Syntaxin-1A binds the nucleotide-binding folds of sulphonylurea receptor 1 to regulate the KATP channel.

ATP-sensitive potassium (KATP) channels in neuron and neuroendocrine cells consist of a pore-forming Kir6.2 and regulatory sulfonylurea receptor (SUR1) subunits, which are regulated by ATP and ADP. SNARE protein syntaxin 1A (Syn-1A) is known to mediate exocytic fusion, and more recently, to also bind and modulate membrane-repolarizing voltage-gated K+ channels. Here we show that Syn-1A acts as an endogenous regulator of KATP channels capable of closing these channels when cytosolic ATP concentrations were lowered. Botulinum neurotoxin C1 cleavage of endogenous Syn-1A in insulinoma HIT-T15 cells resulted in the increase in KATP currents, which could be subsequently inhibited by recombinant Syn-1A. Whereas Syn-1A binds both nucleotide-binding folds (NBF-1 and NBF-2) of SUR1, the functional inhibition of KATP channels in rat islet beta-cells by Syn-1A seems to be mediated primarily by its interactions with NBF-1. These inhibitory actions of Syn-1A can be reversed by physiologic concentrations of ADP and by diazoxide. Syn-1A therefore acts to fine-tune the regulation of KATP channels during dynamic changes in cytosolic ATP and ADP concentrations. These actions of Syn-1A on KATP channels contribute to the role of Syn-1A in coordinating the sequence of ionic and exocytic events leading to secretion.

cells by treatment with 0.015% trypsin in Ca 2ϩ -and Mg 2ϩ -free phosphate-buffered saline as described previously (14). Islet cells were plated on glass coverslips in 35-mm dishes and cultured in 2.8 mM glucose (with 7.5% fetal calf serum, 0.25% sodium, 100 g/ml streptomycin). For intracellular dialysis experiments, islet cells were cultured for 1-2 days before electrophysiological recordings.
Electrophysiology-Single islet ␤-cells and HIT-T15 cells were studied with the standard whole-cell patch voltage clamp technique as reported previously (14,18). Thin-walled (1.5 mm) borosilicate glass tubes were pulled with a two-stage Narishige (Tokyo, Japan) micropipette puller and heat-polished. The typical tip resistance was 2-4 megaohms. The external solution contained 140 mM NaCl, 4 mM KCl, 1 mM MgCl 2 , 2 mM CaCl 2 , 10 mM HEPES, pH 7.3, and as indicated, glybenclamide or diazoxide. The internal patch pipette solution contained 140 mM KCl, 1 mM MgCl 2 , 10 mM HEPES, 1 mM EGTA, and the indicated amounts of MgATP and MgADP; pH 7.3. The indicated recombinant proteins were added to the intracellular solution for dialysis into ␤-cells via the patch pipette. All electrophysiological experiments were done at room temperature (22-24°C). Currents were elicited with 250-ms voltage steps of 20 mV from Ϫ140 mV to Ϫ20 mV from a holding potential of Ϫ50 mV using an EPC-9 amplifier and Pulse software (HEKA Electronik, Lambrecht, Germany). Data were presented as mean Ϯ S.E. Data were compared by Student's t test.
Immunoprecipitation Assays-Protein A-sephorose beads (40 l; 50% slurry, Sigma) were washed with saline (pH 7.4) and incubated with 3 g of rabbit anti-Syn-1A antibody in 200 l of immunoprecipitation (IP) buffer (25 mM HEPES, pH 7.4, 100 mM KCl, 2 mM EDTA, 2% Triton X-100, 20 M NaF, 1 mM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, 10 g/ml aprotinin) at 4°C for 1 h followed by washing twice with IP buffer. 200 l of IP buffer and 500 pmol of recombinant Syn-1A were then added and incubated at 4°C for 1 h followed by another wash. The immunoprecipitated Syn-1A was incubated with GST (negative control), GST-NBF-1, or GST-NBF-2 (500 pmol) in 200 l of IP buffer at 4°C for 2 h, and then washed three times. The co-precipitated proteins were separated on 15% PAGE, and GST-NBF proteins were identified on the gel by Coomassie Blue staining.
