivity can assume non-redundant functions. Recent examples arehe distinct roles of two PIP 5-kinase isoforms, both localized inhagocytic cups, in actin polymerization and phagocytosis [103],r the non-redundant role of PI 4-kinase III� and PI4KII�, bothocalized to the Golgi, in supporting the transfer function of theeramide transfer protein [104]. Although this level of specificityas not been clearly demonstrated for Ca2+ conductive channelshere are some indications that such specific control mechanismslso exists in ion channel regulation. Interaction of ion channelsith enzymes that control phosphoinositides has been reported.ne of the TRP channels, TRPM7 (TRP-PLIK) was identified as an

nteractor with the C-terminal tail of PLC�1 [105]. Subsequentlyt was shown that TRPM7 associates with PLC�1 and is regu-ated by PLC-activating agonists [106], although the direction ofegulation remains controversial [107]. In another example, pro-eomic analysis of proteins associated with the purinergic P2X7hannel has identified PI4KIII� as part of the channel-associatedomplex [108]. The recently described voltage-regulated inosi-ide 5-phosphatase isolated from ciona intestinalis (ci-VSP) alsouggests that rapid localized changes in phosphoinositide levelsegulated by voltage could be directly linked to ion channel function109].

Unfortunately, it is extremely difficult to analyze the exis-ence and the importance of direct interaction of inositide kinasesr phosphatases with ion channels. RNAi-mediated depletion, orxpression of dominant negative versions of these enzymes, hasrave consequences on the vesicular trafficking of the cells likelyltering the distribution of these channels. Interruption of the asso-iation between the two proteins or acute inhibition of the enzymesight be a better approach to analyze the significance of these

rotein–protein interactions leading to localized delivery of phos-hoinositides to ion channels.

. Concluding remarks

Postulating and revealing the connection between phospho-nositides and calcium signaling [2,110] have been one of thereatest milestones in the last 50 years in signal transductionesearch. Yet, none of us who witnessed these developments couldave foreseen the complexity, depth and variety of inositide-egulation of cellular Ca2+ homeostasis. This short summaryighlighted three aspects of Ca2+ channel regulation, namely thelassical link between PLC-mediated Ins(1,4,5)P3 production, Ca2+

elease and the store-operated Ca2+ entry, the direct effect of PMhosphoinositides on channel gating, and the regulation of channelrafficking by phosphoinositides. This, however, is an artificial sepa-ation, because these regulatory means are closely interrelated andork in concert all the time. Appreciating this complexity is cru-

ial for the right interpretation of our experimental data. Often thenly way to change phosphoinositides is to express or knock downhe enzymes that either form them (the kinases) or eliminate themthe phosphatases). However, these manipulations require a longime period before the effects can be analyzed. During this timehe assembly, trafficking or elimination of the channels may havemore important and lasting effects on apparent channel activity

han the actual change in the phosphoinositides in the membrane.his is one of the many reasons why development of specific inosi-ide kinase inhibitors is so desirable and also why we and othersave been working on methods by which inositides can be changed

ithin the intact cell acutely in a matter of seconds [56,111,112].

hese new tools should facilitate our understanding on the impor-ance of each of the inositide-dependent regulatory processes forach of the channels in a real cellular setting. Such studies are stilleeded because the fundamental question of what makes these

(2009) 527–534

lipids so versatile and universally adaptable signaling cues remainsto be answered.

Acknowledgement

This research was supported by the Intramural Research Pro-gram of the Eunice Kennedy Shriver National Institute of ChildHealth and Human Development of the National Institutes ofHealth.

References

[1] D.J. Miller, Sydney Ringer; physiological saline, calcium and the contraction ofthe heart, J. Physiol. 555 (2004) 585–587.

[2] R.H. Michell, Inositol phospholipids and cell surface receptor function,Biochim. Biophys. Acta 415 (1975) 81–147.

