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625 Knowledge of signaling mechanisms has increased dramatically during the past decade, particularly in the areas of development, biochemical signaling cascades, synaptic transmission and ion channel biophysics. Addresses *Howard Hughes Medical Institute, University of California (San Francisco), 533 Parnassus Avenue, San Francisco, CA 94143-0725, USA; e-mail: [email protected] Howard Hughes Medical Institute, Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA; e-mail: [email protected] Current Opinion in Neurobiology 2000, 10:625–630 0959-4388/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations AMPA α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid CaMK calcium/calmodulin-dependent kinase GABA γ-aminobutyric acid Kir channel inwardly rectifying potassium channel Kv channel voltage-gated potassium channel LTD long-term depression LTP long-term potentiation NMDA N-methyl-D-aspartate P pore region S transmembrane segment SNAP-25 synaptosome-associated protein of 25 kDa SNARE SNAP receptor protein TM transmembrane Trk tyrosine kinase receptor tSNARE target-membrane-associated SNARE VAMP vesicle-associated membrane protein vSNARE vesicle-associated SNARE Introduction How did the Decade of the Brain begin? Bestowed with patch-clamp methods to examine single channels from all kingdoms of life, we were awed with the bounty and variety. Eyebrows were still raised, at the first Decade of the Brain symposium, by the notion that the lowly fly and worm carry molecules not that different from our own, but a partnership between neurophysiology and molecular biology was in full swing. Central neurons were within experimental reach thanks to brain slices and neuronal cultures. Cloned recep- tors for neurotrophins and transmitters, as well as voltage-gated ion channels, could be studied in heterolo- gous expression systems, proteins were isolated from the synapses, and there was much excitement about the first described structure of a protein kinase. Hopes were high in tackling challenging questions of neuronal signaling, synap- tic transmission and plasticity, as well as issues concerning how nervous systems form and interact with their surround- ings (see the other reviews in this anniversary issue). The past ten years have witnessed the molecular charac- terization of both neurotransmitter transporters [1–4] and new families of ion channels and receptors [5–16], their linkage to human diseases [17–20], structural and func- tional analysis of channels, G proteins and anchor proteins [21–35], as well as newer and better ways to monitor elec- trical and chemical changes at the synapse [36–38]. Taken now for granted is the conservation of molecular machinery for membrane trafficking and second-messenger pathways from yeast to humans [39], with ample support from genome studies. It may be useful to pause and take stock. What have we learned over the past decade? What chal- lenges can we look forward to? Although signaling mechanisms potentially covers a rather wide range of topics, we have restricted our review to a few research areas most active during the past decade, includ- ing synaptic plasticity, kinases and phosphatases in signaling cascades, the SNARE hypothesis for exocytosis, neurotransmitter transporters, ion channels, and mecha- nisms for forming subcellular clusters of functionally related proteins. Plasticity A preoccupation in neurobiology during the past decade has been synaptic plasticity. Most neuroscientists enter the field hoping to solve some big problem — such as: “What is memory?” — but end up working on esoteric issues that are hard to justify to nonscientific friends at cocktail par- ties. Many in the field now recognize, however, that we are within striking distance of understanding the cellular and molecular basis for one of the biggies, memory, and this recognition has generated an enormous amount of research on two forms of synaptic plasticity — long-term potentia- tion (LTP) and its inverse, long-term depression (LTD): processes that are widely believed to play a key role in forming memories. This intense interest in synaptic plasticity — together with remarkable advances in the ability to alter or delete genes in mice — has led to a lot of studies in which the elimination of one or another gene has produced changes in LTP/LTD and in the animal’s ability to perform specific learning tasks [40,41]. The first of these studies led to great excitement, both because of the dazzling technology and what appeared to be the great promise of the approach. Calcium/calmodulin-dependent kinase (CaMK) had been hypothesized to be crucial for LTP, and animals lacking this gene indeed exhibit normal synaptic transmission together with defective LTP (and LTD) and the anticipat- ed learning deficits. This initial study seemed to show that LTP was a basis for at least some types of learning, and that a CaMK is essential for plasticity. Many gene deletion studies have ensued, and have presented us with an embarrassment of riches: it seems that almost any deletion of a gene expressed in the brain causes changes in LTP Signalling mechanisms A decade of signalling Lily Yeh Jan* and Charles F Stevens

Signalling mechanisms: A decade of signalling

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625

Knowledge of signaling mechanisms has increaseddramatically during the past decade, particularly in the areas ofdevelopment, biochemical signaling cascades, synaptictransmission and ion channel biophysics.

