Latrotoxin Receptor Signaling Engages the UNC-13-Dependent Vesicle-Priming Pathway in C

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector Current Biology, Vol. 14, 1374–1379, August 10, 2004, 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cub.2004.07.056 Latrotoxin Receptor Signaling Engages the UNC-13-Dependent Vesicle-Priming Pathway in C. elegans

James Willson,1 Kiran Amliwala,1 Andrew Davis,1 tent with an ␣-latrotoxin (LTX)-like action [6], and func- Alan Cook,1 Matthew F. Cuttle,1 Neline Kriek,1 tional studies indicate that the predominant target site Neil A. Hopper,1 Vincent O’Connor,1 of emodepside is presynaptic [7]. To directly test for a Achim Harder,2 Robert J. Walker,1 LTX-like action of emodepside on C. elegans neurons, and Lindy Holden-Dye1,* we dissected pharyngeal muscle and the embedded 1Neurosciences Research Group enteric neurons free of the body of the animal, making School of Biological Sciences the tissue accessible for dye loading with the styryl en- University of Southampton docytotic marker FM4-64 [8]. FM4-64 labeling was activ- Bassett Crescent East ity-dependent, i.e., increased in high potassium (data not Southampton SO16 7PX shown) and localized to neuronal puncta consistent with United Kingdom its accumulation into synaptic regions (Figures 1A–1C). 2 Business Group Animal Health In the absence of emodepside (pharynxes treated with Research and Development Agricultural vehicle only), fluorescence was stable for up to 30 min Centre Monheim (n ϭ 4). However, addition of emodepside (100 nM) trig- 51368 Leverkusen gered loss of FM4-64 fluorescence (Figures 1C and 1D; Germany 10 out of 12 preparations) over a time course of 10 min. As a functional correlate of this image analysis, we observed that emodepside (0.125 nM to 1 ␮M) paralyzed Summary the pharynx of intact adult animals. Emodepside also had marked effects on tissues other than the pharynx. ␣-latrotoxin (LTX), a 120 kDa protein in black widow It immobilized mature adults and inhibited egg laying. spider venom, triggers massive neurotransmitter exo- Interestingly, at this range of concentrations, emodep- cytosis. Previous studies have highlighted a role for side had little if any effect on larval animals. both intrinsic pore-forming activity and receptor bind- The potent effect of emodepside on C. elegans phar- ing in the action of this toxin [1]. Intriguingly, activation ynx enabled us to take advantage of established tech- of a presynaptic G protein-coupled receptor, latrophi- niques for electrophysiological analysis (electropha- lin, may trigger release independent of pore-formation ryngeogram, EPG) of this muscle in order to quantify [2]. Here we have utilized a previously identified ligand its action [9]. Emodepside inhibited basal pharyngeal of nematode latrophilin, emodepside [3], to define a pumping rate (IC50 2.2 nM; data not shown) and the latrophilin-dependent pathway for neurotransmitter response of the pharynx to two stimulatory neurotrans- release in C. elegans. In the pharyngeal nervous sys- mitters [10], either 5-HT (IC50 4.1 nM; Figures 2A and 2B) tem of this animal, emodepside (100 nM) stimulates or AF1 (Figure S4). Therefore, emodepside inhibits both exocytosis and elicits pharyngeal paralysis. The phar- the tonically-active pharynx, and pharyngeal pumping ynxes of animals with latrophilin (lat-1) gene knock- stimulated by pharmacological activation of the enteric outs are resistant to emodepside, indicating that emo- neuronal circuit. The cyclic structure of emodepside pre- depside exerts its high-affinity paralytic effect through sents the possibility that its action may derive from a LAT-1. The expression pattern of lat-1 supports the nonspecific, receptor-independent pore-forming capa- hypothesis that emodepside exerts its effect on the bility. However, PF1022-001, a structural isomer of the pharynx primarily via neuronal latrophilin. We build lead compound for emodepside that has the same pore- on these observations to show that pharynxes from forming capability [11], has no inhibitory action on the ␮ ϭ animals with either reduction or loss of function mu- pharynx at concentrations up to 10 M(n 6; data not tations in Gq, phospholipaseC-␤, and UNC-13 are shown). As a caveat to this, it should be noted that the resistant to emodepside. The latter is a key priming pore-forming capability of LTX in biological membranes molecule essential for synaptic vesicle-mediated re- requires tethering of LTX to the membrane by binding lease of neurotransmitter [4, 5]. We conclude that the to specific receptors [12]. Therefore, we cannot discount small molecule ligand emodepside triggers latrophilin- the possibility that the lack of effect of PF1022-001 may mediated exocytosis via a pathway that engages UNC- reflect an inability of the isomer to bind to a membrane 13-dependent vesicle priming. receptor prior to pore formation. However, previous studies have shown that a linear form of cyclooctadepsi- peptide has biological activity and inhibits muscle in the Results and Discussion large parasitic nematode, Ascaris suum [11], providing further weight to the contention that the paralytic action Emodepside belongs to a new class of drugs, the of emodepside does not derive from pore-forming capa- cyclooctadepsipeptides [6] (Figure S1 in the Supple- bility. mental Data available with this article online). These The studies with FM4-64 indicate emodepside may compounds bind to nematode latrophilin-like receptors inhibit the pharynx by stimulation of neurotransmitter [3]. Emodepside causes paralysis in nematodes, consis- release. To test for this, we made intracellular recordings from pharyngeal muscle and monitored effects on mem- *Correspondence: [email protected] brane potential. Emodepside (100 nM) elicited a slow Latrophilin Action through G␣q, PLC-␤ and UNC-13 1375