[3] R.H. Michell, Inositol lipids and their role in receptor function: history andgeneral principles, in: J.W. Putney Jr. (Ed.), Phosphoinositides and ReceptorMechanisms, Alan R. Liss, Inc., New York, 1986, pp. 1–24.

[4] T. Pozzan, P. Magalhaes, R. Rizzuto, The comeback of mitochondria to calciumsignalling, Cell Calcium 28 (2000) 279–283.

[5] J.A. Creba, C.P. Downes, P.T. Hawkins, G. Brewster, R.H. Michell, C.J. Kirk,Rapid breakdown of phosphatidylinositol 4-phosphate and phosphatidylinos-itol 4,5-bisphosphate in rat hepatocytes stimulated by vasopressin and otherCa2+-mobilizing hormones, Biochem. J. 212 (1983) 733–747.

[6] M.J. Berridge, Rapid accumulation of inositol trisphosphate reveals thatagonists hydrolyze polyphosphoinositides instead of phosphatidylinositol,Biochem. J. 212 (1983) 849–858.

[7] H. Streb, R.F. Irvine, M.J. Berridge, I. Schulz, Release of Ca2+ from anonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate, Nature 306 (1983) 67–68.

[8] A. Spät, P.G. Bradford, J.S. McKinney, R.P. Rubin, J.W. Putney, A saturablereceptor for 32P-inositol-1,4,5-trisphosphate in hepatocytes and neutrophils,Nature 319 (1986) 514–516.

[9] T. Furuichi, S. Yoshikawa, A. Miyawaki, K. Wada, N. Maeda, K. Mikoshiba, Pri-mary structure and functional expression of the inositol 1,4,5-trisphosphate-binding protein P400, Nature 342 (1989) 32–38.

[10] J.W. Putney Jr., A model for receptor-regulated calcium entry, Cell Calcium 7(1986) 1–12.

[11] A.B. Parekh, J.W. Putney Jr., Store-operated calcium channels, Phys. Rev. 85(2005) 757–810.

[12] C. Montell, G.M. Rubin, Molecular characterization of the Drosophila trp locus:a putative integral membrane protein required for phototransduction, Neuron2 (1989) 1313–1323.

[13] X. Zhu, M. Jiang, M. Peyton, G. Boulay, R. Hurst, E. Stefani, L. Birnbaumer, Trp,a novel mammalian gene family essential for agonist-activated capacitativeCa2+ entry, Cell 85 (1996) 661–671.

[14] M. Hoth, R. Penner, Depletion of intracellular calcium stores activates a calciumcurrent in mast cells, Nature 355 (1992) 353–356.

[15] A. Zweifach, R.S. Lewis, Mitogen-regulated Ca2+ current of T-lymphocytes isactivated by depletion of intracellular Ca2+ stores, Proc. Natl. Acad. Sci. U.S.A.90 (1993) 6295–6299.

[16] M. Berridge, Conformational coupling: a physiological calcium entry mecha-nism, Sci. STKE (2004) pe33.

[17] C. Randriamampita, R.Y. Tsien, Emptying of intracellular Ca2+ stores releases anovel small messenger that stimulates Ca2+ influx, Nature 364 (1993) 809–814.

[18] R.S. Lewis, The molecular choreography of a store-operated calcium channel,Nature 446 (2007) 284–287.

[19] J.W. Putney Jr., Recent breakthroughs in the molecular mechanism of capac-itative calcium entry (with thoughts on how we got here), Cell Calcium 42(2007) 103–110.

[20] J. Soboloff, M.A. Spassova, M.A. Dziadek, D.L. Gill, Calcium signals mediated bySTIM and Orai proteins—a new paradigm in inter-organelle communication,Biochim. Biophys. Acta 1763 (2006) 1161–1168.

[21] G.N. Huang, W. Zeng, J.Y. Kim, J.P. Yuan, L. Han, S. Muallem, P.F. Worley, STIM1carboxyl-terminus activates native SOC, I(crac) and TRPC1 channels, Nat. CellBiol. 8 (2006) 1003–1010.

