Α-LATROTOXIN and ITS RECEPTORS: Neurexins

P1: FQP April 4, 2001 18:17 Annual Reviews AR121-30

-LATROTOXIN AND ITS RECEPTORS: Neurexins and CIRL/Latrophilins

ThomasCSudhof¨ Howard Hughes Medical Institute, Center for Basic Neuroscience, and the Department of Molecular Genetics, The University of Texas Southwestern Medical Center at Dallas, Texas 75390-9111, e-mail: [email protected]

Key Words neurotransmitter release, synaptic vesicles, exocytosis, membrane fusion, synaptic cell adhesion ■ Abstract -Latrotoxin, a potent neurotoxin from black widow spider venom, triggers synaptic vesicle exocytosis from presynaptic nerve terminals. -Latrotoxin is a large protein toxin (120 kDa) that contains 22 ankyrin repeats. In stimulating exocytosis, -latrotoxin binds to two distinct families of neuronal cell-surface receptors, neurexins and CLs (Cirl/latrophilins), which probably have a physiological function in synaptic cell adhesion. Binding of -latrotoxin to these receptors does not in itself trigger exocytosis but serves to recruit the toxin to the synapse. Receptor-bound -latrotoxin then inserts into the presynaptic plasma membrane to stimulate exocytosis by two distinct transmitter-speciﬁc mechanisms. Exocytosis of classical neurotransmitters (glutamate, GABA, acetylcholine) is induced in a calcium-independent manner by a direct intracellular action of -latrotoxin, while exocytosis of catecholamines requires extracellular calcium. Elucidation of precisely how -latrotoxin works is likely to provide major insight into how synaptic vesicle exocytosis is regulated, and how the release machineries of classical and catecholaminergic neurotransmitters differ. by SCELC Trial on 09/09/11. For personal use only.

INTRODUCTION

Annu. Rev. Neurosci. 2001.24:933-962. Downloaded from www.annualreviews.org Black and brown widow spiders of the genus Latrodectus produce a potent venom that contains a mixture of toxins referred to as latrotoxins. Most latrotoxins target insects or crustaceans, which are the natural prey of black widow spiders. However, one of the spider toxins, -latrotoxin, is specific for vertebrates. Clinically, black widow spider bites are not a significant health problem for humans because they rarely cause serious disease. A black widow spider is occasionally referred to as “the elegant lady who inflicts pain” (Simmons 1991). Only in the most severe cases do black widow spider bites cause latrodectism, a syndrome consisting of gener- alized muscle pain, abdominal cramps, profuse sweating, raised blood pressure, and tachycardia (Muller 1993, Zukowski 1993). Scientifically, -latrotoxin was revealed to be extremely useful. -Latrotoxin has become one of the prime tools

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for studying synaptic transmission in neurobiology because it acts selectively on presynaptic nerve terminals to stimulate synaptic vesicle exocytosis by what turns out to be a very interesting mechanism. Examination of this mechanism has given important insights into the release process. Among others, studies on -latrotoxin led to the discovery of two families of synaptic receptors, whose functions are now being elucidated, and to a better understanding of the various stages of synaptic vesicle exocytosis. In this review, I provide an overview of how -latrotoxin works and what its receptors do in the framework of our current understanding of synaptic transmission.

STRUCTURE OF -LATROTOXIN

-Latrotoxin is a 120-kDa protein, an unusually large size for a toxin (Frontali & Grasso 1964, Frontali et al 1976). Its cDNA sequence predicts an even larger protein (approximately 160 kDa), which suggests that -latrotoxin is synthesized as a protein precursor in the venom gland and is cleaved by endoproteases to generate the mature toxin (Kiyatkin et al 1990). Besides -latrotoxin, three additional latrotoxins that act on invertebrates have been purified and cloned from black widow spiders: insect-specific - and -latroinsectotoxins, and crustacean- specific -latrocrustotoxin (Kiyatkin et al 1993, Dulubova et al 1996, Danilevich et al 1999). These three invertebrate latrotoxins are also proteins of more than 100 kDa that are synthesized as larger precursors similar to -latrotoxin. All three invertebrate latrotoxins are homologous to each other and to -latrotoxin over their entire length. The venom of black widow spiders probably contains additional toxins that act on insects, mollusks, and crustaceans and that remain to be characterized but that are likely to be also homologous. Thus far, all latrotoxins trigger neurotransmitter release in the various organisms in which they are active

by SCELC Trial on 09/09/11. For personal use only. (Fritz et al 1980, Knipper et al 1986, Magazanik et al 1992, Elrick & Charlton 1999). Thus, black widow spider venom consists of a rich mixture of toxins that are homologous to each other and similarly trigger neurotransmitter release but that differ in their species speciﬁcity.

Annu. Rev. Neurosci. 2001.24:933-962. Downloaded from www.annualreviews.org -Latrotoxin and other latrotoxins are composed of four domains that are schematically illustrated for -latrotoxin in Figure 1 (Kiyatkin et al 1990, 1993; Dulubova et al 1996; Danilevich et al 1999). Domain I is a cleaved signal peptide. Although the published cDNA sequence of -latrotoxin lacks a signal peptide (Kiyatkin et al 1990), other latrotoxins contain a signal peptide, and the secretion of -latrotoxin suggests that it requires a signal peptide. Domain II is a conserved N-terminal domain of 431 residues. This domain contains three invariant cysteine residues and two hydrophobic sequences of 20–26 amino acids, which could potentially span a membrane. However, the secreted toxin is highly soluble, which suggests that the hydrophobic sequences are not exposed on the surface of the soluble toxin but rather folded into the interior core of the toxin. Domain III is a central domain composed of 22 imperfect, ankyrin-like repeats covering 745 residues. P1: FQP April 4, 2001 18:17 Annual Reviews AR121-30

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Figure 1 Domain structure of -latrotoxin. The four domains of the -latrotoxin precursor are depicted schematically, with an N-terminal cleaved signal peptide (SP) (domain I), an N- terminal conserved domain with three critical cysteine residues (domain II), a central domain composed of 22 ankyrin-like repeats (domain III), and a C-terminal domain that is proteolytically removed during maturation of the toxin (domain IV). The conserved cysteine residues in domain II that were shown by mutagenesis to be essential for -latrotoxin activity are marked by asterisks. The boundary between the N-terminal domain II and the ankyrin repeats (domain III) at which four amino acids were inserted in the mutant LtxN4C is identiﬁed with an arrow labeled N4C. The boundary between the central ankyrin-like repeats and the C-terminal domain IV where proteolytic cleavage occurs during the maturation of the toxin is also marked by an arrow. [Modiﬁed from Ichtchenko et al (1998).]

This domain is highly homologous among different latrotoxins, although the number of ankyrin repeats varies between toxins. The presence of 22 ankyrin repeats in -latrotoxin is remarkable because such repeats are usually only found in intracellular proteins. With 22 repeats, -latrotoxin contains the largest number of such repeats after ankyrin among currently known proteins. Domain IV is a C- terminal domain of 206 residues that is less conserved between latrotoxins and is presumably cleaved during the maturation of -latrotoxin. In addition to the proteolytic cleavage during the maturation of the toxin, -latrotoxin also appears to be modiﬁed by intramolecular disulﬁde bonds because sulfhydryl reduction abolishes the toxicity of the toxin. No other posttranslational

by SCELC Trial on 09/09/11. For personal use only. modiﬁcations of -latrotoxin are known; in particular, -latrotoxin does not appear to be glycosylated. When puriﬁed from venom glands, -latrotoxin is isolated in a complex with a low-molecular-weight peptide of 70 residues (Kiyatkin et al 1992). This small, secreted protein contains a cleaved signal peptide and six cysteine

Annu. Rev. Neurosci. 2001.24:933-962. Downloaded from www.annualreviews.org residues that form three disulﬁde bonds. The low-molecular-weight component exhibits distant homology to members of the family of crustacean hyperglycemic hormones, which suggests that it is evolutionarily derived from an invertebrate neuropeptide (Gasparini et al 1994). Puriﬁed -latrotoxin from black widow spider venom (including the small peptide component) forms a high-molecular-weight complex in solution, as judged by sucrose gradient centrifugation (Petrenko et al 1993). This complex was shown by cryoelectronmicroscopy to consist of a dimer in the absence of divalent cations, or a tetramer in the presence of divalent cations (Orlova et al 2000). Insight into the relationship between the structure and function of -latrotoxin was gained in studies of recombinant -latrotoxin (Ichtchenko et al 1998, Volynski et al 1999). When the full-length -latrotoxin precursor was synthesized in insect P1: FQP April 4, 2001 18:17 Annual Reviews AR121-30

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cells using baculovirus vectors, no active toxin was obtained. However, recombinant toxin that was truncated at the predicted C-terminal proteolytic cleavage site after the last ankyrin repeat was fully active and as potent in neurotransmitter release as native toxin (Ichtchenko et al 1998). Toxicity of the recombinant toxin did not require the small peptide that copurifies with native toxin. Thus, neither the C-terminal region (domain IV) of the -latrotoxin precursor nor the small peptide copurifying with -latrotoxin are required for the synthesis, folding, or activity of -latrotoxin. Substitution of each of the three conserved cysteine residues in domain II of -latrotoxin for serine residues abolished toxin activity, which testifies to the importance of disulfide bonds in the conserved N-terminal domain. In addition, a mutant that carries an insertion of four amino acids between the N-terminal cysteine- rich domain and the central ankyrin repeats (referred to as LtxN4C) was unable to trigger neurotransmitter release, although it still bound to the two -latrotoxin receptors (Ichtchenko et al 1998). This suggests that the exact distance between N-terminal and central domains in the toxin are of critical importance in activity, an idea that receives additional support from the high degree of conservation between different latrotoxins in the connecting region between the N-terminal domain and ankyrin repeats.