Figure 1. The Effect of Emodepside on Exo- cytosis from C. elegans Pharyngeal Neurons Pharynxes, with their embedded enteric neurons, were dissected from a strain with pan- neuronal GFP expression (NW1229); and loaded with FM4-64 [37]. The pharynxes were incubated with 5 ␮M FM4-64 in the presence of high potassium Dent’s (mM: 144 NaCl, 10

MgCl2, 1 CaCl2 25 KCl, and 5 Hepes, [pH 7.4]) for 5 min followed by a wash in 6 mM KCl Dent’s. (A) Diagram showing the position of the isthmus within the C. elegans pharynx (dotted rectangle) where FM4-64 labeling was im- aged. Images represent Z-series of 50 slices, total depth 23.9 ␮m. (B) Colocalization of FM4-64 staining with GFP labeled neurons (left), FM4-64 staining (middle), and merged image (right). Yellow indicates colocalization of FM4-64 and GFP. (C) FM4-64 staining following loading in high potassium (which marks regions with high vesicle recycling prior to drug addition, left). FM4-64 staining following 10 min addition 0.1% ethanol vehicle (middle). FM4-64 staining following 10 min 100 nM emodepside addition (right). (D) The same preparation as in (C), showing the overlay of red and green fluorescence. This confirms that, despite some movement of the pharynx during the experiment, there is loss of red FM4-64 fluorescence from the GFP labeled neuronal regions. Prolonged application of emodepside (Ͼ 1 hr) did not lead to a disruption in the integrity of the dissected pharynx or associated enteric neurons. Furthermore, in another series of experiments we noted that primary cultures of GFP expressing C. elegans neurons maintained their integrity in the continued presence of emodepside (100 nM for 24 hr; data not shown). These observations support the conclusion that emodepside does not cause physical disruption of biological membranes. depolarization (5.8 Ϯ 2.6 mV; n ϭ 4) and inhibited potent effect of emodepside on C. elegans pharynx in- pharyngeal action potential generation (Figure 2C). Glu- volves latrophilin. To achieve this, we tested the effect tamate depolarizes the pharynx [13]; however, a putative of emodepside on a putative null gene deletion mutant, null allele for the glutamate receptor, avr-15(ad1051) lat-1(ok379), and on animals treated with double- [14], was not resistant to emodepside (n ϭ 10; data stranded RNA (dsRNA) for lat-1 to induce RNA interfer- not shown). Therefore, it is unlikely that the action of ence (RNAi). Interestingly, both the lat-1 knockout and emodepside requires glutamate release alone. Perhaps lat-1 RNAi animals had a prolonged pharyngeal pump this is not surprising, because the pharyngeal nervous duration compared to wild-type. For lat-1(ok379), this system expresses a wide range of neurotransmitters was 220 Ϯ 19 ms (n ϭ 11), and for lat-1 RNAi, this was that have effects on the pharyngeal muscle [10], any or 225 Ϯ 23 ms (n ϭ 21) compared with a wild-type dura- all of which could be released by an LTX-like action of tion of 162 Ϯ 16 ms [n ϭ 22; p Ͻ 0.05 with respect to emodepside. We conclude that the effect of emodep- lat-1(ok379); Figure 3A] and a duration of 180 Ϯ 13 ms side on pharyngeal membrane potential is likely to be (n ϭ 17) in the RNAi control (zero IPTG control; p Ͻ 0.05 the net result of release of multiple neurotransmitters. with respect to lat-1 RNAi). Extended pharyngeal pump Previous studies have shown that emodepside inter- duration is also observed in C. elegans mutants with acts with nematode latrophilin [3]. There are two candi- defective neurotransmission [16], although the effect date latrophilins in C. elegans through which emodep- observed in the lat-1 knockouts was not as marked. side may exert its effect, lat-1 (B0457.1) and lat-2 These observations are consistent with the idea that (B0286.2). Both lat-1 and lat-2 encode seven transmem- latrophilin has a physiological role in facilitating neuro- brane domain, Family 2, G protein-coupled receptors transmitter release. Support for this also comes from a with characteristics of latrophilins, including large ex- study in Drosophila showing that mutations in the G tracellular and intracellular domains, a lectin-like se- protein-coupled receptor Methuselah (Mth), related to quence, a G protein proteolysis site domain (GPS), and latrophilin, results in a 50% decrease in evoked transmit- a short cysteine-rich sequence [15] (Figure S2). LAT-1 ter release [17]. We also observed that lat-1(ok379) were has 22, 23, and 21% amino acid identities to rat, bovine, constipated, similar to an earlier report that RNAi for and human latrophilin, respectively. LAT-1 and LAT-2 lat-1 has this effect [18]. Importantly, these observations have 21% amino acid identity with each other. are indicative of physiological regulation of latrophilin We performed experiments to determine whether the by an endogenous ligand. Current Biology 1376