[22] W. Zeng, J.P. Yuan, M.S. Kim, Y.J. Choi, G.N. Huang, P.F. Worley, S. Muallem,STIM1 gates TRPC channels, but not Orai1, by electrostatic interaction, Mol.Cell 32 (2008) 439–448.

[23] M.S. Kim, W. Zeng, J. Yuan, D.M. Shin, P. Worley, S. Muallem, Native store-operated Ca2+ influx requires the channel function of Orai1 and TRPC1, J. Biol.Chem. 284 (2009) 9733–9741.

[24] D.W. Hilgemann, S. Feng, C. Nasuhoglu, The complex and intriguing lives ofPIP2 with ion channels and transporters, Sci. STKE (2001) RE19.

[25] N. Gamper, M.S. Shapiro, Regulation of ion transport proteins by membranephosphoinositides, Nat. Rev. Neurosci. 8 (2007) 921–934.

[26] B.C. Suh, B. Hille, Regulation of ion channels by phosphatidylinositol 4,5-bisphosphate, Curr. Opin. Neurobiol. 15 (2005) 370–378.

[27] B.C. Suh, B. Hille, PIP2 is a necessary cofactor for ion channel function: howand why? Annu. Rev. Biophys. 37 (2008) 175–195.

um 45

T. Balla / Cell Calci

[28] S. Cayouette, G. Boulay, Intracellular trafficking of TRP channels, Cell Calcium42 (2007) 225–232.

[29] R.M. Luik, R.S. Lewis, New insights into the molecular mechanisms ofstore-operated Ca2+ signaling in T cells, Trends Mol. Med. 13 (2007) 103–107.

[30] T. Hewavitharana, X. Deng, J. Soboloff, D.L. Gill, Role of STIM and Orai pro-teins in the store-operated calcium signaling pathway, Cell Calcium 42 (2007)173–182.

[31] J. Liou, M. Fivaz, T. Inoue, T. Meyer, Live-cell imaging reveals sequentialoligomerization and local plasma membrane targeting of stromal interactionmolecule 1 after Ca2+ store depletion, Proc. Natl. Acad. Sci. U.S.A. 104 (2007)9301–9306.

[32] M.M. Wu, J. Buchanan, R.M. Luik, R.S. Lewis, Ca2+ store depletion causes STIM1to accumulate in ER regions closely associated with the plasma membrane, J.Cell. Biol. 174 (2006) 803–813.

[33] Z. Li, J. Lu, P. Xu, X. Xie, L. Chen, T. Xu, Mapping the interacting domains of STIM1and Orai1 in Ca2+ release-activated Ca2+ channel activation, J. Biol. Chem. 282(2007) 29448–29456.

[34] J.P. Yuan, W. Zeng, M.R. Dorwart, Y.J. Choi, P.F. Worley, S. Muallem, SOAR andthe polybasic STIM1 domains gate and regulate Orai channels, Nat. Cell Biol.11 (2009) 337–343.

[35] P. Varnai, B. Toth, D.J. Toth, L. Hunyady, T. Balla, Visualization and manipulationof plasma membrane-endoplasmic reticulum contact sites indicates the pres-ence of additional molecular components within the STIM1–Orai1 complex,J. Biol. Chem. 282 (2007) 29678–29690.

[36] S. Nakanishi, K.J. Catt, T. Balla, A wortmannin-sensitive phosphatidylinositol 4-kinase that regulates hormone-sensitive pools of inositolphospholipids, Proc.Natl. Acad. Sci. U.S.A. 92 (1995) 5317–5321.

[37] G.B. Willars, S.R. Nahorski, R.A. Challiss, Differential regulation of mus-carinic acetylcholine receptor-sensitive polyphosphoinositide pools andconsequences for signaling in human neuroblastoma cells, J. Biol. Chem. 273(1998) 5037–5046.