STIMULATION OF NEUROTRANSMITTER RELEASE BY -LATROTOXIN: A Dual Mode of Action

-Latrotoxin causes massive neurotransmitter release by synaptic vesicle exocytosis when applied to nerve terminals. This appears to occur by a two-stage process whereby the toxin ﬁrst binds to highly speciﬁc receptors on the terminals and then triggers synaptic vesicle exocytosis. The actions of -latrotoxin in exocytosis

by SCELC Trial on 09/09/11. For personal use only. were ﬁrst studied in detail in the frog neuromuscular junction (Longenecker et al 1970, Clark et al 1970), and later in synaptosomes (Grasso & Senni 1979, Nicholls et al 1982) and cultured hippocampal neurons (Capogna et al 1996, Augustin et al 1999). Furthermore, the toxin stimulates exocytosis of peptidergic vesicles from

Annu. Rev. Neurosci. 2001.24:933-962. Downloaded from www.annualreviews.org neuroendocrine cells such as PC12 cells (Grasso et al 1980, Meldolesi et al 1983), and studies on these cells have provided additional insight into its mechanism of action. Applications of increasing concentrations of -latrotoxin to presynaptic nerve terminals under controlled conditions leads to discrete dose-dependent effects on synaptic transmission. All these effects, independent of the toxin concentration, set in after a lag period of approximately 1 min following addition of the toxin. At low concentrations (subnanomolar), -latrotoxin stimulates continuous exocytosis of synaptic vesicles. This becomes electrophysiologically manifest by an increase in the rate of spontaneous miniature postsynaptic currents (“minis”), which cor- respond to individual vesicle fusion events (Capogna et al 1996). The enhanced mini rate is sustained for more than 30 min even when the toxin is applied for P1: FQP April 4, 2001 18:17 Annual Reviews AR121-30

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only a few seconds and subsequently washed out. Morphologically, low doses of -latrotoxin have no discernable effect on the nerve terminal, and the typical clusters of synaptic vesicles that normally fill the cytoplasm of a resting terminal are unchanged. Addition of higher toxin concentrations (nanomolar) causes a burst of neurotransmitter release followed by continuous steady state release that is also maintained for a considerable time (Ceccarelli et al 1979). At these concentrations, -latrotoxin not only stimulates fusion of docked synaptic vesicles at the active zone (the “readily-releasable pool”), it also mobilizes vesicles in the reserve and resting pools in the backfield of the synapse. This effect does not depend on 2 Ca + (see discussion below) and is quite different from the effects of hypertonic 2 sucrose [a secretagog that also induces Ca +-independent neurotransmitter release (Rosenmund & Stevens 1996)] or even very strong electrical stimulation. In addition, stimulation by nanomolar concentrations of -latrotoxin results in discrete morphological changes. The vesicle cluster shrinks, synaptic vesicles are depleted from the terminal, endocytosis lags behind, and the plasma membrane expands (Pumplin & Reese 1977, Gorio et al 1978, Ceccarelli et al 1979, Duchen et al 1981, Matteoli et al 1988). The total mobilization of vesicles by -latrotoxin suggests that the toxin activates extensive signal transduction cascades. Application of high concentrations of -latrotoxin (>10 nM) overwhelms the metabolic and secretory apparatus of the nerve terminal. High toxin concentrations not only induce massive synaptic vesicle exocytosis, they also lead to a dramatic drop in ATP levels of the nerve terminal that causes disintegration of the plasma membrane with release of cytoplasmic markers (McMahon et al 1990). Under these conditions, -latrotoxin action loses specificity and behaves almost like a detergent. Morphologically, all vesicles are lost from nerve terminals, mitochondria swell, and the plasma membrane becomes grossly extended, especially when stimula- 2 tions are carried out in the presence of Ca + (Pumplin & Reese 1977, Henkel & Betz 1995). Overall, the dose dependence of the -latrotoxin effects reminds us

by SCELC Trial on 09/09/11. For personal use only. that -latrotoxin is a toxin that will provide only interpretable effects in studying neurotransmitter release at submaximal doses. A striking feature of -latrotoxin is that it stimulates neurotransmitter re- 2 lease in the absence of extracellular Ca +, at least in the case of the majority

Annu. Rev. Neurosci. 2001.24:933-962. Downloaded from www.annualreviews.org of neurotransmitters. This was observed in the initial studies of -latrotoxin- triggered acetylcholine release at the frog neuromuscular junction (Clark et al 1970, Longenecker et al 1970, Ceccarelli et al 1979). No inhibition of acetylcholine se- 2 cretion after application of -latrotoxin was observed when Ca + was removed, 2 whereas secretion stimulated by action potentials under the same Ca +-free condi- 2 tions was abolished (Gorio et al 1978). In fact, Ca + removal was protective in that nerve terminal integrity was better after large doses of toxin, and mitochondrial swelling was not observed (Gorio et al 1978, Pumplin & Reese 1977, Henkel & Betz 1995). It is interesting that endocytosis was inhibited when -latrotoxin was 2 applied to neuromuscular nerve terminals in the absence of Ca +, which suggests 2 a role for Ca + in regulating endocytosis, a concept that has since received much experimental support (Ceccarelli & Hurlbut 1980, Robinson et al 1993, Henkel & P1: FQP April 4, 2001 18:17 Annual Reviews AR121-30

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Betz 1995, Brodin et al 2000). These initial experiments suggested that -latrotoxin 2 may be able to trigger exocytosis by bypassing the exocytotic Ca + sensor. In neuroendocrine PC12 cells, however, efficient stimulation of dopamine or nore- 2 pinephrine secretion by -latrotoxin clearly requires Ca + (Grasso et al 1980, Meldolesi et al 1983). Similar observations were made with synaptosomes, cast- 2 ing doubt on the ability of -latrotoxin to work in the absence of Ca + (Davletov et al 1997). In hippocampal cultures, conversely, stimulation of glutamate release (similar to acetylcholine release in neuromuscular synapses) did not require extra- 2 cellular Ca + (Capogna et al 1996). The reason for these apparent contradictions probably is that there are dif- 2 ferences in the Ca + dependence of -latrotoxin action between various neurotransmitters. This is illustrated in a comparison of the release of different neurotransmitters from synaptosomes triggered by -latrotoxin in the absence and 2 presence of Ca + (Figure 2). Various transmitters respond dramatically differently 2 to -latrotoxin as a function of Ca + (Figure 2) (Khvotchev et al 2000). Glu- tamate and GABA are released equally well by -latrotoxin in the presence or 2 absence of Ca + (Figure 2). These neurotransmitters are used by the majority of central synapses in morphologically typical synaptic connections, with clusters of vesicles adjacent to an active zone. In contrast, vesicles containing transmitters that are not exocytosed at traditional synapses (e.g. neuropeptides and monoamines) 2 require Ca + for -latrotoxin-stimulated release (Figure 2) (Khvotchev & Südhof 2000a). This result suggests that -latrotoxin has a dual, transmitter-specific mode 2 of action: Ca + appears to be necessary for -latrotoxin to trigger exocytosis of dense-core vesicles containing catecholamines and/or neuropeptides, but it appears to be dispensible for stimulation of exocytosis of small clear vesicles containing acetylcholine, glutamate, and GABA (Khvotchev et al 2000). In a beautiful example of this phenomenon, stimulation of neuromuscular junctions by high doses 2 of -latrotoxin in the absence of Ca + leads to a complete depletion of synaptic

by SCELC Trial on 09/09/11. For personal use only. vesicles from the motor nerve terminals while neuropeptide-containing dense-core vesicles are left untouched (Matteoli et al 1988). 2 It is interesting that the requirement for Ca + in neurotransmitter release triggered by -latrotoxin precisely parallels the necessity of phosphatidylinositolphos-

Annu. Rev. Neurosci. 2001.24:933-962. Downloaded from www.annualreviews.org phate biosynthesis (Khvotchev & S¨udhof 1998). When phosphatidylinositolphos- 2 phate synthesis is inhibited with phenylarsine oxide, Ca +-dependent release of norepinephrine is inhibited, whereas glutamate and GABA are secreted normally. This suggests that the organization of the synapse may be the decisive factor in 2 allowing -latrotoxin to trigger release in the absence of Ca +. According to this idea, classical synapses containing characteristic vesicle clusters and active zones 2 (e.g. glutamatergic, GABAergic, and cholinergic synapses) do not require Ca + for -latrotoxin action, whereas nonclassical synapses without predocked vesicles 2 2 require Ca +. This Ca + requirement may be due to the same mechanism that dictates the need of these synapses for phosphatidylinositolphosphate synthesis in 2 exocytosis. One possible explanation is that Ca + and phosphatidylinositolphosphates are involved in the efﬁcient transport and docking of the vesicles in a manner P1: FQP April 4, 2001 18:17 Annual Reviews AR121-30