Addresses*Howard Hughes Medical Institute, University of California(San Francisco), 533 Parnassus Avenue, San Francisco,CA 94143-0725, USA; e-mail: [email protected]†Howard Hughes Medical Institute, Salk Institute, 10010 North TorreyPines Road, La Jolla, CA 92037, USA; e-mail: [email protected]

Current Opinion in Neurobiology 2000, 10:625–630

0959-4388/00/$ — see front matter© 2000 Elsevier Science Ltd. All rights reserved.

AbbreviationsAMPA α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acidCaMK calcium/calmodulin-dependent kinaseGABA γ-aminobutyric acidKir channel inwardly rectifying potassium channelKv channel voltage-gated potassium channelLTD long-term depressionLTP long-term potentiationNMDA N-methyl-D-aspartateP pore regionS transmembrane segmentSNAP-25 synaptosome-associated protein of 25 kDaSNARE SNAP receptor proteinTM transmembraneTrk tyrosine kinase receptortSNARE target-membrane-associated SNAREVAMP vesicle-associated membrane proteinvSNARE vesicle-associated SNARE

IntroductionHow did the Decade of the Brain begin? Bestowed withpatch-clamp methods to examine single channels from allkingdoms of life, we were awed with the bounty and variety.Eyebrows were still raised, at the first Decade of the Brainsymposium, by the notion that the lowly fly and worm carrymolecules not that different from our own, but a partnershipbetween neurophysiology and molecular biology was in fullswing. Central neurons were within experimental reachthanks to brain slices and neuronal cultures. Cloned recep-tors for neurotrophins and transmitters, as well asvoltage-gated ion channels, could be studied in heterolo-gous expression systems, proteins were isolated from thesynapses, and there was much excitement about the firstdescribed structure of a protein kinase. Hopes were high intackling challenging questions of neuronal signaling, synap-tic transmission and plasticity, as well as issues concerninghow nervous systems form and interact with their surround-ings (see the other reviews in this anniversary issue).

The past ten years have witnessed the molecular charac-terization of both neurotransmitter transporters [1–4] andnew families of ion channels and receptors [5–16], their

linkage to human diseases [17–20], structural and func-tional analysis of channels, G proteins and anchor proteins[21–35], as well as newer and better ways to monitor elec-trical and chemical changes at the synapse [36–38]. Takennow for granted is the conservation of molecular machineryfor membrane trafficking and second-messenger pathwaysfrom yeast to humans [39], with ample support fromgenome studies. It may be useful to pause and take stock.What have we learned over the past decade? What chal-lenges can we look forward to?

Although signaling mechanisms potentially covers a ratherwide range of topics, we have restricted our review to a fewresearch areas most active during the past decade, includ-ing synaptic plasticity, kinases and phosphatases insignaling cascades, the SNARE hypothesis for exocytosis,neurotransmitter transporters, ion channels, and mecha-nisms for forming subcellular clusters of functionallyrelated proteins.

PlasticityA preoccupation in neurobiology during the past decadehas been synaptic plasticity. Most neuroscientists enter thefield hoping to solve some big problem — such as: “Whatis memory?” — but end up working on esoteric issues thatare hard to justify to nonscientific friends at cocktail par-ties. Many in the field now recognize, however, that we arewithin striking distance of understanding the cellular andmolecular basis for one of the biggies, memory, and thisrecognition has generated an enormous amount of researchon two forms of synaptic plasticity — long-term potentia-tion (LTP) and its inverse, long-term depression (LTD):processes that are widely believed to play a key role informing memories.