Confocal Microscopy-As described previously (18), HIT-T15 cells were plated on polylysine-coated coverslips, transfected as described above, and then fixed in 2% formaldehyde for 0.5 h at room temperature followed by blocking with 5% normal goat serum (0.1% saponin, 0.5 h, room temperature) and then immunolabeled with primary antibodies (1:50, overnight, 4°C) against Syn-1 (Sigma) or SNAP-25 (Sternberger Monoclonal, Lutherville, MD). After rinsing with 0.1% saponin/ phosphate-buffered saline, the coverslips were incubated with the appropriate rhodamine-labeled secondary antiserum for 1 h, mounted on slides in a fading retarder (0.1% p-phenylenediamine in glycerol), and then examined by a laser scanning confocal imaging system (LSM510, Carl Zeiss, Oberkochen, Germany). Transfected cells were identified by visualization of the co-expressed EGFP.

Syntaxin-1A Inhibits K ATP Channel
Opening-Using the whole-cell patch clamp technique (14,18), we first identified the K ATP channels in the insulinoma HIT-T15 cells by using glybenclamide (Glyb), the sulfonylurea K ATP channel inhibitor. Glyb (0.1 M) greatly inhibited the HIT-T15 K ATP currents (Fig.  1, A and B) recorded at low ATP (0.3 mM) concentration (n ϭ 3). Dialysis of GST-Syn-1A (1 M) into the HIT-T15 cells also greatly inhibited the K ATP currents (Fig. 1, A and B; n ϭ 10). As a control, we used HIT-T15 cells dialyzed with GST (1 M), which had no effect on K ATP currents when compared with the pipette solution (data not shown). Fig. 1C shows the time course of GST-Syn-1A inhibitory effect on the K ATP currents under the low ATP (0.3 mM) concentration. Just after the formation of the whole-cell configuration (t ϭ 0 min), the K ATP currents remained low for ϳ2 min (asterisk) for both control and GST-Syn-1A-treated cells. After 2 min, control currents increased steadily as the cell interior equilibrated with the low ATP pipette solution so that the K ATP channels would open. At t ϭ 10 min, the control K ATP current amplitude exhibited a vigorous increase to 169.0 Ϯ 15.9 pA (n ϭ 5), whereas the current amplitude of the cells dialyzed with GST-Syn-1A showed only a small increase to 65.4 Ϯ 9.7 pA (n ϭ 5). Fig. 1D shows the summary bar graph in which the K ATP currents were normalized to cell capacitance (pA/pF) to eliminate the variations in cell size. Glyb (0.1 M) inhibited K ATP current amplitude by ϳ85% (n ϭ 3), whereas GST-Syn-1A (1 M) reduced K ATP currents by ϳ65% (n ϭ 5) of the control current (61.9 Ϯ 8.8 pA/pF; n ϭ 5).
To determine whether the effect of Syn-1A is at the plasma membrane or cytosolic level, we examined HIT-T15 cells overexpressing the full-length Syn-1A (aa 1-288) (20), which included the transmembrane domain, thereby specifically targeting Syn-1A to the plasma membrane (18). This Syn-1A overexpression (Ͼ3-fold by Western blot) exhibited very similar inhibition of the K ATP whole-cell currents (data not shown) as the dialysis of cytosolic Syn-1A (aa 1-266) shown in Fig. 1. These results indicate that the active Syn-1A domain that inhibits this K ATP channel involves only the cytoplasmic domain (aa 1-266) and not the transmembrane domain (aa 267-288) of Syn-1A.