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2 Figure 2 Ca + requirement for -latrotoxin (-Ltx) action in release of various neurotransmitters from synaptosomes. Dose/response curves show the amount of release of different neurotransmitters stimulated by the indicated -latrotoxin concentrations. [Modiﬁed from Khvotchev et al (2000).] Release triggered from synaptosomes by -latrotoxin was measured in the presence and

by SCELC Trial on 09/09/11. For personal use only. 2 absence of Ca +, demonstrating that there is no difference in the amount of GABA and glutamate release triggered by -latrotoxin under these two conditions, whereas major inhibition of release 2 is observed for norepinephrine and dopamine in the absence of Ca +. The norepinephrine release 2 observed in the absence of Ca + is probably a lytic artifact due to high -latrotoxin concen- 2 trations, whereas dopamine release likely has a Ca +-independent component, possibly because Annu. Rev. Neurosci. 2001.24:933-962. Downloaded from www.annualreviews.org dopaminergic vesicles are observed both in varicosities outside of classical synapses and in classical synapses.

similar to what has been observed in PC12 cells (Martin et al 1997), and that the transport and docking of the vesicles occur by a different mechanism in classical synapses containing clouds of vesicles at the active zone. 2 The precise point at which Ca + is required for -latrotoxin to trigger norepinephrine release was examined in synaptosomes (Khvotchev et al 2000). When 2 -latrotoxin is applied in the absence of Ca + and the synaptosomes are then 2 2 washed in Ca +-free buffer, subsequent addition of Ca + potently triggers norepinephrine release. Thus, -latrotoxin reacts with synaptosomes in the absence of P1: FQP April 4, 2001 18:17 Annual Reviews AR121-30

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2 2 Ca + but requires Ca + for stimulus-secretion coupling in norepinephrine release. 2 This is an important ﬁnding because it shows that the Ca + dependence operates not at the level of receptor binding of -latrotoxin but at the level of stimulation of release. The ﬁnding agrees well with the idea that differences in the mechanism of exocytosis, such as the requirement for phosphatidylinositolphosphates, determine 2 the differential Ca + sensitivity of norepinephrine release vs GABA and glutamate release induced by -latrotoxin.

CHANNEL FORMATION BY -LATROTOXIN

A very inﬂuential observation on how -latrotoxin might stimulate secretion was made by Finkelstein et al (1976). These authors showed that -latrotoxin added to the solution on one side of black lipid membranes spontaneously inserts into the membranes and forms a stable cation channel. Extensive characterization of the channel revealed that it requires incorporation of -latrotoxin into the lipid bilayer, 2 2 is nonselective with highest conductance for the divalent cations Ca + and Mg +, 2 2 and is inhibited by such transition metals as Cd + and Ni + (Mironov et al 1986, Robello et al 1987, Lishko et al 1990). However, -latrotoxin does not readily insert into the membranes of cultured cells when added to the medium. It appears to require high-afﬁnity receptors on the cell surface in order to insert into the membrane, and -latrotoxin-induced channels were probably only detectable in black lipid membranes because of the exquisite sensitivity of this detection technique. For example, in neuroendocrine cells containing -latrotoxin receptors, the toxin causes opening of cation channels (Wanke et al 1986, Hurlbut et al 1994, Hlubek et al 2000). At least the channels observed in the latter two studies were similar to those recorded with black lipid membranes, which suggests that the toxin spontaneously inserts into membranes to form a channel in vivo. However, in Xenopus

by SCELC Trial on 09/09/11. For personal use only. oocytes that do not express -latrotoxin receptors, application of -latrotoxin by 2 itself does not cause Ca + channels to open. Only after brain proteins have been ex- 2 pressed by injection of total brain mRNA are -latrotoxin-induced Ca + channels observed in the plasma membranes, presumably because -latrotoxin receptors

Annu. Rev. Neurosci. 2001.24:933-962. Downloaded from www.annualreviews.org were synthesized from the brain mRNA (Filipov et al 1990, Umbach et al 1990). Based on these studies, it seems likely that binding of -latrotoxin to receptors is necessary for the toxin to insert into biological membranes, where one of its actions then is to form a nonselective cation channel (Khvotchev & Südhof 2000). This mechanism, schematically depicted in Figure 3, would also explain the approximately 1-min lag period that is required for -latrotoxin to act on a synapse. 2 The finding that -latrotoxin induces formation of Ca + channels provides a ready explanation for how -latrotoxin triggers exocytosis from chromaffin 2 cells, PC12 cells, and noradrenergic nerve terminals in a Ca +-dependent manner (Meldolesi et al 1983, Khvotchev et al 2000). For this type of release, 2 -latrotoxin appears to act primarily as a Ca + channel, although even here, -latrotoxin may have an additional direct effect on the secretory apparatus (Liu & P1: FQP April 4, 2001 18:17 Annual Reviews AR121-30

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Figure 3 Model for the sequential interactions of -latrotoxin with receptors followed by membrane insertion, channel formation, and intracellular activation of exocytosis. The model proposes that -latrotoxin first binds to one or both of its receptors [CIRL/latrophilins (CLs) and neurexins] followed by a conformational change with subsequent membrane insertion. Receptor binding and the conformational change occur at 4C, but membrane insertion requires a higher temper- ature. The lectin concanavalin A (Con A) does not inhibit receptor binding but interferes with 2 the conformational change. Membrane insertion results in the formation of Ca + channels and the translocation of the N-terminal domain of -latrotoxin into the presynaptic intracellular space. Exocytosis can subsequently be triggered by two mechanisms that apply differentially to differ- 2 ent neurotransmitters (see text): Ca + influx via -latrotoxin channels stimulates norepinephrine, 2 dopamine, and neuropeptide release, whereas a direct Ca +-independent action of the N-terminal domain induces secretion of glutamate, GABA, and acetylcholine. The N and C termini of the various proteins are indicated by N and C, respectively. Note that only a single subunit of the -latrotoxin multimer is shown. [Modified from Khvotchev & Südhof (2000).] by SCELC Trial on 09/09/11. For personal use only.

2 Misler 1998, Bittner et al 1998). Curiously, it has been suggested that Ca + inﬂux is not responsible for triggering norepinephrine release by -latrotoxin. Instead, it 2

Annu. Rev. Neurosci. 2001.24:933-962. Downloaded from www.annualreviews.org was proposed that Ca + has to be mobilized by -latrotoxin from internal stores in order for release to occur (Davletov et al 1997, Rahman et al 1999). This conclusion was based entirely on the effect of high doses of thapsigargin, an 2 2 inhibitor of the endoplasmic reticulum Ca +-ATPase that empties internal Ca + stores. However, thapsigargin is unable to trigger norepinephrine release by itself and does not inhibit -latrotoxin action at more moderate doses that com- 2 pletely block Ca +-ATPase (Khvotchev et al 2000). This suggests that internal 2 Ca + stores are probably not involved in -latrotoxin action (Khvotchev et al 2 2000). Furthermore, the need for Ca + release from internal stores is difﬁcult 2 to understand under conditions that involve large Ca + inﬂux from the external medium via -latrotoxin-created channels. In addition, most nerve terminals lack extensive endoplasmic reticulum and mitochondria (Shepherd & Harris 1998) and P1: FQP April 4, 2001 18:17 Annual Reviews AR121-30

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2 do not have large internal Ca + stores, which argues against an essential role for 2 internal Ca + stores in neurotransmitter release. Therefore, in the case of cat- 2 echolamines and neuropeptides, -latrotoxin probably acts as a Ca + channel (Rosenthal et al 1990, Khvotchev et al 2000). The discovery of channel formation by -latrotoxin raised the question of whether there really is a dual mode of action of -latrotoxin whereby at least 2 some neurotransmitters are released in a Ca +-independent fashion. Is it possible that the channels could explain the action of -latrotoxin in triggering classical 2 neurotransmitter release in the absence of Ca +? It was suggested that release of 2 acetylcholine, glutamate, and GABA in the absence of extracellular Ca + could 2 be due to leaky membranes, membrane depolarization, and mobilization of Ca + from internal stores, all mediated directly or indirectly by -latrotoxin channels (Finkelstein et al 1976, Pumplin & Reese 1977). For example, in the absence of ex- 2 2 tracellular Ca +,Mg +and/or Na could flow through the -latrotoxin channels to + 2 trigger release by depolarizing the terminal and/or mobilizing Ca + from binding sites and internal stores. A number of findings strongly argue against this hypothesis and suggest that -latrotoxin truly does have a direct mode of action in the terminal. First, if the channel hypothesis was true, it is unclear why norepinephrine 2 release requires Ca +—why can it not be stimulated by the same mechanism as 2 glutamate and GABA? Second, application of Ca + ionophores, such as A23187, 2 2 that are not specific for Ca + but also conduct Mg + does not mimic the effect of -latrotoxin (Khvotchev & Südhof 1998). If these ionophores are applied in the 2 2 absence of Ca + (but with Mg +), no stimulation of release is observed. Even in 2 the presence of Ca +, these ionophores cause less neurotransmitter release than does -latrotoxin, indicating that -latrotoxin is better able to mobilize vesicles 2 2 than are ionophores. Third, the requirement for Mg + in Ca +-free solutions for -latrotoxin action can be partly abolished by increasing the molarity of the solution (Misler & Hurlbut 1979), which suggests that divalent cations are not abso-

by SCELC Trial on 09/09/11. For personal use only. lutely required. Fourth, monoclonal antibodies to -latrotoxin have been described 2 that inhibit -latrotoxin-stimulated Ca + ﬂuxes completely but have only a par- tial effect on GABA release, thereby uncoupling the two processes (Pashkov et al 2 1993). Fifth, Cd +, which is known to inhibit the conductance of the -latrotoxin