[38] L.M. Broad, F.J. Braun, J.P. Lievremont, G.S. Bird, T. Kurosaki, J.W. Putney Jr.,Role of the phospholipase C-inositol 1,4,5-trisphosphate pathway in calciumrelease-activated calcium current and capacitative calcium entry, J. Biol. Chem.276 (2001) 15945–15952.

[39] J.A. Rosado, S.O. Sage, Phosphoinositides are required for store-mediated cal-cium entry in human platelets, J. Biol. Chem. 275 (2000) 9110–9113.

[40] H. Watanabe, Q.K. Tran, K. Takeuchi, M. Fukao, M.Y. Liu, M. Kanno, T. Hayashi,A. Iguchi, M. Seto, K. Ohashi, Myosin light-chain kinase regulates endothelialcalcium entry and endothelium-dependent vasodilation, FASEB J. 15 (2001)282–284.

[41] A. Balla, Y. Ju Kim, P. Varnai, Z. Szentpetery, Z. Knight, K.M. Shokat, T. Balla,Maintenance of hormone-sensitive phosphoinositide pools in the plasmamembrane requires phosphatidylinositol 4-kinase III{alpha}, Mol. Biol. Cell19 (2007) 711–721.

[42] V. Niggli, E.S. Adunyah, E. Carafoli, Acidic phospholipids, unsaturated fattyacids, and limited proteolysis mimic the effect of calmodulin on the purifiederythrocyte Ca2+-ATPase, J. Biol. Chem. 256 (1981) 8588–8592.

[43] M. Varsanyi, H.G. Tolle, M.G. Heilmeyer Jr., R.M. Dawson, R.F. Irvine, Activa-tion of sarcoplasmic reticular Ca2+ transport ATPase by phosphorylation of anassociated phosphatidylinositol, EMBO J. 2 (1983) 1543–1548.

[44] S. Nakanishi, K.J. Catt, T. Balla, Inhibition of agonist-stimulated inositol 1,4,5-trisphosphate production and calcium signaling by the myosin light chainkinase inhibitor, wortmannin, J. Biol. Chem. 269 (1994) 6528–6535.

[45] J.A. Rosado, S.O. Sage, Regulation of plasma membrane Ca2+-ATPase by smallGTPases and phosphoinositides in human platelets, J. Biol. Chem. 275 (2000)19529–19535.

[46] R. Vemuri, K.D. Philipson, Phospholipid composition modulates the Na+–Ca2+

exchange activity of cardiac sarcolemma in reconstituted vesicles, Biochim.Biophys. Acta 937 (1988) 258–268.

[47] D.W. Hilgemann, R. Ball, Regulation of cardiac Na+, Ca2+ exchange and KATPpotassium channels by PIP2, Science 273 (1996) 956–959.

[48] C. Nasuhoglu, S. Feng, Y. Mao, I. Shammat, M. Yamamato, S. Earnest, M. Lem-mon, D.W. Hilgemann, Modulation of cardiac PIP2 by cardioactive hormonesand other physiologically relevant interventions, Am. J. Physiol. Cell Physiol.283 (2002) C223–C234.

[49] D.W. Hilgemann, On the physiological roles of PIP(2) at cardiac Na+ Ca2+

exchangers and K(ATP) channels: a long journey from membrane biophysicsinto cell biology, J. Physiol. 582 (2007) 903–909.

[50] L. Wu, C.S. Bauer, X.G. Zhen, C. Xie, J. Yang, Dual regulation of voltage-gatedcalcium channels by PtdIns (4,5)P2, Nature 419 (2002) 947–952.

[51] I.E. Michailidis, Y. Zhang, J. Yang, The lipid connection-regulation of voltage-gated Ca(2+) channels by phosphoinositides, Pflugers Arch. 455 (2007)147–155.

[52] T. Rohacs, B. Nilius, Regulation of transient receptor potential (TRP) channelsby phosphoinositides, Pflugers Arch. 455 (2007) 157–168.