The pharynxes of lat-1(ok379) animals were resistant to emodepside and continued to pump at concentrations up to 100 nM emodepside (Table 1; Figure 3B). This is corroborated by a similar observation for lat-1 RNAi (Table 1; Figure 3B). In contrast, emodepside was still effective at inhibiting locomotion in the lat-1 knockout, with a potency indistinguishable from wild-type (data not shown). This suggests that some other receptor, perhaps lat-2, may be involved in mediating the inhibitory action of emodepside on locomotion. In support of this, dsRNA targeted at lat-2 and a lat-2 gene deletion mutant were both resistant to the effects of emodepside on locomotion (data not shown). Overall, the data support the conclusion that in C. elegans pharynx, LAT-1 plays a pivotal role in the high- affinity effect of emodepside. We found support for this when examining the expression pattern of lat-1, by using a lat-1::DsRed2 reporter construct. In larval stages, expression was most evident in the pharyngeal muscle, consistent with earlier reports that used immunostaining [6] (Figures 3C and 3D). There may also have been neuronal expression in the larva, but this was hard to discern in the pharynx region. However, in adults, lat-1 expression appeared to be lost from the muscle but was expressed in anterior neurons, both extra-pharyngeal and pharyngeal, including one neuron in the terminal bulb and neurons of the corpus, with projections into the isthmus (Figures 3E and 3F). Notably, in the pharynx of adult animals, which are most susceptible to emodepside, lat-1 is expressed in neurons rather than muscle. These observations, derived from using pharyngeal activity in C. elegans to determine the effect of emodepside, are consistent with the idea that it acts via a neuronal latrophilin-signaling pathway. We gained further insight into this pathway by screening for emodepside resistance in several strains with mutations in genes encoding candidate effector molecules. As latrophilin copurifies with Gq␣ and Go␣ in mammalian cells [19], Figure 2. The Effect of Emodepside on Pharyngeal Pumping we first tested emodepside in animals with mutations Extracellular recordings of the pharyngeal muscle (EPG) were made in genes encoding these G proteins. A C. elegans mu- as described in [9]. Individual worms were placed in a Petri dish tant, egl-30(ad806), which has a reduction-of-function containing modified Dent’s saline (mM: 144 NaCl, 10 MgCl2, 1 CaCl2 ␣ 6 KCl, and 5 Hepes [pH 7.4]). A razor blade was used to cut the mutation in the Gq protein [20], was emodepside resis- worm just posterior to the pharynx, which caused the cuticle to tant (Table 1; Figure 4A), whereas a gain-of-function retract, exposing the isthmus and terminal bulb. This semi-intact mutant, egl-30(tg26) [21], was hypersensitive (Table 1; worm preparation consisted of the pharynx, the nerve ring, and the Figure 4B). A Go␣ reduction-of-function mutant [22], enteric nervous system. goa-1(n1134), also showed hypersensitivity to emodep- (A) Each vertical line represents the electrical activity associated side (Table 1; Figure 4B). In this context, it is interesting with a single muscle pump; therefore, this provides a readout of the activity of the muscle. The protocol involved applying 5-HT for 2 to note that at the body wall neuromuscular junction in min, to stimulate pumping, followed by a 10 min application of either C. elegans,Go␣ acts antagonistically to Gq␣ [23, 24] with vehicle control (0.1% ethanol) or emodepside, and lastly, a second the latter facilitating neurotransmitter release [25, 26]. application of 5-HT for 2 min. The top trace shows a typical result Gq␣ couples to the signaling molecule PLC-␤, which from a control experiment in which the pharynx continues to pump is a key modulator of pathways that regulate vesicular- throughout the entire time-course of the experiment. The bottom mediated release in C. elegans [26, 23]. To further track trace shows the effect of 100 nM emodepside on pharyngeal pumping. Note the disappearance of the pumps during the period of the mechanism of action of emodepside, we utilized two emodepside application and the failure of the muscle to respond C. elegans mutants, egl-8(md1971) and egl-8(n488), that to the second application of 5-HT. encode PLC-␤ [23]. These two alleles have disruptions (B) Concentration-response curve for the effect of varying concentrations of emodepside on pharyngeal pumping rates in wild-type C. elegans. “% control response” is the rate of pumping during the second application of 5-HT (i.e., following emodepside application) as ing was stimulated by the addition of 5-HT (500 nM), and the bar a percentage of pumping rate elicited by the first application of 5-HT. indicates the duration of application of emodepside. Subsequent (C) The effect of emodepside on the membrane potential was deter- application of 5-HT failed to elicit pumping (not shown). Note the mined by making intracellular recordings from the terminal bulb, as small depolarizing shift from baseline membrane potential (indicated described in [13]. The trace shows an example. Pharyngeal pump- by dotted line) upon application of emodepside. Latrophilin Action through G␣q, PLC-␤ and UNC-13 1377