This intense interest in synaptic plasticity — togetherwith remarkable advances in the ability to alter or deletegenes in mice — has led to a lot of studies in which theelimination of one or another gene has produced changesin LTP/LTD and in the animal’s ability to perform specificlearning tasks [40,41]. The first of these studies led togreat excitement, both because of the dazzling technologyand what appeared to be the great promise of the approach.Calcium/calmodulin-dependent kinase (CaMK) had beenhypothesized to be crucial for LTP, and animals lackingthis gene indeed exhibit normal synaptic transmissiontogether with defective LTP (and LTD) and the anticipat-ed learning deficits. This initial study seemed to show thatLTP was a basis for at least some types of learning, and thata CaMK is essential for plasticity. Many gene deletionstudies have ensued, and have presented us with anembarrassment of riches: it seems that almost any deletionof a gene expressed in the brain causes changes in LTP

Signalling mechanismsA decade of signallingLily Yeh Jan* and Charles F Stevens†

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and/or LTD, usually together with changes in learning.The situation is similar to that for ‘epilepsy’ genes, wherethe deletion of many apparently unrelated genes leads toseizures. The lesson seems to be that synaptic plasticityand learning are complex phenomena that are affected bymany genes, probably mostly through regulatory pathways.

Perhaps the most influential concept in LTP/LTD duringthe past decade is the ‘silent synapse’ hypothesis [42–44].The idea is that many synapses have NMDA but notAMPA receptors and that these NMDA-only synapses pro-duce currents only if the neuron is depolarized (because ofthe voltage dependence of the NMDA receptor channel).If one records synaptic currents near the resting potential,these synapses are silent (i.e. they generate no current)because they lack AMPA receptors. With the conditionsthat produce LTP (i.e. calcium influx through NMDAreceptor channels), AMPA receptors become available atpreviously silent synapses, either through the unmaskingof already resident receptors or the insertion of new recep-tors into the postsynaptic membrane, and the synapsebecomes functional, that is, no longer silent. This notionhas generated much research and is currently the dominanttheory for how LTP/LTD is produced. Despite its wideacceptance, the silent synapse hypothesis has not yet beenconfirmed directly and some doubt its validity.

The debate about the mechanism of LTP/LTD continues,but the interest in how to account for memory is shifting tolonger-term changes in synapse properties [45,46]. The tra-ditionally studied LTP/LTD persists only for a relativelyshort time — a matter of hours at most — whereas memo-ries can last a lifetime. Most researchers in the field believethat the more permanent changes responsible for long-last-ing memories must involve structural modifications ofsynapses or the addition of new synapses, and this is whererecent interest has concentrated. A lot of the recent researchhas focused on the insertion of postsynaptic neurotransmit-ter receptors, but presynaptic alterations in synapticstructure presumably also contribute to the regulation ofsynaptic strength responsible for storing information.

Kinases and phosphatasesSignaling systems that switch protein function by phos-phorylation and dephosphorylation have remained acentral concern throughout biology, including neurobiolo-gy, during the past decade. Some of the most interestingfindings from a neurobiological point of view relate tomembers of the receptor tyrosine kinase family, and to theneurotrophin/Trk and the ephrin/Eph signaling pathways.

Because the organization of the brain is so orderly, thequestion of how maps form — for example, the map of thevisual world on primary visual cortex — has long beenimportant. It was almost universally believed that thesemaps would require a spatial concentration gradient ofsome signaling molecules, but for a long time such gradi-ents remained more hypothetical than real. In 1994, two

papers demonstrated the existence of gradients involvingEph receptors and their ligands (the ephrins), and thiswork has initiated a rapid increase in our understanding ofhow maps are formed (see [47] for a review). The Ephreceptors are a large subfamily of receptor tyrosine kinases,with more than a dozen members, whose many types ofligands, the ephrins, are also transmembrane signalingmolecules. This ephrin/Eph pair provides for specificlabels on cell pairs and for two-way signaling. To form amap, axon growth cones from one structure (the retina, forexample) express a concentration of Eph receptors thatdepends on the location of the axon’s cell body in a gradedway, and the target structure (the tectum, for example)expresses a complementary spatial concentration gradientof ephrin receptors. The high-Eph-expressing axons go tothe part of the target with fewest ephrins, whereas the low-Eph-expressing axons prefer the target region with thehighest concentration of ephrins. Through this repulsionmechanism, orderly maps are formed.