[53] I.E. Michailidis, T.D. Helton, V.I. Petrou, T. Mirshahi, M.D. Ehlers, D.E.Logothetis, Phosphatidylinositol-4,5-bisphosphate regulates NMDA receptoractivity through alpha-actinin, J. Neurosci. 27 (2007) 5523–5532.

[54] L.P. Bernier, A.R. Ase, S. Chevallier, D. Blais, Q. Zhao, E. Boue-Grabot, D.Logothetis, P. Seguela, Phosphoinositides regulate P2X4 ATP-gated channelsthrough direct interactions, J. Neurosci. 28 (2008) 12938–12945.

[55] L.F. Horowitz, W. Hirdes, B.C. Suh, D.W. Hilgemann, K. Mackie, B. Hille,Phospholipase C in living cells: activation, inhibition, Ca2+ requirement, andregulation of M current, J. Gen. Physiol. 126 (2005) 243–262.

(2009) 527–534 533

[56] B.C. Suh, T. Inoue, T. Meyer, B. Hille, Rapid chemically induced changes ofPtdIns(4,5)P2 gate KCNQ ion channels, Science 314 (2006) 1454–1457.

[57] T. Rohacs, C.M. Lopes, I. Michailidis, D.E. Logothetis, PI(4,5)P2 regulates theactivation and desensitization of TRPM8 channels through the TRP domain,Nat. Neurosci. 8 (2005) 626–634.

[58] R.L. Daniels, Y. Takashima, D.D. McKemy, Activity of the neuronalcold sensor TRPM8 is regulated by phospholipase C via the phospho-lipid phosphoinositol 4,5-bisphosphate, J. Biol. Chem. 284 (2009) 1570–1582.

[59] H.H. Chuang, E.D. Prescott, H. Kong, S. Shields, S.E. Jordt, A.I. Basbaum, M.V.Chao, D. Julius, Bradykinin and nerve growth factor release the capsaicinreceptor from PtdIns(4,5)P2-mediated inhibition, Nature 411 (2001) 957–962.

[60] B. Liu, C. Zhang, F. Qin, Functional recovery from desensitization ofvanilloid receptor TRPV1 requires resynthesis of phosphatidylinositol 4,5-bisphosphate, J. Neurosci. 25 (2005) 4835–4843.

[61] V. Lukacs, B. Thyagarajan, P. Varnai, A. Balla, T. Balla, T. Rohacs, Dual regulationof TRPV1 by phosphoinositides, J. Neurosci. 27 (2007) 7070–7080.

[62] D.B. van Rossum, R.L. Patterson, S. Sharma, R.K. Barrow, M. Kornberg, D.L. Gill,S.H. Snyder, Phospholipase Cgamma1 controls surface expression of TRPC3through an intermolecular PH domain, Nature 434 (2005) 99–104.

[63] B. Nilius, G. Owsianik, T. Voets, Transient receptor potential channels meetphosphoinositides, EMBO J. 27 (2008) 2809–2816.

[64] Y. Kwon, T. Hofmann, C. Montell, Integration of phosphoinositide- andcalmodulin-mediated regulation of TRPC6, Mol. Cell 25 (2007) 491–503.

[65] C.L. Huang, S. Feng, D.W. Hilgemann, Direct activation of inward rectifier potas-sium channels by PIP2 and its stabilization by Gbetagamma, Nature 391 (1998)803–806.

[66] T. Baukrowitz, U. Schulte, D. Oliver, S. Herlitze, T. Krauter, S.J. Tucker, J.P. Rup-persberg, B. Fakler, PIP2 and PIP as determinants for ATP inhibition of KATPchannels, Science 282 (1998) 1141–1144.

[67] G. Di Paolo, P. De Camilli, Phosphoinositides in cell regulation and membranedynamics, Nature 443 (2006) 651–657.