Annu. Rev. Neurosci. 2001.24:933-962. Downloaded from www.annualreviews.org channel (Lishko et al 1990), enhances -latrotoxin action (Ichtchenko et al 1998). Finally, -latrotoxin is still capable of triggering quantitatively unchanged glu- 2 tamate release in two mouse mutants in which normal Ca + triggered release is severely inhibited (Geppert et al 1994, Augustin et al 1999). In these mutants, the synaptic proteins synaptotagmin I or munc13-1 have been deleted, leading to a 2 block in the normal ability of Ca + to stimulate release. Because these mouse mu- 2 tants are Ca + unresponsive but -latrotoxin responsive, -latrotoxin must act at a 2 point upstream of Ca +. Together these ﬁndings establish that although -latrotoxin forms a channel in the membrane, it also directly stimulates the secretory apparatus of a synapse, presumably by direct action of sequences that have translocated into the cytosol (Figure 3). P1: FQP April 4, 2001 18:17 Annual Reviews AR121-30

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RECEPTOR-BASED INSERTION OF -LATROTOXIN INTO BIOLOGICAL MEMBRANES

In order to stimulate exocytosis, -latrotoxin needs to bind to specific neuronal high-affinity receptors (Tzeng & Siekevitz 1979). These receptors appear to be localized close to the active zone in the presynaptic plasma membrane (Valtorta et al 1984). Two classes of -latrotoxin receptors were found in brain that dif- 2 fer in their Ca + dependence. Both classes exhibit a similar abundance and bind -latrotoxin with the same nanomolar affinity. However, one receptor class is 2 2 Ca + dependent, whereas the second class is Ca + independent (Rosenthal et al 2 1990, Geppert et al 1998). As discussed below, the Ca +-dependent class of -latrotoxin receptors is constituted by neurexins, a large family of neuron-specific cell-adhesion molecules that were discovered in the search for -latrotoxin recep- 2 tors, whereas the Ca +-independent receptor class is composed of proteins called CLs (for CIRL/latrophilins). It should be noted that there is no connection between 2 the Ca +-dependent and -independent classes of -latrotoxin receptors and the 2 Ca +-dependent and -independent classes of neurotransmitters whose release is triggered by -latrotoxin. At first glance, there appears to be a striking similarity between the two classes of -latrotoxin receptors and the dual mode of neurotransmitter release triggered by -latrotoxin because both are defined in terms of their 2 2 Ca + dependence. However, the requirement for Ca + in norepinephrine release 2 triggered by -latrotoxin occurs after receptor binding, and Ca +-independent re- 2 ceptors can still mediate Ca +-dependent neurotransmitter release (e.g. see Sugita et al 1998); thus, the two phenomena are unrelated. As discussed above, formation of ion channels by -latrotoxin depends on the presence of receptors and occurs only with a delay after -latrotoxin application to nerve terminals. This suggests that -latrotoxin first binds to receptors and then inserts into the presynaptic plasma membrane (Figure 3). This notion was recently by SCELC Trial on 09/09/11. For personal use only. confirmed biochemically when it was shown that -latrotoxin becomes partially resistent to proteases after it has been incubated with synaptosomes (Khvotchev & Südhof 2000). Two conformational changes were detected on the basis of protease resistance patterns. The changes were distinguished because they required differ-

Annu. Rev. Neurosci. 2001.24:933-962. Downloaded from www.annualreviews.org ent incubation temperatures. At 4C, the toxin binds to receptors and undergoes a conformational change that alters its protease digestion pattern without causing true protease resistance. This limited conformational change was interpreted as a receptor-induced change at the membrane, possibly an exposure of the hydrophobic sequences in the N-terminal domain of -latrotoxin (Figure 1), in preparation for membrane insertion. However, receptor binding alone was not sufﬁcient for the initial conformational change because the change could be inhibited by the lectin concanavalin A, which had no effect on receptor binding (Khvotchev & S¨udhof 2000). When synaptosomes with receptor-bound -latrotoxin were warmed to 37C, the pattern of protease resistance was altered dramatically, with nearly complete protection of part of the toxin from digestion. This observation was taken as P1: FQP April 4, 2001 18:17 Annual Reviews AR121-30

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evidence for membrane insertion because protease protection was independent of the type of protease used, could be reversed on lysis of the synaptosomes, and was associated with the conversion of the toxin from a peripheral membrane protein to a detergent-insoluble form (Khvotchev & Südhof 2000). Because only parts and not the whole -latrotoxin was protected, the toxin is not endocytosed in toto but remains partly surface accessible. Mapping of the regions of the toxins that become protease resistant, and thus presumably translocated into the cytosol, resulted in a surprise (Khvotchev & Südhof 2000). The entire N-terminal domain was found to be protected during prolonged exposure to proteases, whereas only the N-terminal few ankyrin repeats were not proteolysed (see Figure 3). This was an unexpected result because ankyrin-like repeats are generally found only in intracellular sequences, which suggests that these repeats would most likely be intracellularly active. The protected N-terminal domain that translocates into the synaptosomal membrane includes two hydrophobic sequences (Dulubova et al 1996), indicating that this domain may form part of the channel in addition to being translocated into the cytosol (Figure 3). In solution, -latrotoxin forms a multimer that is dependent on divalent cations (Petrenko et al 1993, Orlova et al 2000). Although the oligomerization state of -latrotoxin in the membrane is unknown, it seems likely that it will form a multimer primarily composed of the N-terminal domain, with the hydrophobic segments assemblying into a channel (Figure 3). Here, the requirement for divalent cations, 2 such as Mg +, for -latrotoxin-triggered release of classical neurotransmitters 2 (which does not require Ca +) may reflect a need for tetramerization that is mediated by these divalent cations (Orlova et al 2000). In this respect it is interesting that 2 the divalent cation Cd + enhances -latrotoxin-induced neurotransmitter release (Ichtchenko et al 1998) but blocks the cation channels formed by -latrotoxin (Mironov et al 1986, Robello et al 1987, Lishko et al 1990). This suggests the possibility that divalent cations are generally required for -latrotoxin action be-

by SCELC Trial on 09/09/11. For personal use only. cause they multimerize the toxin in the membrane. According to this hypothesis, 2 Cd + and other divalent cations enter the mouth of the -latrotoxin channels to perform their action in triggering release but do not actually have to pass through the channel. Annu. Rev. Neurosci. 2001.24:933-962. Downloaded from www.annualreviews.org

PROPERTIES OF NEUREXINS

The first insight into the nature of -latrotoxin receptors was gained in affinity chromatography experiments by the purification of binding proteins on immobi- lized -latrotoxin (Scheer & Meldolesi 1985, Scheer et al 1986, Petrenko et al 1990). It is confusing, however, that a large number of different proteins were purified, many of which turned out to be presumably irrelevant intracellular components, for example a mitochondrial protein (Petrenko et al 1993). Nevertheless, one particular class of proteins with the properties of -latrotoxin receptors was discovered (Ushkaryov et al 1992). These proteins, called neurexins, constitute P1: FQP April 4, 2001 18:17 Annual Reviews AR121-30

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a highly polymorphic family of neuron-specific cell-surface proteins. Vertebrates contain at least three neurexin genes (neurexins 1, 2, and 3). Each gene has two independent promoters that direct transcription of the larger -neurexins and the shorter -neurexins (Ushkaryov et al 1992, 1994; Ushkaryov & Südhof 1993). Structurally, neurexins resemble cell-surface receptors with extended extracellular sequences that include O-linked sugar attachment sites, a single transmembrane region, and a short intracellular sequence (Figure 4). - and -neurexins differ only in their extracellular N-terminal domains and contain identical transmembrane regions and intracellular C-terminal domains. The extracellular sequences of -neurexins are composed of three overall repeats, each of which consists of a central epidermal growth factor (EGF)-like domain flanked by distantly related LNS domains (Figure 4). The LNS domains were named after laminin A, neurexins, and sex-hormone binding globulin because this domain was first identified as a repeat in the G-domain of laminin, was first shown to constitute an independently folding functional domain in neurexins, and was independently identified as a sequence motif in sex-hormone binding globulin (for a review, see Missler & Südhof 1998a). LNS domains are found in a large number of extracellular sequences, including agrin, slit, protein S, and perlecan (Ushkaryov et al 1992). They constitute protein-protein interaction domains that in neurexins, among others, bind -latrotoxin (see below). The three-dimensional structure of the sixth LNS domain of neurexin 1, simultaneously the only LNS domain of neurexin 1, has been characterized by crystallography (Rudenko et al 1999). The structure revealed that the LNS domain is composed of a 14-stranded -sandwich with a concave/convex overall shape. Although not identical to any known previously determined structure, the LNS domain structure is similar to that of pentraxin and lectins, which suggests even a possible evolutionary relationship (G Rudenko, E Hohenester, YA Muller, submitted for publication). The primary transcripts of neurexins are subject to extensive alternative splicing,

by SCELC Trial on 09/09/11. For personal use only. resulting in potentially thousands of isoforms (Ushkaryov et al 1992, Ullrich et al 1995). The highly variable nature of neurexins is interesting because expression of distinct neurexins on the neuronal cell surface could provide a possible mechanism by which different neurons are identiﬁed. To be physiologically meaningful,