We have postulated that the closed state of this K ATP channel is attributed not only to cytosolic ATP levels but possibly also to the levels of endogenous Syn-1A. To examine whether Syn-1A acts as an endogenous inhibitor of the K ATP channel, we expressed botulinum neurotoxin C1 (BoNT/C1) in HIT-T15 cells, which would specifically cleave the endogenous Syn-1A (21). Since HIT-T15 cells very reliably pick up multiple plasmids (Ͼ90%), we co-expressed GFP to identify the BoNT/C1-expressing cells (18). Confocal microscopy showed that the membrane Syn-1A signal in the BoNT/C1-expressing cells (GFP-containing, indicated by arrows) was greatly reduced (Fig. 1E, lower panels) when compared with the endogenous Syn-1A levels of the adjacent cells that did not express BoNT/C1. As the BoNT/ C1-expressing cells did not show an increase in the cytoslic Syn-1A signal, this suggests that the cleaved Syn-1A fragments likely undergo cytosolic proteolysis since the Syn-1A monoclonal antibody (Sigma) was generated against the full-length protein and would have recognized either the membrane-bound or the cytosolic fragments of Syn-1A. Since BoNT/C1 could also cleave SNAP-25 in some cell types (22), we also labeled these cells with anti-SNAP-25 antibody (Fig. 1E, upper panels) but saw no difference in the membrane SNAP-25 signals between the BoNT/C1-expressing cells (GFP-containing, indicated by arrows) and the cells that did not express GFP. This result indicates specific proteolysis of endogenous Syn-1A by BoNT/ C1. Next, we examined the whole-cell K ATP currents of these BoNT/C1-expressing cells (Fig. 1, A and B), which was ϳ155% (n ϭ 5) of the control cells (Fig. 1D), indicating that the K ATP currents were indeed being inhibited by the endogenous Syn-1A. Dialysis of GST-Syn-1A (1 M) into these BoNT/C1-expressing cells (Fig. 1A, BoNT/C1ϩSyn-1A) reduced the K ATP current by ϳ70% (n ϭ 8) as compared with the BoNT/C1-expressing cells, a reduction similar to the one observed with GST-Syn-1A

Syntaxin-1A Regulates K ATP Channels
in the control cells. The cytosolic ATP concentration in these cells was lowered to 0.3 mM ATP to reduce ATP blockade of the K ATP channel. The observed Syn-1A inhibition of the K ATP channels may be due to an increase in the sensitivity to ATPmediated inhibition, or it may be independent of ATP inhibition. More studies would be required to distinguish these possibilities.
Syntaxin 1A Binds SUR1 at its NBF-1 and NBF-2-Syn-1A actions on these K ATP channels could be mediated by binding to the SUR1 or Kir6.2 subunits. GST-Syn-1A bound to agarose beads pulled down SUR1 from solubilized rat brain synaptic membranes, which was identified with a specific antibody generated against the SUR1 C terminus (19) (Fig. 2A). The major cytoplasmic domains of SUR1 are NBF-1 (aa 696 -893) and C-terminal NBF-2 (aa 1358 -1544) (3). Fig. 2B shows that GST-NBF-1 and GST-NBF-2 bound to agarose beads pulled down native Syn-1A from rat brain synaptic membranes. To negate the possibility of a coprecipitation of unknown ternary proteins present in the brain, we demonstrated that GST-NBF-1 and GST-NBF-2 pulled down recombinant Syn-1A but not control GST or recombinant SNAP-25 (data not shown). Fig. 2C shows the reciprocal study of immunoprecipitation of Syn-1A (GST cleaved off by thrombin), which pulled down GST-NBF-1 (lane 3) and GST-NBF-2 (lane 5) and not GST (lane 1). The coprecipitated NBFs were identified by Coomassie Blue staining as the only proteins eluting at the same sizes as the control proteins (lanes 4 and 6). Syn-1A could inhibit the K ATP channels by acting directly on the cytoplasmic N or C termini of the Kir6.2 subunit on which the ATP acts (8,9). This is not the case since neither recombinant cytoplasmic N-(aa 1-70) nor C-terminal (aa 263-390) domains of Kir6.2 bound to agarose beads pulled down recombinant Syn-1A (data not shown). Furthermore, dialysis of these Kir6.2 domain proteins into rat islet ␤-cells did not influence GST-Syn-1A effects on K ATP activity (data not shown). Since SNAP-25 partners with Syn-1A to bind and regulate Ca 2ϩ channels (23), SNAP-25 might also directly modulate K ATP channels. However, GST-SNAP-25 (1 M) dialyzed into HIT-T15 cells (and rat islet ␤-cells) had no effect on K ATP activity (data not shown), and neither GST-NBF-1 nor GST-NBF-2 pulled down recombinant SNAP-25 (data not shown).