Figure 3. Latrophilin Signaling in the Pharynx (A) lat-1(ok379) has a pharyngeal phenotype. The traces show an electropharyngeogram (EPG) [9] recorded from a wild-type and lat- 1(ok379) animal. Each recording represents a single muscle pump, with the first upward deflection corresponding to contraction and the last downward deflection corresponding to relaxation. The small potentials that appear during the pump have been associated with neuronal activity in the pharyngeal neuron M3 [38]. (B) The role of LAT-1 in the inhibitory effect of emodepside on the pharynx. Assays for pharyngeal activity were performed as for Figure 2A. The lat-1 control group for RNAi was rrf-3(pk1426) [36] fed on bacteria trans- formed with vectors for dsRNA but in the absence of IPTG so that the expression of dsRNA was not induced. The graphs show concentration-response curves for the effect of emodepside on pharyngeal pumping in wild-type

with an IC50 of 4.1 nM (95% confidence limits

1.2 to 14.7 nM); lat-1(ok379) with an IC50 of 53 nM (95% confidence limits 26 to 109 nM);

rrf-3(pk1426) (dsRNA control) with an IC50 of 2.9 nM (95% confidence limits 0.3 to 10 nM); and lat-1 RNAi with an IC50 of 70 nM (95% confidence limits 22 to 219 nM). Each point is the mean Ϯ S.E.M. of “n” determinations. *p Ͻ 0.05 and **p Ͻ 0.01 with respect to control. (C) The expression pattern of lat-1 was determined using a lat-1::DsRed2 reporter construct for expression of DsRed2 fused to the N-terminal domain (51 amino acids) of LAT-1. Expression was only observed in the anterior region of the animal. (C) is a fluorescence image from the anterior region of L3 stage larval animals. (D) A fluorescence image from L3 superimposed on a bright field background. Note the distinctive labeling in the pharyngeal muscle (m). (E) and (F) show images from the anterior of adult animals. (F) is a dissected pharynx showing a neuronal pattern of expression. One neuron is marked “n” as an example. Confocal images were obtained on a Zeiss Axioskop 2 LSM 510 Meta laser scanning microscope consisting of a Z-series of 24 sections, total depth 22.7 ␮m. in the catalytic Y domain of the enzyme. The pharynxes PLC-␤ hydrolyses phosphatidylinositolbisphosphate of these animals have a significantly reduced sensitivity to generate inositol 1,4,5-trisphosphate (IP3) and diacyl- to emodepside (Table 1; Figure 4C). This supports a glycerol (DAG). Either or both IP3 and DAG may be signaling pathway for emodepside through Gq␣ and involved in the downstream signaling from PLC-␤.In PLC-␤. The relative reduction in sensitivity to emodep- mammals, IP3 is implicated in LTX receptor-mediated side was similar for the lat-1 knockout, the Gq␣ reduc- activation of neurotransmitter release [27]. However, in tion of function mutant, and the PLC-␤ mutant (Table C. elegans, IP3 receptors have a very limited expres- 1), consistent with the hypothesis that they are in the sion pattern in the pharyngeal nervous system [28], sug- same signaling pathway for emodepside, which is nega- gesting a subsidiary role in neuronal signaling. Alterna- tively regulated by Go␣ (Figure S3). tively, there is extensive evidence from the stimulatory