The first neurotrophin identified, nerve growth factor(NGF), also activates a receptor tyrosine kinase, theTrkA receptor. Now, several additional members of thisfamily, and the corresponding receptors (TrkB and TrkC)have been identified; for example, brain-derived neu-rotrophic factor (BDNF) is a ligand for TrkB receptors.The members of this family, which signal through con-ventional tyrosine kinase cascades, are produced byneurons in an activity-dependent manner and, it hasbeen discovered, are responsible for modulating theform of dendritic trees and the formation and function ofsynapses (see [48] for a review).

The SNARE hypothesisAmong the most striking advances in neurobiology overthe past decade was the identification of proteins responsi-ble for neurotransmitter release, and the realization thatexocytosis at the synapse is just a highly regulated form ofthe constitutive vesicle fusion events that play such a cen-tral role in cell biology. Cell biology’s SNARE hypothesis[49] proposes that certain vesicle proteins, the vSNAREs(‘v’ for vesicular), bind to complementary proteins, thetSNAREs (‘t’ for target), in a target membrane to form amolecular complex that is responsible for membranefusion. (SNARE officially stands for SNAP receptor, butreally is a catchy name selected to connote the mecha-nisms that are used to snare vesicles at their targetmembrane for docking and exocytosis.) Neurotransmitterrelease employs a version of this general mechanism[50,51]. The vSNARE used by synaptic vesicles is calledVAMP/synaptobrevin, and it pairs with the tSNAREs syn-taxin and SNAP-25 in the active zone of the presynapticterminal membrane to form a core complex needed formembrane fusion and the release of the vesicle’s contents.A calcium-sensing regulatory molecule, presumably asynaptotagmin, prevents vesicles at the active zone fromfusing, even though their core complex has formed, untilthis sensor undergoes a calcium-driven conformational

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change. After the conformational change has taken place,membrane fusion is permitted to proceed rapidly. Thesame molecular machinery that yeast and all other eukary-otic cells use to move material from one membranecompartment to the next, or to make the cell grow byincreasing its membrane area, has been specialized in thebrain to permit the highly regulated and very rapid — lessthan a millisecond — release of neurotransmitter.Interestingly, the tSNAREs are not limited to the activezone, so they cannot be the molecular species responsiblefor insuring that vesicles dock and release only in this spe-cific location. What still needs to be determined is thedetailed molecular mechanisms that underlie exocytosisand what determines the number and location of dockingsites for synaptic vesicles.

By combining electrophysiological and imaging tech-niques, it has recently been possible to study in detail theevents involved in vesicle fusion. Mainly on the basis ofstudying large, dense-core vesicles of neuroendocrinecells, we now picture exocytosis as beginning with the for-mation of a fusion pore that can flicker open andclosed — neurotransmitter escapes through the openpore — before finally permitting the vesicle membrane tofuse with the cell’s surface membrane [52].

Transmitter transportersFor a long time, it has been known that vesicular trans-porters package transmitters into synaptic vesicles,whereas plasma membrane transporters remove transmit-ters that have been released and then recycle them[1–3,53]. Only in the past ten years have the molecularentities of these transporters become known. The relativeease to assay for the latter allowed them to be purified orcloned by functional expression [1], facilitating studies oftheir regulation and modification in response to psycho-stimulants [2]. Alternative approaches were required forcloning vesicular transporters [3], first taking advantage ofthe likely detoxification function of vesicular dopaminetransporters and then relying on the expected mutant phe-notypes for other transporters in the nematodeCaenorhabditis elegans. It turns out that these vesiculartransporters are rather slow; their activities limit vesiclefilling and may regulate quantal size [3]. The vesiculartransporter for glutamate, the major excitatory transmitterin the brain, has not yet been identified; cloning of thistransporter will facilitate further characterizations (see,however, Note added in proof).