Syntaxin-1A Inhibition of the K ATP Channel Is Mediated by Its Binding to NBF-1-The binding of Syn-1A to NBF-1 and NBF-2 of SUR1 suggests that Syn-1A inhibition of the HIT-T15 K ATP channels may be mediated by its interactions with either one of the NBFs. Therefore, we examined the functional interactions of Syn-1A with each NBF using rat islet ␤-cells, which would be a better physiologic model. The rat islet ␤-cell K ATP channels (Fig. 3, A-C) behaved very similarly as the HIT-T15 cell K ATP channels (Fig. 1, A-D). Specifically, under low ATP Syntaxin-1A Regulates K ATP Channels inhibited the K ATP currents (Fig. 3A). The time course of GST-Syn-1A inhibition of rat islet ␤-cell K ATP channels (Fig. 3B) was in fact very similar in pattern to those observed in the HIT cell study (Fig. 1C). When normalized to cell capacitance (pA/pF; Fig. 3C), Glyb (0.1 M) and GST-Syn-1A (1 M) inhibited rat islet ␤-cell K ATP control currents (65.4 Ϯ 9.7 pA/pF; n ϭ 12) by 85% (9.7 Ϯ 1.7 pA/pF, n ϭ 3) and 61% (25.6 Ϯ 5.7 pA/pF, n ϭ 10), respectively. These values are remarkably similar to the HIT cell studies (Fig. 1).
Next we examined the effects of GST-NBF-1 and GST-NBF-2 on the inhibitory actions of Syn-1A (Fig. 3, A and C). GST-NBFs would be expected to bind the endogenous Syn-1A, which would either prevent the formation of or disrupt the complex already formed by endogenous Syn-1A with the ␤-cell SUR1, both of which would increase K ATP currents. Since GST-NBF-1 (1 M) and GST-NBF-2 (1 M) alone only slightly but not significantly increased ␤-cell K ATP currents to 77.8 Ϯ 14.6 pA/pF (n ϭ 6) and 72.6 Ϯ 16.3 pA/pF (n ϭ 8), respectively, this supports the latter possibility, that the pre-existing endogenous Syn-1A may have formed stable complexes with the NBFs of many of the endogenous SUR1 proteins, and these complexes may be resistant to disruption by the exogenous GST-NBFs. BoNT/C1 (Fig. 1) would disrupt such complexes upon cleavage of the endogenous Syn-1A. The exogenous NBFs could still pull down Syn-1A from the rat brain (as in Fig. 2A) since Syn-1A is more abundant and generally distributed in the brain than SUR1, and hence, an excess of brain Syn-1A would be available to bind the exogenous NBF proteins. More importantly, when dialyzed together with GST-Syn-1A (1 M), GST-NBF-1 (NBF-1ϩSyn-1A) com-pletely blocked the inhibitory effect of GST-Syn-1A on the K ATP currents (90.2 Ϯ 16.6 pA/pF; n ϭ 10) (Fig. 3C), which was not significantly different from NBF-1 alone. This is because GST-NBF-1 was premixed with GST-Syn-1A in the pipette solution, which would block the exogenous GST-Syn-1A from binding the free endogenous ␤-cell SUR1 to inhibit the K ATP channel. However, GST-NBF-2 did not prevent the inhibitory effects of GST-Syn-1A (NBF-2ϩSyn-1A), whose K ATP currents remained reduced at 31.9 Ϯ 7.4 pA/pF (n ϭ 4), a value close to the inhibition caused by GST-Syn-1A used alone. These results suggest that Syn-1A seems to interact functionally more with NBF-1 than with NBF-2 to inhibit the ␤-cell K ATP channels, at least under these experimental conditions. However, NBF-1 and NBF-2 exist as a heterodimer in the intact cell (1,5), which, when taken with our result in Fig. 2 showing that Syn-1A binds NBF-1 and NBF-2 with similar affinity, supports an important role of NBF-2, either by its direct interaction with Syn-1A and/or by its influence on NBF-1 interactions with Syn-1A.