Table 1. A Summary of the Effect of Emodepside on the Pharyngeal Pumping Frequency of Wild-Type and Mutant C. elegans % Inhibition with 10 nM

Strain Emodepside IC50 nM emodepside egl-30(tg26) 0.2 92 Ϯ 3 (5)* goa-1(n1134) 0.4 85 Ϯ 5 (5)* rrf-3(pk1426) 2.9 75 Ϯ 7 (5) wild-type 4.1 60 Ϯ 8 (5) egl-8(n488) 59 18 Ϯ 15 (9)* egl-8(md1971) 79 4 Ϯ 4 (6)*** snb-1(md247) 123 1 Ϯ 8 (5)* lat-1 RNAi 70 11 Ϯ 23 (5)** lat-1(ok379) 53 8 Ϯ 8 (5)** egl-30(ad806) 78 0 Ϯ 9 (5)** unc-13(e1091) 203 12 Ϯ 27 (5)* unc-13(s69) 360 20 Ϯ 20 (5)*

The mutants tested are listed in the left-hand column. The assay used is the same as that described in the legend for Figure 2A. Emodepside was tested at five concentrations and each concentration was tested between 2 and 22 times (see Figure 4). These data were pooled to construct inhibition curves, fitted to the modified logistic equation (GraphPad Prism, San Diego) from which IC50 values (the concentration of emodepside that caused a 50% reduction in pharyngeal pumping frequency) were estimated. The right-hand column gives the % inhibition observed at 10 nM emodepside (Ϯ S.E.M.; n; *p Ͻ 0.05, **p Ͻ 0.01; ***p Ͻ 0.001 with respect to wild-type or rrf-3(pk1426), as appropriate). Current Biology 1378

Figure 4. The Role of G Proteins, Phospholi- paseC-␤, and Synaptic Proteins in the Action of Emodepside (A) Concentration-response curves showing the effect of emodepside on pharyngeal pumping in an egl-30 reduction of function

mutant (ad806;IC50 ϭ 78 nM, 95% confidence limits 7.4 to 829 nM). (B) Concentration-response curves showing the hypersensitivity of the gain of function

mutant egl-30 (tg26;IC50 ϭ 0.2 nM, 95% confi-

dence limits 0.05 to 0.9 nM; control IC50 ϭ 4.1 nM, 95% confidence limits 1.2 to 14.7 nM)

and goa-1 (reduction of function n1134;IC50 ϭ 0.4 nM, 95% confidence limits 0.1 to 1.4 nM;

control IC50 ϭ 4.1 nM 95% confidence limits 1.2 to 14.7 nM) to emodepside. (C) Concentration-response curves showing the effect of emodepside on pharyngeal pumping on egl-8 (md1971 reduction of func-

tion; IC50 ϭ 79 nM, 95% confidence limits 32

to 192 nM; control IC50 ϭ 4.1 nM 95% confidence limits 1.2 to 14.7 nM). Dashed line shows results for the effect of emodepside on pharyngeal pumping on egl-8 (n488; re-

duction of function; IC50 ϭ 59 nM, 95% confidence limits 23 to 147 nM).

(D) Concentration-response curves showing the effect of emodepside on pharyngeal pumping of unc-13 (reduction of function; e1091;IC50 ϭ