Annu. Rev. Neurosci. 2001.24:933-962. Downloaded from www.annualreviews.org however, the alternative splicing of neurexins would have to fullfill the following criteria: (a) Alternative splicing should be similar for different neurexin genes; (b) it should be evolutionarily conserved; (c) it should be regulated and not random (i.e. different neurons should express distinct combinations of splice variants); (d ) alternative splicing should determine the interactions of neurexins with other cell-surface proteins; and (e) it should be instrumental for the in vivo functions of neurexins. All these criteria except for the last have been confirmed at least partially for neurexins. The last criterion, a demonstration that alternative splicing regulates the in vivo functions of neurexins, has not been demonstrated because the in vivo functions of neurexins remain to be clarified. The transcripts of all three neurexin genes are alternatively spliced, confirm- ing the first criterion (numbered arrows in Figure 4) (Ullrich et al 1995). For P1: FQP April 4, 2001 18:17 Annual Reviews AR121-30

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Figure 4 Domain structures of neurexins. The domain structures of - and -neurexins are illustrated and compared with those of neurexin-like proteins. Neurexins 1–3 and 1–3 are characterized by distinct extracellular N-terminal domain structures but share the same C-terminal domains, including the carbohydrate attachment sequence (CH), transmembrane region (TMR), and cytoplasmic tail. -Neurexins are composed of three overall repeats (labeled I, II, and III) containing a central epidermal growth factor (EGF)-like sequence that is flanked by LNS domains. Although significant homology exists between all LNS domains in the repeats, the LNS-A domains N-terminal to the EGF-like sequence form a separate class from the LNS-B domains C-terminal to the EGF-like sequence. -Neurexins lack most of the N-terminal domains found in -neurexins by SCELC Trial on 09/09/11. For personal use only. and are composed only of a -neurexin-specific sequence at the very N terminus (N) followed by a single LNS-B domain that corresponds to the sixth overall LNS domain in -neurexins. - and - neurexins are subject to extensive alternative splicing at five canonical positions for -neurexins (splices sites numbers 1–5), the last two of which are also found in -neurexins, as indicated

Annu. Rev. Neurosci. 2001.24:933-962. Downloaded from www.annualreviews.org by the labeled arrows. Neurexin-like proteins share some of the LNS- and EGF-like sequences found in -neurexins but in addition contain an N-terminal discoidin-like domain (Disc.), a central ﬁbrinogen / -related sequence (Fibr.), and a PGY-motif (P). [Modiﬁed from Missler et al (1998a).]

some splice sites, the alternatively spliced inserts consist of more than 10 distinct variants. The positions of alternative splicing and at least some of the splice variants are evolutionarily conserved, as predicted by the second criterion (Ullrich et al 1995, Patzke & Ernsberger 2000). In situ hybridizations demonstrated that neurexins are expressed in overlapping differential patterns, with regional regulation of at least some of the alternative splicing, satisfying the third criterion (Ullrich P1: FQP April 4, 2001 18:17 Annual Reviews AR121-30

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et al 1995). This was particularly striking for splice site 4, which is expressed in only two variants, which are differentially expressed in various types of neurons (Ichtchenko et al 1995). The different sites of alternative splicing are used independently of each other, resulting in probably thousands of isoforms that are present in different brain regions in a characteristic combination of multiple variants. With this pattern, neurexins have the potential to represent a combinatorial code. As regards the fourth criterion, only two classes of endogenous ligands for neurexins are known, neuroligins and neurexophilins (Ichtchenko et al 1995, 1996; Petrenko et al 1996; Missler & Südhof 1998b; Missler et al 1998b). Of these, neuroligins are neuronal cell-surface proteins that only bind to -neurexins when these lack an insert in splice site 4. Thus, the interaction of neuroligins with neurexins is tightly regulated by alternative splicing, satisfying the fourth criterion (Ichtchenko et al 1995, 1996). Neurexin binding to neuroligins forms an intercellular junction, which suggests that these proteins function as cell adhesion molecules (Nguyen & Südhof 1997). Neuroligins, when expressed in heterologous cells, can induce the generation of presynaptic specializations in neurons, indicating a function for neuroligins in synapse formation (Scheiffele et al 2000). In contrast to neuroligins, neurexophilins are secreted proteins, only bind to -neurexins, and interact with neurexins independently of alternative splicing (Missler & Südhof 1998a,b; Missler et al 1998a,b). Although in situ hybridizations indicate that neurexins are expressed only in neurons, no precise ultrastructural localization is available. Indirect evidence suggests that neurexins are concentrated in synapses. For example, subcellular frac- tionations revealed a high degree of enrichment of neurexins with active zones (Butz et al 1999), and the identification of neurexins as functional presynaptic -latrotoxin receptors suggests a relation to synapses (see below) (Sugita et al 1999). Also, immunogold localization of CASK and neuroligin I, interacting part- ners for neurexins, to synaptic complexes supports a localization to synapses (Song

by SCELC Trial on 09/09/11. For personal use only. et al 1999, Hsueh et al 1998). However, these indirect clues do not supplant the need for a direct localization of neurexins. Indeed, immunoﬂuorescence studies in Torpedo have suggested that some neurexins may be axonal instead of synaptic (Russell & Carlson 1997), although the resolution of these studies was so low that

Annu. Rev. Neurosci. 2001.24:933-962. Downloaded from www.annualreviews.org it was not possible to determine the precise localization of neurexins. Neurexins are evolutionarily conserved in vertebrates and in invertebrates, as revealed by the Caenorhabditis elegans and Drosophila genome sequences (Rubin et al 2000). However, these classical neurexins should not be confused with neurexin-like proteins (Figure 4). In Drosophila, a neurexin-like protein that is required for the formation and maintenance of septate junctions was called “neurexin IV” (Baumgartner et al 1996). Neurexin IV has caused confusion in the literature because its naming erroneously suggests that it belongs to the same family as vertebrate neurexins, whereas in fact it is the Drosophila homolog of vertebrate CASPR/paranodin proteins that exhibit a distant resemblance to neurexins (Peles et al 1997a,b; Menegoz et al 1997; Poliak et al 1999). CASPR/neurexin IV and vertebrate neurexins are similar cell surface receptor-like proteins containing, P1: FQP April 4, 2001 18:17 Annual Reviews AR121-30

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among others, LNS and EGF-like domains (Figure 3) (reviewed in Missler & S¨udhof 1998a). However, there are considerable differences between these proteins. Vertebrate neurexins are neuron speciﬁc, whereas Drosophila neurexin IV is expressed primarily outside of neurons; the domain structures of neurexins and CASPR/neurexin IV differ, and CASPR/neurexin IV are not polymorphic and not subject to alternative splicing. Thus, CASPR and neurexin IV should be more accurately thought of as neurexin-like proteins (Figure 4).

NEUREXINS AS -LATROTOXIN RECEPTORS

The initial discovery of neurexins as -latrotoxin-binding proteins during affinity chromatography suggested that they may constitute cell surface receptors for the toxin (Ushkaryov et al 1992). This was confirmed in studies demonstrating that the extracellular sequences of recombinant neurexins bind to -latrotoxin with nanomolar affinity (Davletov et al 1995). Both - and -neurexins bind to -latrotoxin directly, although only -neurexins were initially identified because their binding is much more resistant to salt washes (Sugita et al 1999). Binding of 2 -latrotoxin requires micromolar concentrations of Ca + and is tightly regulated by alternative splicing of neurexins at splice site 4 in the middle of the last LNS domain (Figure 4) (Sugita et al 1999), the same splice site that also regulates binding of -neurexins to neuroligins (Ichtchenko et al 1995, Nguyen & Südhof 1997). These data suggest that -latrotoxin binds to the last LNS domain of neurexin close to the membrane. Because alternative splicing of this LNS domain is differentially regulated, this binding may help explain the regional differences in -latrotoxin binding sites in brain (Malgaroli et al 1989). The fact that neurexins are high-affinity cell surface binding proteins for -latrotoxin, however, does not necessarily establish their function as -latrotoxin

by SCELC Trial on 09/09/11. For personal use only. receptors. Because -latrotoxin stimulates the release of classical neurotransmit- 2 2 ters without extracellular Ca + and neurexins require extracellular Ca + for binding to -latrotoxin, the candidacy of neurexins as physiological -latrotoxin receptors was dubious for some time. However, a series of experiments established that

Annu. Rev. Neurosci. 2001.24:933-962. Downloaded from www.annualreviews.org neurexins are “true” -latrotoxin receptors. Analysis of neurexin 1-knockout mice showed that -latrotoxin binding to brain membranes was reduced approx- 2 imately twofold in neurexin 1-deﬁcient mice in the presence of Ca + but was 2 unchanged in the absence of Ca + (Geppert et al 1998). In synaptosomes from neurexin 1-deﬁcient mice, -latrotoxin-stimulated glutamate release was normal 2 2 in the absence of Ca + but severely depressed in the presence of Ca + (Geppert et al 1998). These results showed that in vivo, neurexin 1 is responsible for approxi- 2 mately half of the -latrotoxin binding sites in the presence of Ca + and is required for -latrotoxin to elicit neurotransmitter release. The function of neurexins as -latrotoxin receptors was also studied in PC12 cells in which various neurexins were cotransfected with human growth hormone as a reporter molecule (Sugita et al 1999). In “naive” PC12 cells that have not been transfected with a neurexin, P1: FQP April 4, 2001 18:17 Annual Reviews AR121-30