Different ions drive transmitter transport across plasmamembrane and into vesicles, and may be linked to the ionpumps on these membranes. Like prokaryotes, eukaryoticvesicles and other intracellular organelles utilize protonpumps to generate pH gradients and possibly membranepotential differences across their membranes. These differ-ences, in turn, drive vesicular transmitter transporters [3].Eukaryotic plasma membrane contains sodium–potassiumATPase, which pumps sodium out and potassium into the

cell. Hence, plasma membrane transmitter transporters aretypically symporters that import both sodium and transmit-ter [1], though the stoichiometry could be complex,involving other ions such as protons and potassium [54].Surprisingly, a close relative of vesicular transporters is onthe plasma membrane of astrocytes and resembles sodi-um–proton exchangers [55], which probably serve differentfunctions in prokaryotes and eukaryotes.

Ion channelsEvolutionary kinship among a surprisingly wide array ofion channels and transporters has recently become evi-dent. By 1990, we had come to recognize that differentvoltage-gated cation channels belong to the same super-family. Voltage-gated sodium channels and calciumchannels each have four repeats that resemble an alphasubunit of the tetrameric voltage-gated potassium (Kv)channels. In the past decade, more distantly related rela-tives began to emerge as inwardly rectifying potassium(Kir) channels [5]. Compared to the six transmembrane(6TM) segments (S1–S6) in each Kv subunit, the 2TMsegments flanking a P (H5) pore region present in each Kirsubunit correspond to the second half (S5–P–S6) of the Kvsubunit. Interestingly, the chlorella virus encodes a potas-sium channel composed of just the two transmembranesegments flanking a P region, which is important for virusreplication [56]. There are two of these basic pore-formingdomains (2TM) in each subunit of the ‘two-pore’ potassi-um channels [6], and four 2TM domains are recognizablein prokaryotic and eukaryotic potassium transporters[57,58]. How might transporters have evolved differentlyfrom channels to be more refrained in their transport activ-ities? Some plasma membrane transporters exhibitsubstrate-induced ion channel activities [4]. Does thishave to do with their relations to channels?

Surprisingly, an ‘upside down’ 2TM domain forms the poreof glutamate receptors in both eukaryotes and prokaryotes,and the prokaryotic glutamate receptor is actually selectivefor potassium [59,60]. The sequences flanking this invert-ed 2TM domain are similar to prokaryotic periplasmicbinding proteins [59,61]; glutamate binding to this clam-like structure may then trigger conformational changes inthe pore-forming 2TM domain. Structurally distinct fromthese glutamate receptors are ligand-gated channels acti-vated by acetylcholine, glycine, GABA and serotonin [62].Electron microscopic analysis of acetylcholine receptorstructure suggests that it may resemble pentameric bacter-ial toxins that are related to cholera toxin [62,63]. Dramaticrotation of the pore-lining segments is propelled by acetyl-choline binding to open the channel [63].

Even more dramatic conformational changes must accompa-ny the opening of a prokaryotic mechanosensitive channel.Sensitive to stretch in the lipid bilayer, the mechanosensitivechannel of large conductance (i.e. the MscL channel) in theclosed state has a narrow constriction but must open to a porediameter of 40 Å to allow thio-reductase to go through [64].

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How might a potassium channel allow potassium but not thesmaller sodium ions to go through at a rate of millions of ionsper second? Recent biophysical studies reveal that thesechannels have a multi-ion single-file pore; the presence ofmultiple ions leads to their brief interaction with their bind-ing sites. It is gratifying to see that three ions can be resolvedin the pore of a bacterial potassium channel, KcsA, albeit inthe presumed closed conformation at pH 7 [32,65]. Channelopening at reduced pH involves rotation and movements ofthe pore-lining transmembrane segment M2 [66].