[68] P. Viard, A.J. Butcher, G. Halet, A. Davies, B. Nurnberg, F. Heblich, A.C. Dolphin,PI3K promotes voltage-dependent calcium channel trafficking to the plasmamembrane, Nat. Neurosci. 7 (2004) 939–946.

[69] L.A. Blair, J. Marshall, IGF-1 modulates N and L calcium channels in a PI 3-kinase-dependent manner, Neuron 19 (1997) 421–429.

[70] M.B. Butterworth, R.S. Edinger, R.A. Frizzell, J.P. Johnson, Regulation of theepithelial sodium channel by membrane trafficking, Am. J. Physiol. Renal Phys-iol. 296 (2009) F10–F24.

[71] O. Pochynyuk, J. Medina, N. Gamper, H. Genth, J.D. Stockand, A. Staruschenko,Rapid translocation and insertion of the epithelial Na+ channel in response toRhoA signaling, J. Biol. Chem. 281 (2006) 26520–26527.

[72] F. Lang, C. Bohmer, M. Palmada, G. Seebohm, N. Strutz-Seebohm, V. Vallon,(Patho)physiological significance of the serum- and glucocorticoid-induciblekinase isoforms, Physiol. Rev. 86 (2006) 1151–1178.

[73] R.L. Patterson, D.B. van Rossum, D.L. Ford, K.J. Hurt, S.S. Bae, P.G. Suh, T.Kurosaki, S.H. Snyder, D.L. Gill, Phospholipase C-gamma is required for agonist-induced Ca2+ entry, Cell 111 (2002) 529–541.

[74] V.J. Bezzerides, I.S. Ramsey, S. Kotecha, A. Greka, D.E. Clapham, Rapid vesicu-lar translocation and insertion of TRP channels, Nat. Cell Biol. 6 (2004) 709–720.

[75] M. Kanzaki, Y.Q. Zhang, H. Mashima, L. Li, H. Shibata, I. Kojima, Translocation ofa calcium-permeable cation channel induced by insulin-like growth factor-I,Nat. Cell Biol. 1 (1999) 165–170.

[76] X. Zhang, J. Huang, P.A. McNaughton, NGF rapidly increases membrane expres-sion of TRPV1 heat-gated ion channels, EMBO J. 24 (2005) 4211–4223.

[77] M. Bahner, S. Frechter, N. Da Silva, B. Minke, R. Paulsen, A. Huber, Light-regulated subcellular translocation of Drosophila TRPL channels induceslong-term adaptation and modifies the light-induced current, Neuron 34(2002) 83–93.

[78] N.E. Meyer, T. Joel-Almagor, S. Frechter, B. Minke, A. Huber, Subcellular translo-cation of the eGFP-tagged TRPL channel in Drosophila photoreceptors requiresactivation of the phototransduction cascade, J. Cell Sci. 119 (2006) 2592–2603.

[79] L. Fang, B.-Y. Kim, J.B. Wade, P.A. Welling, Molecular mechanism of ROMKchannel endocytosis, FASEB J. 22 (2008) 1158.9.

[80] A. Yaradanakul, S. Feng, C. Shen, V. Lariccia, M.J. Lin, J. Yang, T.M. Kang, P.Dong, H.L. Yin, J.P. Albanesi, D.W. Hilgemann, Dual control of cardiac Na+

Ca2+ exchange by PIP(2): electrophysiological analysis of direct and indirectmechanisms, J. Physiol. 582 (2007) 991–1010.

[81] B. Sarkadi, A. Tordai, G. Gardos, Membrane depolarization selectively inhibitsreceptor-operated calcium channels in human T (Jurkat) lymphoblasts,Biochim. Biophys. Acta 1027 (1990) 130–140.

[82] A.J. Patel, M. Lazdunski, E. Honore, Lipid and mechano-gated 2P domain K(+)channels, Curr. Opin. Cell Biol. 13 (2001) 422–428.