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-latrotoxin triggers exocytosis only at relatively high toxin concentrations, probably because the levels of endogenous -latrotoxin receptors in PC12 cells are very low. Transfection of PC12 cells with neurexin 1 or 1 makes the PC12 cells 10- to 100-fold more sensitive to -latrotoxin (Sugita et al 1999). Neurexin 2 1 was as potent in this assay as the Ca +-independent receptor CL1 (discussed below), which suggests that both receptors can equally mediate -latrotoxin action in transfected PC12 cells. As -latrotoxin receptors, neurexins are presumably close to the point of exocytosis in the presynaptic nerve terminal. The discovery that the putative synaptic 2 vesicle Ca +-sensor synaptotagmin 1 and the synaptic adaptor protein CASK bind to the cytoplasmic tail of neurexins initially suggested that neurexins transduce an exocytotic signal of -latrotoxin to the intracellular space (Hata et al 1993a, 1996). As mentioned above, however, -latrotoxin is fully active in synaptotagmin 2 1 knockout mice even though in the same mice Ca +-stimulated glutamate release is severely impaired (Geppert et al 1994). This result demonstrates that synaptotagmin is not essential for -latrotoxin action. Furthermore, deletion of the entire cytoplasmic tail of neurexin 1 has no effect on its function as an -latrotoxin receptor in transfected PC12 cells (Sugita et al 1999). These results argue against the possibility of a direct coupling of receptor binding to activation of exocytosis and suggest that after neurexin binding, -latrotoxin performs a second independent action in triggering neurotransmitter release.

CIRL/LATROPHILINS AS -LATROTOXIN RECEPTORS

2 The puriﬁcation and cloning of a Ca +-independent receptor for -latrotoxin was simultaneously described by two groups (Davletov et al 1996; Krasnoperov et al 1996, 1997; Lelianova et al 1997). In a long-standing tradition of work on synaptic

by SCELC Trial on 09/09/11. For personal use only. proteins, the two groups gave the receptor different names, CIRL and latrophilin. Further cloning revealed that at least three closely related forms of CIRL/latrophilin are expressed in vertebrates (Sugita et al 1998, Ichtchenko et al 1999, Matsushita et al 1999). To avoid confusion between the names of these receptors (CIRL and

Annu. Rev. Neurosci. 2001.24:933-962. Downloaded from www.annualreviews.org latrophilin), we follow the nomenclature of Sugita et al (1998) and abbreviate the two designations into a single name, CL. The three isoforms are therefore referred to as CL1, CL2, and CL3. All CLs represent unusual G-protein-linked receptors with very large extra- and intracellular domains and are composed of the same domain structure (Sugita et al 1998): At the N terminus, a cleaved signal peptide is followed by a series of individual domains that cover approximately 500 amino acids (Figure 5). These domains include a lectin-like sequence; a region homologous to olfactomedins and myocilin; a long, previously undefined homology region that CLs share with a protein family of unknown function, referred to as BAI1-3 (brain-specific angiogenesis inhibitors) (Shiratsuchi et al 1997); and a short, cysteine-rich sequence. The short, cysteine-rich sequence outside of the first transmembrane region in CLs P1: FQP April 4, 2001 18:17 Annual Reviews AR121-30

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Figure 5 Domain structures of CIRL/latrophilins (CLs). CLs are G-protein-coupled receptors with unusually large extra- and intracellular sequences. The extracellular sequences are composed of ﬁve domains, shown above the plasma membrane, whereas the intracellular sequences have no discernable domain structure. The transmembrane regions of the CLs are similar to those of the calcitonin/secretin receptor family; in addition, the extracellular domains share homology with those of the BAI1-3 and the EMR1 family of G-protein-coupled receptors. [Modiﬁed from Sugita et al (1998).]

is highly homologous to sequences found in several G-protein-linked receptors

by SCELC Trial on 09/09/11. For personal use only. (Sugita et al 1998). In CD97 and in CL1, the cysteine-rich domain probably directs the cleavage of the receptors after the last cysteine residue just N-terminal to the ﬁrst transmembrane region (Gray et al 1996, Krasnoperov et al 1997). This sequence may represent a proteolytic cleavage signal during the maturation of G-protein-

Annu. Rev. Neurosci. 2001.24:933-962. Downloaded from www.annualreviews.org linked receptor and has been termed GPS domain for G-protein-coupled receptor proteolytic site domain (Gray et al 1996, Krasnoperov et al 1997, Ichtchenko et al 1999). However, copies of this domain are also present in other membrane proteins, such as polycystin-1, which suggests that it may be a general motif present outside of G-protein-linked receptors (Ponting et al 1999). In CL1, the cleaved N-terminal extracellular domain largely remains attached to the transmembrane portion of the receptor by an unknown mechanism, although some of the cleaved protein may also be shed into the medium. The sequences of the seven transmembrane regions and connecting linkers of CLs are signiﬁcantly related to those of the secretin family of G-protein-coupled receptors, placing CLs into this general class of G-protein-coupled receptors (Figure 5). Following the seven transmembrane regions, CLs feature an unusually long cytoplasmic tail of approximately P1: FQP April 4, 2001 18:17 Annual Reviews AR121-30

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500 residues that is subject to extensive alternative splicing (Sugita et al 1998, Matsushita et al 1999). CL1, 2, and 3 are differentially expressed in a pattern that suggests a widespread function not directly related to neurons. Although CL1 was initially thought to be brain specific (Krasnoperov et al 1997, Lelianova et al 1997), more-sensitive RNA blots revealed low levels of CL1 in all tissues (Sugita et al 1998). CL2 is produced primarily outside of brain, with low levels in brain, whereas CL3 is dectectable only in brain (Sugita et al 1998, Ichtchenko et al 1999, Matsushita et al 1999). Both the initially discovered CL1 and CL2 bind -latrotoxin with nanomolar affinity, whereas CL3 is unable to bind (Ichtchenko et al 1999). This suggests that CL1 and CL2 can function as -latrotoxin receptors, a surprising result considering the ubiquitous distribution of CL2. This result does agree, however, with the observation that -latrotoxin is capable of stimulating glutamate release from such nonneuronal cells as astrocytes (Parpura et al 1995). Based on their structure, it seems likely that CLs have a physiological function as cell-adhesion molecules coupled to signal transduction. This hypothesis is supported by the binding of PDZ-domain proteins called SHANKs to the C terminus of all CLs (Tobaben et al 2000). However, the in vivo roles of CLs or their natural ligands are unknown. Clearly, in view of their tissue distributions, CLs do not perform a synapse-specific, let alone neuron-specific, function. Recent studies employing a CL1 knockout mouse demonstrated that CL1 is not essential for normal mouse development, survival, or cage life but confirms its function as an -latrotoxin receptor (S Tobaben, TC Südhof, B Stahl, submitted for publication). Challenges of synaptosomes lacking CL1 with -latrotoxin revealed that the majority of -latrotoxin-evoked glutamate release was abolished, whereas depolarization-stimulated release was normal. -Latrotoxin-induced release was 2 impaired both in the presence and absence of Ca +, in contrast to the neurexin 1-deficient synaptosomes, in which release was only decreased in the presence 2 2 by SCELC Trial on 09/09/11. For personal use only. of Ca + (Geppert et al 1998). These findings confirm that CL1 is the major Ca +- independent -latrotoxin receptor in vivo. How do CLs function as receptors for -latrotoxin in triggering release? Af- ter the discovery of CL1 as a G-protein-linked receptor, the initial expectation

Annu. Rev. Neurosci. 2001.24:933-962. Downloaded from www.annualreviews.org had been that -latrotoxin activates an intracellular G-protein signal that triggers exocytosis (Krasnoperov et al 1997, Lelianova et al 1997). This expectation was enhanced by the observation of the copuriﬁcation of syntaxin, a SNARE protein involved in fusion, with the receptor (Krasnoperov et al 1997). However, transfection experiments with PC12 cells and chromafﬁn cells revealed that CL1 lacking all cytoplasmic sequences functioned as an excellent -latrotoxin receptor, demonstrating that intracellular signal transduction is not required (Sugita et al 1998, Hlubek et al 2000). This result parallels data obtained for neurexins (Sugita et al 1999), which suggest that at least in transfected PC12 cells, both receptors presumably serve to recruit -latrotoxin to the membrane at the appropriate point (i.e. close to the active zone at the presynaptic plasma membrane) without in fact transducing the intracellular effect of the toxin. P1: FQP April 4, 2001 18:17 Annual Reviews AR121-30