ADP and Diazoxide Can Antagonize Syntaxin-1A Inhibition of K ATP Channels-Since ADP is the physiologic antagonist to ATP-mediated inhibition of the K ATP channels (25), we examined whether ADP could antagonize the inhibitory effect of Syn-1A on islet ␤-cell K ATP channels (Fig. 4, A and B). MgADP (0.3 mM ADP in the pipette solution with 0.3 mM ATP) elicited a ϳ135% increase (154.3 Ϯ 48.0 pA/pF, n ϭ 8) in K ATP currents from control values (65.4 Ϯ 9.7 pA/pF, n ϭ 12), consistent with a similar study performed on Xenopus oocytes (26). Additional ADP could also be further generated from the hydrolysis of the 0.3 mM ATP. K ATP current amplitude resulting from dialysis of FIG. 3. Syntaxin-1A inhibition of pancreatic islet ␤-cell K ATP channels is mediated by its binding to NBF-1 but not NBF-2. A, representative K ATP whole-cell currents under control conditions and after dialysis with GST-Syn-1A and NBF-1 or NBF-2. B, time course of K ATP currents (mean Ϯ S.E.) in control (n ϭ 12) and GST-Syn-1A treated (n ϭ 10) cells (at Ϫ120 mV). C, bar graph (mean Ϯ S.E.) of K ATP currents normalized to cell capacitance (pA/pF) in control (cont) cells (n ϭ 12) and in cells treated with glybenclamide (n ϭ 3), GST-Syn-1A (n ϭ 10), GST-NBF-1 (n ϭ 6), GST-NBF-1 plus GST-Syn-1A (n ϭ 10), GST-NBF2NBF-2 (n ϭ 8), and GST-NBF2NBF-2 plus GST-Syn-1A (n ϭ 4). Cells were stimulated from Ϫ140 to Ϫ20 mV from a holding potential of Ϫ50 mV. Currents were recorded 10 min after the formation of the whole-cell configuration with the pipette solution containing low (0.3 mM) ATP concentration. *, p Ͻ 0.05; NS, not significant. MgADP (0.3 mM) along with GST-Syn-1A (1 M) was reduced to 60.6 Ϯ 16.2 pA/pF (n ϭ 8), which is similar to the control currents. This is in contrast to a much more reduced K ATP current caused by Syn-1A, where cytosolic ADP levels were not raised (Fig. 3). These results indicate that Syn-1A reduced the sensitivity of the K ATP channel to opening by MgADP, or vice versa, that MgADP reduced the sensitivity of the K ATP channel to closing by Syn-1A. A possible explanation for these results is that ADP could displace ATP binding to NBF-1 (6,24). Even at 0.3 mM ATP, there is sufficient ATP to bind the NBFs since ATP has been shown to bind the NBFs at micromolar concentrations (6,24). Taken together, these results suggest that the Syn-1A interactions with the NBFs might include the binding to the Walker A and B motifs or at least be influenced by these domains (25,26).
The K ATP channel opener, diazoxide, opens K ATP channels even under high cytosolic ATP concentrations by its high affinity interaction with SUR1 (5,(25)(26)(27). In Fig. 4C (open bars) (n ϭ 4). The subsequent addition of diazoxide (200 M) to these cells (Fig. 4C, solid bars) opened the K ATP channels to the same extent either in the absence or in the presence of GST-Syn-1A (1 M), whether the cytosolic ATP concentration was high (3 mM ATP) or low (0.3 mM ATP). Specifically, at 3 mM ATP, diazoxide increased K ATP currents to 30.7 Ϯ 12.8 pA/pF (n ϭ 8) and 33.2 Ϯ 8.1 pA/pF (n ϭ 8) in the absence and in the presence of GST-Syn-1A, respectively. At 0.3 mM ATP, diazoxide caused the K ATP currents to increase to 91.8 Ϯ 10.8 pA/pF (n ϭ 3) and 80.3 Ϯ 12.9 pA/pF (n ϭ 4) in the absence and in the presence of GST-Syn-1A, respectively. Diazoxide can therefore completely prevent GST-Syn-1A inhibition of the ␤-cell SUR1/K ATP channel, but higher ATP concentrations could reduce this diazoxide effect. This effect of diazoxide to fully reverse the Syn-1A inhibition is contrasted to only by a reduced sensitivity of the Syn-1Ainhibited K ATP channels to ADP-mediated opening (Fig. 4, A  and B). DISCUSSION Our studies show that Syn-1A binds to the NBF-1 of neuroendocrine ␤-cell SUR1 (Fig. 1), which leads to the inhibition of the K ATP channel even when cytosolic concentrations of ATP are low. However, as cytosolic ATP concentrations rise, the inhibitory effect of Syn-1A is small relative to direct ATP inhibition of the Kir6.2 subunit (8,9). This inhibition of the K ATP channel by Syn-1A may reduce the efficacy of the rising cytosolic ADP concentrations (Fig. 4) 4) (iv). Cells were stimulated from Ϫ140 to Ϫ20 mV from a holding potential of Ϫ50 mV. Currents were measured at 10 min after the formation of the whole-cell configuration at Ϫ120 mV. *, p Ͻ 0.05; NS, not significant. possibility that Syn-1A does not simply inhibit the K ATP channel but may also provide a "braking" mechanism on the K ATP channel opening during dynamic changes of ATP and ADP concentrations. This regulation may be lost when Syn-1A levels are reduced, as is the case in a number of diseases (28 -31).