[83] J. Barhanin, F. Lesage, E. Guillemare, M. Fink, M. Lazdunski, G. Romey, K(V)LQT1and lsK (minK) proteins associate to form the I(Ks) cardiac potassium current,Nature 384 (1996) 78–80.

[84] B.C. Suh, B. Hille, Regulation of KCNQ channels by manipulation of phospho-inositides, J. Physiol. 582 (2007) 911–916.

[85] S. Cockcroft, N. Carvou, Biochemical and biological functions of class Iphosphatidylinositol transfer proteins, Biochim. Biophys. Acta 1771 (2007)677–691.

[86] C.J. Mousley, K.R. Tyeryar, P. Vincent-Pope, V.A. Bankaitis, The Sec14-superfamily and the regulatory interface between phospholipid metabolismand membrane trafficking, Biochim. Biophys. Acta 1771 (2007) 727–736.

5 ium 45

[

[

[

[

[

[

[

[

[

[

34 T. Balla / Cell Calc

[87] P.V. Schu, K. Takegawa, M.J. Fry, J.H. Stack, M.D. Waterfield, S.D. Emr, Phos-phatidylinositol 3-kinase encoded by yeast VPS34 gene essential for proteinsorting, Science 260 (1993) 88–91.

[88] A. Simonsen, R. Lippe, S. Christoforidis, J.M. Gaullier, A. Brech, J. Callaghan,B.H. Toh, C. Murphy, M. Zerial, H. Stenmark, EEA1 links PI(3)K function to Rab5regulation of endosome fusion, Nature 394 (1998) 494–498.

[89] A. Balla, G. Tuymetova, M. Barshishat, M. Geiszt, T. Balla, Characterizationof type II phosphatidylinositol 4-kinase isoforms reveals association of theenzymes with endosomal vesicular compartments, J. Biol. Chem. 277 (2002)20041–22050.

[90] Y.J. Wang, J. Wang, H.Q. Sun, M. Martinez, Y.X. Sun, E. Macia, T. Kirschhausen,J.P. Albanesi, M.G. Roth, H.L. Yin, Phosphatidylinositol 4 phosphate regulatestargeting of clathrin adaptor AP-1 complexes to the Golgi, Cell 114 (2003)299–310.

[91] S. Minogue, M.G. Waugh, M.A. De Matteis, D.J. Stephens, F. Berditchevski, J.J.Hsuan, Phosphatidylinositol 4-kinase is required for endosomal traffickingand degradation of the EGF receptor, J. Cell Sci. 119 (2006) 571–581.

[92] A. Balla, T. Balla, Phosphatidylinositol 4-kinases; old enzymes with emergingfunctions, Trends Cell Biol. 16 (2006) 351–361.

[93] S.R. Shaikh, M.A. Edidin, Membranes are not just rafts, Chem. Phys. Lipids 144(2006) 1–3.

[94] L.J. Pike, L. Casey, Localization and turnover of phosphatidylinositol 4,5-bisphosphate in caveolin-enriched membrane domains, J. Biol. Chem. 271(1996) 26453–26456.

[95] S. Minogue, J.S. Anderson, M.G. Waugh, M. dosSantos, S. Corless, R. Cramer,J.J. Hsuan, Cloning of a human type II phosphatidylinositol 4-kinase reveals anovel lipid kinase family, J. Biol. Chem. 276 (2001) 16635–16640.

[96] M.J. Aman, K.S. Ravichandran, A requirement for lipid rafts in B cell receptorinduced Ca(2+) flux, Curr. Biol. 10 (2000) 393–396.

[97] R. Rodriguez, M. Matsuda, A. Storey, M. Katan, Requirements for distinct stepsof phospholipase Cgamma2 regulation, membrane-raft-dependent targetingand subsequent enzyme activation in B-cell signalling, Biochem. J. 374 (2003)

269–280.

[98] J. van Rheenen, E.M. Achame, H. Janssen, J. Calafat, K. Jalink, PIP2 signaling inlipid domains: a critical re-evaluation, EMBO J. 24 (2005) 1664–1673.