203 nM, 95% confidence limits 28 nM to 1454 nM; control IC50 ϭ 4 nM, 95% confidence limits 1.7 to 9.8 nM), unc-13 (s69, severe reduction of function; IC50 ϭ 360 nM, 95% confidence limits 68 to 1905 nM) and snb-1 (reduction of function; md247;IC50 ϭ 123 nM, 95% confidence limits 33 to 460 nM). Each point is the mean Ϯ S.E.M. of (n) determinations. *p Ͻ 0.05, **p Ͻ 0.01, and ***p Ͻ 0.001, with respect to wild-type. action of ␤-phorbol esters on synaptic transmission that In conclusion, our results reinforce the functional sig- implicates DAG in the regulation of neurotransmitter re- nificance of latrophilin in the regulation of neurotransmit- lease [26, 29]. A major presynaptic DAG receptor is ter release [17]. Furthermore, we have linked latrophilin (m)UNC-13 [30]. (m)UNC-13 is an active-zone-specific to a signaling pathway involving Gq␣ and PLC-␤, which plasma membrane-associated protein essential for syn- requires UNC-13 in order to facilitate vesicle-mediated aptic vesicle-mediated neurotransmitter release in Dro- release of neurotransmitter (Figure S3). This provides sophila, mammals, and C. elegans [4, 5, 25]. In C. ele- further insight into the receptor-mediated modulation of gans, UNC-13 is regulated by DAG via a pathway transmission via nerve-terminal signaling cascades. In involving cholinergic and serotonergic control of motor addition, the possibility to direct small molecular weight neurons [26, 31]. Furthermore, it has been shown that compounds at latrophilin might provide potential thera- a mouse double knockout for (m)UNC-13-1 and (m)UNC- peutic routes to target this pathway in the treatment of 13-2 is resistant to LTX [32]. To determine whether UNC- disease. 13 may be required for the action of emodepside, we Details of Experimental Procedures are provided in looked at two reduction-of-function alleles, unc- the Supplemental Data. 13(e1091) and the more severe unc-13(s69) [33]. Both were emodepside resistant. Notably, unc-13(s69) was Supplemental Data the only mutant that continued to pump strongly in the Supplemental Data including Experimental Procedures and four ad- ␮ ditional figures are available at http://www.current-biology.com/cgi/ presence of 100 nM and 1 M emodepside (Table 1; content/full/14/15/1374/DC1/. Figures 4D and S4) and is approximately five times more resistant than the lat-1 knockout. This indicates that Acknowledgments high concentrations of emodepside, i.e., greater than 100 nM, exert an action on neurotransmitter release that We are grateful to the following: J. Dent provided avr15(ad1051),J. is likely to be independent of the LAT-1 pathway. This Culotti provided NW1229, K. Iwasaki provided egl-30(tg26), L. Sega- lat provided goa-1 (n1134), and A. Rose provided unc-13 (s69). We may not be too dissimilar to the situation observed with are grateful to R. Barstead and the C. elegans gene knockout con- LTX, which can act through different mechanisms de- sortium for providing lat-1 (ok379). All other strains were provided pending on the toxin concentration [1]. Activation of by the C. elegans Genetics Center. K.A. is sponsored jointly by the UNC-13 leads to facilitation of vesicle-mediated neuro- Biotechnology and Biological Sciences Research Council, UK, and transmitter release by promoting the open configuration Bayer AG. of syntaxin [34] ready to engage the SNARE proteins Received: October 14, 2003 synaptobrevin, syntaxin, and SNAP-25 [35]. Synapto- Revised: June 8, 2004 brevin hypomorphic C. elegans mutants snb-1(md-247) Accepted: June 10, 2004 [16] also exhibited reduced sensitivity to emodepside Published: August 10, 2004 (Table 1; Figure 4D). Overall, these data on animals with mutations in synaptic proteins confirm the involvement References of the neurotransmitter release pathway in the action of 1. Sudhof, T.C. (2001). alpha-Latrotoxin and its receptors: neurex- emodepside. ins and CIRL/latrophilins. Annu. Rev. Neurosci. 24, 933–962. Latrophilin Action through G␣q, PLC-␤ and UNC-13 1379