Our results showing the effects of BoNT/C1-induced reduction of HIT-T15 cell levels of Syn-1A on the K ATP currents (Fig.  1) may be of relevance to the pathogenesis of a number of neuro-and neuroendocrine diseases in which the cellular levels of Syn-1A are severely perturbed (28 -31). In Williams syndrome, a multisystem disorder, the observed hemizygous deletion of Syn-1A is believed to cause or at least contribute to the severe neurological symptoms (28). The frontal cortex of patients with schizophrenia and depression, whose cause of death was suicide, were noted to exhibit elevated Syn-1A levels as compared with patients who died of other causes (29). These reports suggest that abnormalities in Syn-1A and the complexes formed with its interacting proteins could provide the molecular substrates for abnormalities of neural connectivity (28,29). Our work suggests that the K ATP channel is one of these Syn-1A-interacting proteins, and through the Syn-1A-K ATP channel interactions, either an excess or a deficiency of Syn-1A would be expected to adversely alter membrane excitability. In the most common neuroendocrine disease, diabetes, Syn-1A levels in the islets of type-2 diabetes Goto-Kakizaki rats are severely reduced (30,31) and become even further reduced by prolonged exposure of the islets to high glucose concentrations (30). This could contribute to the loss of glucose sensitivity of the diabetic islet ␤-cells in the Goto-Kakizaki rat, and in particular, the ␤-cell K ATP channel (32). In fact, raising the islet Syn-1A levels in these Goto-Kakizaki rats, either by better glycemic control for even a few days (30) or by adenoviral gene transfer of Syn-1A (31), rescued the impaired insulin secretion. The fluctuation of islet Syn-1A levels with glycemic control could therefore contribute to the changing patterns of insulin exocytic responses in this type-2 diabetes rat model (30). The impaired closure of the K ATP channels in the neuroendocrine islet ␤-cell in this diabetes model is attributable not only to the abnormal glucose metabolism that results in deficient ATP production (32) but also to the reduced levels of Syn-1A (30,31).
Although we have not yet elucidated the specific action of Syn-1A binding to NBF-2 of SUR1 (Fig. 2), the remarkable similarity of the binding affinity between NBF-2 and NBF-1 to Syn-1A (Fig. 2, D and E), taken with previous studies showing that NBF-1 and NBF-2 exist as heterodimers in the intact cell (1,5), suggest the possibility that Syn-1A might play a role in coordinating the cooperative complex interactions between NBF-2 and NBF-1. Since ADP, acting on NBF-2, is known to regulate NBF-1 function (1, 5, 7), the ability of ADP to reverse the inhibitory actions of Syn-1A on NBF-1 (Fig. 4, A and B) might be mediated by its possible effects on modulating Syn-1A interactions with NBF-2. Further work will be needed to determine the complex actions by which ADP antagonizes Syn-1A inhibition of the K ATP channel.
The SUR family also includes members of the SUR2 family (SUR2A, SUR2B, and SUR2C), whose NBFs-1 and NBFs-2 exhibit sequence and functional heterogeneity as compared with SUR1 (1,27). These NBFs of the SUR family are even more different from the NBFs of the other ATP-binding cassette transporter proteins (16,17). In fact, Syn-1A does not bind the NBFs of CFTR (17). The possibility that Syn-1A might bind the NBFs of the SUR2 isoforms and their resulting effects on the respective tissue SUR2/Kir6.1 or Kir6.2 K ATP channel regulation will have to be explored. Syn-1A, known to act as a platform to which many other proteins bind to and become activated to regulate exocytosis (10,33), may well attract additional novel modulators that may also further regulate the SUR proteins.
This work demonstrating the regulation of the K ATP channels by Syn-1A provides an important additional mechanism by which Syn-1A finely tunes the coordination of the orderly and sequential ionic (12)(13)(14)(15)23) and exocytic (10,11) events involved in secretion. Insights derived from this study and further work could lead to the identification of novel therapeutic targets for drug design to treat neuropsychiatric and neuroendocrine disorders to which abnormalities in Syn-1A expression or function contribute (28 -31).