[99] S.C. Brazer, B.B. Singh, X. Liu, W. Swaim, I.S. Ambudkar, Caveolin-1 contributesto assembly of store-operated Ca2+ influx channels by regulating plasma mem-brane localization of TRPC1, J. Biol. Chem. 278 (2003) 27208–27215.

(2009) 527–534

100] B. Pani, H.L. Ong, X. Liu, K. Rauser, I.S. Ambudkar, B.B. Singh, Lipid raftsdetermine clustering of STIM1 in endoplasmic reticulum-plasma membranejunctions and regulation of store-operated Ca2+ entry (SOCE), J. Biol. Chem.283 (2008) 17333–17340.

101] C.M. Lopes, T. Rohacs, G. Czirjak, T. Balla, P. Enyedi, D.E. Logothetis, PIP2hydrolysis underlies agonist-induced inhibition and regulates voltage gatingof two-pore domain K+ channels, J. Physiol. 564 (2005) 117–129.

102] G.R. Hammond, Y. Sim, L. Lagnado, R.F. Irvine, Reversible binding and rapiddiffusion of proteins in complex with inositol lipids serves to coordinate freemovement with spatial information, J. Cell Biol. 184 (2009) 297–308.

103] Y.S. Mao, M. Yamaga, X. Zhu, Y. Wei, H.Q. Sun, J. Wang, M. Yun, Y. Wang, G.Di Paolo, M. Bennett, I. Mellman, C.S. Abrams, P. De Camilli, C.Y. Lu, H.L. Yin,Essential and unique roles of PIP5K-gamma and -alpha in Fcgamma receptor-mediated phagocytosis, J. Cell Biol. 184 (2009) 281–296.

104] B. Toth, A. Balla, H. Ma, Z.A. Knight, K.M. Shokat, T. Balla, Phosphatidylinositol4-kinase IIIbeta regulates the transport of ceramide between the endoplasmicreticulum and Golgi, J. Biol. Chem. 281 (2006) 36369–36377.

105] L.W. Runnels, L. Yue, D.E. Clapham, TRP-PLIK, a bifunctional protein with kinaseand ion channel activities, Science 291 (2001) 1043–1047.

106] L.W. Runnels, L.X. Yue, D.E. Clapham, The TRPM7 channel ois inactivated byPIP2 hydrolysis, Nat. Cell Biol. 15 (2002) 370–378.

107] M. Langeslag, K. Clark, W.H. Moolenaar, F.N. van Leeuwen, K. Jalink, Activa-tion of TRPM7 channels by phospholipase C-coupled receptor agonists, J. Biol.Chem. 282 (2007) 232–239.

108] M. Kim, L.H. Jiang, H.L. Wilson, R.A. North, A. Surprenant, Proteomic and func-tional evidence for a P2X7 receptor signalling complex, EMBO J. 20 (2001)6347–6358.

109] Y. Murata, H. Iwasaki, M. Sasaki, K. Inaba, Y. Okamura, Phosphoinositide phos-phatase activity coupled to an intrinsic voltage sensor, Nature 435 (2005)1239–1243.

[110] M.J. Berridge, R.F. Irvine, Inositol trisphosphate, a novel second messenger incellular signal transduction, Nature 312 (1984) 315–321.

[111] P. Varnai, B. Thyagarajan, T. Rohacs, T. Balla, Rapidly inducible changes inphosphatidylinositol 4,5-bisphosphate levels influence multiple regulatoryfunctions of the lipid in intact living cells, J. Cell Biol. 175 (2006) 377–382.

[112] W.D. Heo, T. Inoue, W.S. Park, M.L. Kim, B.O. Park, T.J. Wandless, T. Meyer,PI(3,4,5)P3 and PI(4,5)P2 lipids target proteins with polybasic clusters to theplasma membrane, Science 314 (2006) 1458–1461.