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THE TWO CLASSES OF -LATROTOXIN RECEPTORS MAY COOPERATE

-Latrotoxin is unique among toxins because it binds to two families of high- affinity receptors with no sequence similarity or functional resemblance. Why did two receptors evolve for -latrotoxin? Transfections of either neurexins or CLs into PC12 cells demonstrated that they can function as independent -latrotoxin receptors in this system, and cotransfection of the two receptor families does not enhance the -latrotoxin response over that of the singly transfected cells (Sugita et al 1998, 1999). These results show that the two receptors do not functionally cooperate in PC12 cells. However, as discussed above, -latrotoxin has a dual mode of action, and the action of the receptors in PC12 cell exocytosis, a typical example of 2 dense-core vesicle secretion that requires Ca +, may not be representative of receptor action in central synapses. Indeed, several observations suggest that the CL and neurexin receptor families collaborate in -latrotoxin-triggered neurotransmitter release in central synapses, although the nature of their interaction is unclear. One line of evidence for a collaboration between receptors was obtained from 2 2 comparisons of Ca +-dependent and Ca +-independent binding of -latrotoxin to 2 2 brain membranes and of Ca +-dependent and Ca +-independent glutamate and GABA release stimulated by -latrotoxin in synaptosomes. The number of - 2 latrotoxin binding sites is approximately two times higher in the presence of Ca + 2 than in the absence of Ca +, as would be expected if the total binding was the sum 2 2 of Ca +-dependent and Ca +-independent binding (Rosenthal et al 1990, Geppert et al 1998). However, glutamate and GABA release is approximately the same in 2 the presence or absence of Ca +, independent of the -latrotoxin dose (see Figure 2) (Khvotchev et al 2000). If the -latrotoxin receptors acted independently, release 2 2 2 observed in the presence of Ca + should be the sum of Ca +-dependent and Ca +- independent release, similar to binding, which is clearly not the case. by SCELC Trial on 09/09/11. For personal use only. Results from studies with the neurexin 1 knockout mice (Geppert et al 1998) provide further support for the idea that the two classes of receptors functionally cooperate. In these mice, an expected nearly 50% decrease of total -latrotoxin- 2 binding sites in brain was observed, with a majority of the Ca +-dependent binding 2 Annu. Rev. Neurosci. 2001.24:933-962. Downloaded from www.annualreviews.org sites abolished. Furthermore, as expected, Ca +-independent neurotransmitter release triggered by -latrotoxin was unchanged. Unexpected was the finding that in the same mice, -latrotoxin triggered neurotransmitter release was reduced in 2 the presence of Ca +. This result is puzzling because the portion of release that 2 corresponds to the Ca +-independent mechanism should be selectively preserved 2 in the neurexin 1 knockouts. These observations raise the possibility that Ca + 2 inhibits -latrotoxin action via CLs, and that this inhibition is abolished on Ca + withdrawal. Although this hypothesis is currently difficult to understand mechanis- 2 tically, it should be recalled that Ca + is normally always present in the extracellular 2 fluid, and the release in the presence of Ca + represents the more physiological situation. P1: FQP April 4, 2001 18:17 Annual Reviews AR121-30

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Additional evidence for a cooperation between CLs and neurexins as - latrotoxin receptors was provided by the analysis of the CL1 single and the CL1/ neurexin 1 double knockout mice (S Tobaben, TC Südhof, B Stahl, submitted for publication 2001). As described above, the CL1 knockout had the expected 2 phenotype, in that Ca +-independent glutamate release triggered by -latrotoxin 2 was largely abolished, but unexpectedly, Ca +-dependent glutamate release was 2 also largely abolished although neurexin I was still present. Furthermore, Ca +- dependent -latrotoxin release in the double knockout mouse was no more impaired than in either of the two single knockout mice whereas, surprisingly, 2 Ca +-independent glutamate release in the double knockout mouse was even slightly enhanced. It is easy to understand why some -latrotoxin-triggered release was retained in the double knockout mice because the remaining isoforms of neurexins and CLs can also serve as -latrotoxin receptors (Sugita et al 1998, 1999; Ichtchenko et al 1999). However, the relative impairments in -latrotoxin- triggered release by the two knockouts suggest that they do not function as independent receptors but must somehow interact. Because each receptor forms an independent high-affinity -latrotoxin binding site in brain, this interaction must occur downstream of receptor binding at a currently unidentified process. Based on the overall evidence, it is likely that the receptors cooperate in triggering release by -latrotoxin from central synapses. However, this interaction is probably not due simply to receptor activation. In the -latrotoxin mutant LtxN4C described above, four amino acids were inserted between the N-terminal conserved domain and the ankyrin repeats (Figure 1). The LtxN4C mutant binds to -latrotoxin receptors with the same affinity as wild-type -latrotoxin but causes no stimulation of neurotransmitter release (Ichtchenko et al 1998). -Latrotoxin binding to its receptors on neurons and synaptosomes produces a number of intra- 2 cellular effects, some of which are likely caused by intracellular Ca + influx. For example, -latrotoxin stimulates massive hydrolysis of phosphatidylinositolphos-

by SCELC Trial on 09/09/11. For personal use only. phates, presumably by activation of phospholipase C (Vicentini & Meldolesi 1984, Ichtchenko et al 1998). The fact that the LtxN4C mutant still stimulates hydrolysis of phosphatidylinositolphosphates suggests that it is still active as a ligand although unable to trigger release. This mutant therefore uncouples receptor activation from

Annu. Rev. Neurosci. 2001.24:933-962. Downloaded from www.annualreviews.org the stimulation of neurotransmitter release, which suggests that the two processes are mechanistically distinct.

HOW DOES -LATROTOXIN TRIGGER RELEASE AT A CLASSICAL SYNAPSE?

As described above, considerable progress has been made in the understanding of -latrotoxin action. The toxin has been cloned and functionally expressed, allowing analysis of mutants. The receptors for -latrotoxin on neuronal membranes have now been identiﬁed, leading to the discovery of two new families of cell P1: FQP April 4, 2001 18:17 Annual Reviews AR121-30

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surface proteins, neurexins and CLs. The toxin has been shown to insert into the plasma membrane after receptor binding, and the N-terminal parts of the toxin that are involved in the intracellular translocation have been identified. It seems likely that the toxin multimerizes in the membrane in a reaction that requires divalent 2 cations, although not necessarily Ca +, and that the multimerization is essential for all actions of the toxin. The intracellular signaling initiated by the toxin, such 2 as Ca + influx and hydrolysis of phosphatidylinositols, has been uncoupled from its action in stimulated exocytosis of synaptic vesicles containing classical neuro- 2 transmitters, such as GABA and glutamate, that do not require Ca + for release triggered by -latrotoxin. The overall picture of -latrotoxin action that emerges from these studies is similar to that of many other toxins that bind to cell surface receptors, insert into the membrane, and then transduce intracellular signals. -Latrotoxin, however, also has several unusual properties that we currently do not understand. It acts via two classes of dissimilar high-affinity receptors. The rationale for having two receptors is unclear, although recent data suggest that the two receptors may in fact cooperate in triggering exocytosis. The most unusual feature of -latrotoxin may be its dual mode of action. In what could be termed its simple mode, -latrotoxin forms a channel in the membrane that 2 2 conducts Ca +, with the resulting Ca + influx triggering a number of intracellular events, including exocytosis of dense-core vesicles. The second mode of action of -latrotoxin, which could be called the complex mode and is probably more important, is its ability to directly stimulate synaptic vesicle exocytosis without participa- 2 tion of Ca +. Insertion of -latrotoxin into the membrane with translocation of the N-terminal domain is required for this mode of action but is not sufficient because the LtxN4C mutant, which is unable to trigger release, still inserts into the membrane, although with lowered efficiency (Khvotchev & Südhof 2000). In the 2 complex mode, -latrotoxin does not function as a Ca + channel or channel for 2 any other ion because this mode is not inhibited by Cd +, which does inhibit the

by SCELC Trial on 09/09/11. For personal use only. -latrotoxin channel (Ichtchenko et al 1998). Thus, this mode involves a direct stimulation of the secretory apparatus at the synapse. Recent work, primarily based on studies of knockout mice and results obtained with botulinum and tetanus toxins, have allowed a description of key proteins

Annu. Rev. Neurosci. 2001.24:933-962. Downloaded from www.annualreviews.org that function in identified steps in vesicle fusion (Figure 6) (Fernandez-Chacon & Südhof 1999). According to this work, vesicles that are docked at the active zone undergo a priming/prefusion reaction before they are ready to be stimulated for 2 release by Ca +. The priming/prefusion reaction requires initially the SM protein munc18-1 and the SNARE proteins synaptobrevin, syntaxin, and SNAP-25, which are inhibited by the botulinum and tetanus toxins, and then the active zone pro- 2 tein munc13-1 (Jahn & Südhof 1999). The Ca +-triggered fusion reaction then is regulated by synaptotagmin, rab3, and the rab3-effector RIM (Figure 6). Stud- ies with botulinum and tetanus toxins, which interfere with the functions of the SNARE proteins, showed that the SNARE proteins are essential for -latrotoxin action (Dreyer et al 1987, Capogna et al 1997; for a review, see Südhof et al 1993). The fact that these toxins can block -latrotoxin action demonstrates that P1: FQP April 4, 2001 18:17 Annual Reviews AR121-30

-LATROTOXIN AND ITS RECEPTORS 955

Figure 6 Point of action of -latrotoxin in neurotransmitter release in relation to synaptic pro- 2 teins. The final part of synaptic vesicle exocytosis physiologically triggered by Ca + influx during an action potential can be divided into a priming/prefusion reaction that makes the vesicles com- 2 2 petent for a Ca + signal, and the actual Ca +-triggering reaction (top of diagram) (for a review, see Südhof 1995). The point in the reaction where each of the indicated synaptic proteins are required is shown in the middle. Based primarily on knockout studies, the priming/prefusion reaction can be subdivided into an early stage that requires the SNAREs synaptobrevin, SNAP-25, and syntaxin, as well as the SM-protein munc18-1 (Jahn & Südhof 1999), and a later stage that requires munc13-1 (Augustin et al 1999). The action of these proteins is then followed by the need for synaptotag- 2 min I, rab3A, and the rab3-effector RIM in late stages of exocytosis during the Ca +-triggering reaction. Exocytosis can be triggered by -latrotoxin at a stage that precedes munc13-1 action because -latrotoxin stimulates release normally in munc13-1-deficient mice, whereas hypertonic 2 sucrose, another secretagog that triggers release in the absence of Ca + (Rosenmund & Stevens 1996), requires munc13-1 but does not need rab3 or synaptotagmin I.