How do voltage-gated ion channels respond to changes ofelectric potential across the membrane and open? Recentwork suggests that the intrinsic voltage sensor correspondsprimarily to the S4 segment bearing multiple positivelycharged residues. Membrane depolarization propels the S4segment outward, triggering conformational changes thatopen the channel [67–70]. Curiously, some members ofthis superfamily are activated by hyperpolarization. Amongthem, the hyperpolarization-activated cation channel isespecially curious — it contains the potassium channel sig-nature sequence but is not potassium selective [71–73].

The superfamily of the voltage-gated ion channels alsoincludes channels gated by second messengers or evenheat [16,74,75]. Often a channel is regulated by multiplefactors. For example, the voltage dependence of the maxiK calcium-activated potassium channel may be altered bycalcium over several orders of magnitude [76].

Evolutionary conservation of signaling pathways is well illus-trated by the involvement of calmodulin in channelmodulation from Paramecium to humans [77–81], and theprincipal role of G-protein βγ subunits in signaling from yeastto mammals [33,35]. Examples involve cyclic-nucleotide-gated cation channels, potassium channels and calciumchannels. The versatility of calmodulin could derive from itsflexible joint [82], whereas different effectors may settle withdifferent though overlapping binding sites over the expanseof potential interaction surfaces of the G-protein β propellercomposed of WD (tryptophan–aspartate) repeats [35], adesign well suited for protein–protein interactions.

Molecular velcroA decade ago, most of us viewed the synapse as a special-ized structure fitted with specialized molecules that wereeither soluble or membrane bound. We now have come torealize that these specialized molecules are highly orga-nized into functional groups tied together by molecularvelcro [27,83]. First recognized in the postsynaptic density,these structural elements are now known to provide themeans for dividing both the presynaptic and postsynapticelements of the synapse into microdomains. The firstmotif to be identified was the PDZ domain, which pairskinases and neurotransmitter receptors in the postsynapticdensity of excitatory synapses. (PDZ stands for postsynap-tic density protein of 95 kDa, Discs large, zonaoccludens 1 — the first proteins recognized to contain this

binding motif.) As additional binding molecules have beenfound [28], the study of the microdomains of synapses hasbecome an important new research area. Postsynaptically,the two kinds of glutamate receptors as well as kinases arelinked together; presynaptically, calcium channels are keptnear the docking sites for vesicles. Efforts are now focusedon finding all of the molecules used for linking, and therules that are used to establish the various domains.

Over the past decade, it has thus become evident that wecan no longer consider channels, receptors and second mes-sengers as separate entities. Not only are they segregated toremarkably discrete compartments so that the neuronalmembrane appears to be an intricate mosaic, but they arealso organized into macromolecular complexes connectingproteins on the cell membrane with second messengers,cytoskeletal elements, and even extracellular matrix com-ponents [29,30]. Besides considering the combinatorialpossibilities of multimeric channels, the likelihood ofG-protein-coupled receptors actually acting as homodimersor heterodimers further increases the complexity [7,84–87].Whereas ‘compartmentalization’ conceptually offers oneway to limit cross talk, the task in front of us is to under-stand where and how various complexes of macromoleculesare assembled and targeted, and whether regulation ofthese processes contributes to neuronal signaling.

ConclusionsNew technology — molecular biological and various imag-ing methods — has driven most advances made during thepast decade. These new technologies, and still newer onesnow starting to become common (such as gene ‘knock-ins’that can be turned on at will and DNA arrays that permitmonitoring patterns of gene expression for the entiregenome of common laboratory animals), will only continueto accelerate the understanding of signaling mechanisms.We anticipate that the signaling mechanisms discussedabove will become understood in increasing moleculardetail through the next decade. Furthermore, we expectthat the new information will be synthesized into theoriesthat explain the general principles of signaling networks.Signaling mechanisms will, we predict, be one of the mostexciting fields in this golden age of biology.

Note added in proofWhile this manuscript was in preparation, two papersreporting the identification of the vesicular glutamatetransporter have been published [88,89].

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630 A decade of Current neurobiology