2. Volynski, K.E., Capogna, M., Ashton, A.C., Thomson, D., Orlova, G(q)␣ gene disrupt movement, egg-laying, and viability. Neuron E.V., Manser, C.F., Ribchester, R.R., and Ushkaryov, Y.A. (2003). 16, 999–1009. Mutant alpha latrotoxin (LTXN4C) does not form pores and causes 21. Doi, M., and Iwasaki, K. (2002). Regulation of retrograde signal- secretion by receptor stimulation. This action does not require ing at neuromuscular junctions by the novel C2 domain protein neurexins. J. Biol. Chem. 278, 31058–31066. AEX-1. Neuron 33, 249–259. 3. Saeger, B., Schmitt-Wrede, H.P., Dehnhardt, M., Benten, W.P., 22. Segalat, L., Elkes, D.A., and Kaplan, J.M. (1995). Modulation of Krucken, J., Harder, A., Von Samson-Himmelstjerna, G., Wie- serotonin-controlled behaviors by G(o) in Caenorhabditis ele- gand, H., and Wunderlich, F. (2001). Latrophilin-like receptor gans. Science 267, 1648–1651. from the parasitic nematode Haemonchus contortus as a target 23. Miller, K.G., Emerson, M.D., and Rand, J.B. (1999). Goalpha and for the anthelmintic depsipeptide PF1022A. FASEB J. 15, 1332– diacylglycerol kinase negatively regulate the Gq␣ pathway in 1334. C. elegans. Neuron 24, 323–333. 4. Aravamudan, B., Fergestad, T., Davis, W.S., Rodessch, C.K., 24. Hadju-Cronin, Y.M., Chen, W.J., Patikoglou, G., Koelle, M.R., and Broadie, K. (1999). Drosophila UNC-13 is essential for syn- and Sternberg, P.W. (1999). Antagonism between Go␣ and Gq␣ aptic transmission. Nat. Neurosci. 2, 965–971. in Caenorhabditis elegans: the RGS protein EAT-16 is necessary 5. Augustin, I., Rosenmund, C., Sudhof, T.C., and Brose, N. (1999). for Go␣ signaling and regulates Gq␣ actvity. Genes Dev. 13, Munc13–1 is essential for fusion competence of glutamatergic 1780–1793. synaptic vesicles. Nature 40, 457–461. 25. Miller, K.G., Alfonso, A., Nguyen, M., Crowell, J.A., Johnson, 6. Harder, A., Schmitt-Wrede, H.P., Krucken, J., Marinowski, P., C.D., and Rand, J.B. (1996). A genetic selection for Caenorhab- Wunderlich, F., Willson, J., Amliwala, K., Holden-Dye, L., and ditis elegans synaptic transmission mutants. Proc. Natl. Acad. Walker, R.J. (2003). Cyclooctadepsipeptides—an anthelminti- Sci. USA 93, 12593–12598. cally active class of compounds exhibiting a novel mode of 26. Lackner, M.R., Nurrish, S.J., and Kaplan, J.M. (1999). Facilitation action. Int. J. Antimicrob. Agents 22, 318–331. of synaptic transmission by EGL-30 Gqalpha and EGL-8 PLC-␤: 7. Willson, J., Amliwala, K., Harder, A., Holden-Dye, L., and Walker, DAG binding UNC-13 is required to stimulate acetylcholine re- R.J. (2003). The effect of the anthelmintic emodepside at the lease. Neuron 24, 335–346. neuromuscular junction of the parasitic nematode Ascaris 27. Capogna, M., Volynski, K.E., Emptage, N.J., and Ushkaryov, suum. Parasitology 126, 79–86. Y.A. (2003). The alpha-latrotoxin mutant LTXN4C enhances 8. Betz, W.J., Mao, F., and Bewick, G.S. (1992). Activity-dependent spontaneous and evoked transmitter release in CA3 pyramidal fluorescent staining and destaining of living vertebrate motor neurons. J. Neurosci. 23, 4044–4053. nerve terminals. J. Neurosci. 12, 363–375. 28. Baylis, H.A., Furuichi, T., Yoshikawa, F., Mikoshiba, K., and Sat- 9. Avery, L., Raizen, D., and Lockery, S. (1995). Electrophysiologi- telle, D.B. (1999). Inositol 1,4,5-triphosphate receptors are cal methods. Methods Cell Biol. 48, 251–269. strongly expressed in the nervous system, pharynx, intestine, 10. Rogers, C.M., Franks, C.J., Walker, R.J., Burke, J.F., and gonad and excretory cell of Caenorhabditis elegans and are Holden-Dye, L. (2001). Regulation of the pharynx of Caenorhab- encoded by a single gene (itr-1). J. Mol. Biol. 294, 467–476. ditis elegans by 5-HT, octopamine and FMRFamide-like neuro- 29. Majewski, H., and Iannazzo, L. (1998). Protein kinase C: a physi- peptides. J. Neurobiol. 49, 235–244. ological mediator of enhanced transmitter output. Prog. Neuro- 11. Gessner, G., Meder, S., Rink, T., Boheim, G., Harder, A., biol. 55, 463–475. Jeschke, P., Scherkenbeck, J., and Londerhausen, M. (1996). 