-latrotoxin utilizes the same SNARE-dependent mechanism of secretion as does 2 Ca +-triggered neurotransmitter release. In addition, mice lacking munc18-1 are probably unable to respond to -latrotoxin (Verhage et al 2000). However, for the

by SCELC Trial on 09/09/11. For personal use only. munc18-1 mutant, the situation is not totally clear because there appears to be a general lack of synaptic transmission, making it difficult to determine whether there is a true deficiency of -latrotoxin response (Verhage et al 2000). Together these findings suggest that the core fusion proteins, synaptobrevin, syntaxin, SNAP-25,

Annu. Rev. Neurosci. 2001.24:933-962. Downloaded from www.annualreviews.org and munc18-1, are required for -latrotoxin action (Figure 6). It is interesting, however, that mutants in munc13-1 are not impaired in the -latrotoxin response 2 (Augustin et al 1999). In the same mutant, most of the Ca +-triggered release 2 of glutamate and Ca +-independent release stimulated by hypertonic sucrose are severely impaired. These results place the action of -latrotoxin downstream of the munc18/SNARE complex but upstream of munc13-1. The question now arises as to how -latrotoxin acts on the core complex and munc18-1, and what its precise intracellular mode of action is. After many efforts, which may be characterized as scientiﬁc dances—two steps forward, one step back—the promise of -latrotoxin is now beginning to be realized. We now know that (a) the toxin can indeed directly stimulate the secretory apparatus, (b)ithas been demonstrated that this only occurs at classical synapses, and (c) the point at P1: FQP April 4, 2001 18:17 Annual Reviews AR121-30

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which this stimulation occurs has been identiﬁed. This provides a unique oppor- tunity to identify the molecular switch or handle that -latrotoxin uses to trigger release. The most plausible hypothesis is that the toxin acts by releasing a molecular brake that normally blocks vesicle fusion from occurring in classical synapses 2 in the absence of Ca +. Clearly, deﬁnition of the mechanism by which -latrotoxin operates will provide a major step forward in understanding what keeps the vesicles from automatically progressing through the fusion reaction and thus will clarify a key question of how a synapse, as opposed to a nonsecretory system, functions.

ACKNOWLEDGMENTS I would like to thank my colleagues and collaborators Drs. B Stahl and S Tobaben (G¨ottingen) and Drs. M Khvotchev and S Sugita (Dallas) for helpful comments. This work was supported by NIH grant R37-MH52804-06.

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CONTENTS PDZ DOMAINS AND THE ORGANIZATION OF 1 SUPRAMOLECULAR COMPLEXES, Morgan Sheng, Carlo Sala

THE ROLE AND REGULATION OF ADENOSINE IN THE CENTRAL 31 NERVOUS SYSTEM, Thomas V. Dunwiddie, Susan A. Masino LOCALIZATION AND GLOBALIZATION IN CONSCIOUS VISION, 57 S. Zeki GLIAL CONTROL OF NEURONAL DEVELOPMENT, Greg Lemke 87 TOUCH AND GO: Decision-Making Mechanisms in Somatosensation, 107 Ranulfo Romo, Emilio Salinas SYNAPTIC MODIFICATION BY CORRELATED ACTIVITY: Hebb''s 139 Postulate Revisited, Guo-qiang Bi, Mu-ming Poo AN INTEGRATIVE THEORY OF PREFRONTAL CORTEX 167 FUNCTION, Earl K. Miller, Jonathan D. Cohen THE PHYSIOLOGY OF STEREOPSIS, B. G. Cumming, G. C. 203 DeAngelis PARANEOPLASTIC NEUROLOGIC DISEASE ANTIGENS: RNA- Binding Proteins and Signaling Proteins in Neuronal Degeneration, Kiran 239 Musunuru, Robert B. Darnell ODOR ENCODING AS AN ACTIVE, DYNAMICAL PROCESS: Experiments, Computation, and Theory, Gilles Laurent, Mark Stopfer, 263 Rainer W Friedrich, Misha I Rabinovich, Alexander Volkovskii, Henry DI Abarbanel PROTEIN SYNTHESIS AT SYNAPTIC SITES ON DENDRITES, 299 Oswald Steward, Erin M. Schuman

SIGNALING AND TRANSCRIPTIONAL MECHANISMS IN 327 PITUITARY DEVELOPMENT, Jeremy S. Dasen, Michael G. Rosenfeld NEUROPEPTIDES AND THE INTEGRATION OF MOTOR

by SCELC Trial on 09/09/11. For personal use only. 357 RESPONSES TO DEHYDRATION , Alan G. Watts THE DEVELOPMENTAL BIOLOGY OF BRAIN TUMORS, Robert 385 Wechsler-Reya, Matthew P. Scott TO EAT OR TO SLEEP? OREXIN IN THE REGULATION OF Annu. Rev. Neurosci. 2001.24:933-962. Downloaded from www.annualreviews.org FEEDING AND WAKEFULNESS, Jon T. Willie, Richard M. Chemelli, 429 Christopher M. Sinton, Masashi Yanagisawa SPATIAL PROCESSING IN THE BRAIN: The Activity of Hippocampal 459 Place Cells, Phillip J. Best, Aaron M. White, Ali Minai THE VANILLOID RECEPTOR: A Molecular Gateway to the Pain 487 Pathway, Michael J Caterina, David Julius PRION DISEASES OF HUMANS AND ANIMALS: Their Causes and 519 Molecular Basis, John Collinge VIKTOR HAMBURGER AND RITA LEVI-MONTALCINI: The Path to 551 the Discovery of Nerve Growth Factor, W. Maxwell Cowan EARLY DAYS OF THE NERVE GROWTH FACTOR PROTEINS, 601 Eric M. Shooter SEQUENTIAL ORGANIZATION OF MULTIPLE MOVEMENTS: 631 Involvement of Cortical Motor Areas, Jun Tanji INFLUENCE OF DENDRITIC CONDUCTANCES ON THE INPUT- 653 OUTPUT PROPERTIES OF NEURONS, Alex Reyes NEUROTROPHINS: Roles in Neuronal Development and Function, Eric 677 J Huang, Louis F Reichardt CONTRIBUTIONS OF THE MEDULLARY RAPHE AND VENTROMEDIAL RETICULAR REGION TO PAIN MODULATION 737 AND OTHER HOMEOSTATIC FUNCTIONS, Peggy Mason ACTIVATION, DEACTIVATION, AND ADAPTATION IN VERTEBRATE PHOTORECEPTOR CELLS, Marie E Burns, Denis A Baylor 779 ACTIVITY-DEPENDENT SPINAL CORD PLASTICITY IN HEALTH AND DISEASE, Jonathan R Wolpaw, Ann M Tennissen 807 QUANTITATIVE GENETICS AND MOUSE BEHAVIOR, Jeanne M 845 Wehner, Richard A Radcliffe, Barbara J Bowers EARLY ANTERIOR/POSTERIOR PATTERNING OF THE 869 MIDBRAIN AND CEREBELLUM, Aimin Liu, Alexandra L Joyner NEUROBIOLOGY OF PAVLOVIAN FEAR CONDITIONING, Stephen 897 Maren {{alpha}}-LATROTOXIN AND ITS RECEPTORS: Neurexins and CIRL/Latrophilins, Thomas C Südhof 933 IMAGING AND CODING IN THE OLFACTORY SYSTEM, John S 963 Kauer, Joel White THE ROLE OF THE CEREBELLUM IN VOLUNTARY EYE 981 MOVEMENTS, Farrel R Robinson, Albert F Fuchs ROLE OF THE REELIN SIGNALING PATHWAY IN CENTRAL 1005 NERVOUS SYSTEM DEVELOPMENT, Dennis S Rice, Tom Curran HUMAN BRAIN MALFORMATIONS AND THEIR LESSONS FOR 1041 NEURONAL MIGRATION, M Elizabeth Ross, Christopher A Walsh MORPHOLOGICAL CHANGES IN DENDRITIC SPINES ASSOCIATED WITH LONG-TERM SYNAPTIC PLASTICITY, Rafael 1071 Yuste, Tobias Bonhoeffer

STOPPING TIME: The Genetics of Fly and Mouse Circadian Clocks, 1091 Ravi Allada, Patrick Emery, Joseph S. Takahashi, Michael Rosbash NEURODEGENERATIVE TAUOPATHIES, Virginia M-Y Lee, Michel 1121 Goedert, John Q Trojanowski

by SCELC Trial on 09/09/11. For personal use only. MATERNAL CARE, GENE EXPRESSION, AND THE TRANSMISSION OF INDIVIDUAL DIFFERENCES IN STRESS 1161 REACTIVITY ACROSS GENERATIONS, Michael J Meaney NATURAL IMAGE STATISTICS AND NEURAL 1193

Annu. Rev. Neurosci. 2001.24:933-962. Downloaded from www.annualreviews.org REPRESENTATION, Eero P Simoncelli, Bruno A Olshausen Nerve Growth Factor Signaling, Neuroprotection, and Neural Repair, 1217 Michael V Sofroniew, Charles L Howe, William C Mobley FLIES, GENES, AND LEARNING, Scott Waddell, William G Quinn 1283