30. Rhee, J.S., Betz, A., Pyott, S., Reim, K., Varoqueaux, F., Au- Ionophore and anthelmintic activity of PF1022A, a cyclooctade- gustin, I., Hesse, D., Sudhof, T.C., Takahashi, M., Rosenmund, psipeptide, are not related. Pest. Sci. 48, 399–407. C., and Brose, N. (2002). ␤-phorbol ester- and diacylglycerol- 12. Renterghem, C., Iborra, CM.-MN., Lelianova, V., Ushkaryov, Y., induced augmentation of transmitter release is mediated by and Seagar, M. (2000). Alpha-latrotoxin forms calcium-perme- Munc13s and not by PKCs. Cell 108, 121–133. able membrane pores via interactions with latrophilin or neu- 31. Nurrish, S., Segalat, L., and Kaplan, J. (1999). Serotonin inhibi- rexin. Eur. J. Neurosci. 12, 3953–3962. tion of synaptic transmission: G␣o decreases the abundance 13. Pemberton, D., Franks, C., Walker, R., and Holden-Dye, L. of UNC-13 at release sites. Neuron 24, 231–242. (2001). Characterisation of glutamate-gated chloride channels 32. Varoqueaux, F., Sigler, A., Rhee, J.-S., Brose, N., Enk, C., Reim, in the pharynx of wild-type and mutant Caenorhabditis elegans K., and Rosenmund, C. (2002). Total arrest of spontaneous and delineates the role of the subunit GluCl-␣2 in the function of evoked synaptic transmission but normal synaptogenesis in the the native receptor. Mol. Pharmacol. 59, 1037–1043. absence of Munc13-mediated vesicle priming. Proc. Natl. Acad. 14. Dent, J.A., Davis, M.W., and Avery, L. (1997). avr-15 encodes a Sci. USA 99, 9037–9042. chloride channel subunit that mediates inhibitory glutamatergic 33. Kohn, R.E., Duerr, J.S., McManus, J.R., Duke, A., Rakow, T.L., neurotransmission and ivermectin sensitivity in Caenorhabditis Maruyama, H., Moulder, G., Maruyama, I.N., Barstead, R.J., and elegans. EMBO J. 16, 5867–5879. Rand, J.B. (2000). Expression of multiple UNC-13 proteins in 15. Lelianova, V.G., Davletov, B.A., Sterling, A., Rahman, M.A., Gri- the Caenorhabditis elegans nervous system. Mol. Biol. Cell 11, shin, E.V., Totty, N.F., and Ushkaryov, Y.A. (1997). Alpha-latro- 3441–3452. toxin receptor, latrophilin, is a novel member of the secretin 34. Richmond, J.E., Wehner, R.M., and Jorgensen, E.M. (2001). An family of G protein-coupled receptors. J. Biol. Chem. 272, open form of syntaxin bypasses the requirement for UNC-13 in 21504–21508. vesicle priming. Nature 412, 338–341. 16. Nonet, M.L., Saifee, O., Zhao, H.J., Rand, J.B., and Wei, L.P. 35. Sollner, T., Bennett, M.K., Whiteheart, S.W., Scheller, R.H., and (1998). Synaptic transmission deficits in Caenorhabditis elegans Rothman, J.E. (1993). A protein assembly-disassembly pathway synaptobrevin mutants. J. Neurosci. 18, 70–80. in vitro that may correspond to sequential steps of synaptic 17. Song, W., Ranjan, R., Dawson-Scully, K., Bronk, P., Marin, L., vesicle docking, activation and fusion. Cell 75, 409–418. Seroude, L., et al. (2002). Presynaptic regulation of neurotrans- 36. Simmer, F., Tijsterman, M.P., Parrish, S., Koushika, S., Nonet, mission in Drosophila by the G protein-coupled receptor Methu- M., Fire, A., Ahringer, J., and Plasterk, R. (2002). Loss of the selah. Neuron 36, 105–119. putative RNA-directed RNA polymerase RRF-3 makes 18. Mee, C.J., Tomlinson, S.R., Perestenko, P.V., Pomerai, D., Duce, C. elegans hypersensitive to RNAi. Curr. Biol. 12, 1317–1319. I.R., Usherwood, P.N., and Bell, D.R. (2004). Latrophilin is re- 37. Kay, A.R., Alfonso, A., Alford, S., Cline, H.T., Holgado, M., Sak- quired for toxicity of black widow spider venom in Caenorhab- mann, B., Snitsarev, V.A., Stricker, T.P., Takahashi, M., and Wu, ditis elegans. Biochem. J. 378, 185–191. L.G. (1999). Imaging synaptic activity in intact brain and slices 19. Rahman, M.A., Ashton, A.C., Meunier, F.A., Davletov, B.A., Dolly, with FM1–43 in C. elegans, lamprey and rat. Neuron 24, 809–817. J.O., and Ushkaryov, Y.A. (1999). Norepinephrine exocytosis 38. Avery, L. (1993). Motor-neuron M3 controls pharyngeal muscle- stimulated by alpha-latrotoxin requires both external and stored relaxation timing in Caenorhabditis elegans. J. Exp. Biol. 175, calcium and is mediated by latrophilin, G proteins and phospho- 283–297. lipase C. Phil. Trans. Roy. Soc. Lond., B 354, 379–386. 20. Brundage, L., Avery, L., Katz, A., Kim, U.J., Mendel, J.E., Stern- berg, P.W., and Simon, M.I. (1996). Mutations in a C. elegans