Analysis of the synaptotagmin family during reconstituted membrane fusion. Uncovering a class of inhibitory isoforms

Ca(2+)-triggered exocytosis in neurons and neuroendocrine cells is regulated by the Ca(2+)-binding protein synaptotagmin (syt) I. Sixteen additional isoforms of syt have been identified, but little is known concerning their biochemical or functional properties. Here, we assessed the abilities of fourteen syt isoforms to directly regulate SNARE (soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor)-catalyzed membrane fusion. One group of isoforms stimulated neuronal SNARE-mediated fusion in response to Ca(2+), while another set inhibited SNARE catalyzed fusion in both the absence and presence of Ca(2+). Biochemical analysis revealed a strong correlation between the ability of syt isoforms to bind 1,2-dioleoyl phosphatidylserine (PS) and t-SNAREs in a Ca(2+)-promoted manner with their abilities to enhance fusion, further establishing PS and SNAREs as critical effectors for syt action. The ability of syt I to efficiently stimulate fusion was specific for certain SNARE pairs, suggesting that syts might contribute to the specificity of intracellular membrane fusion reactions. Finally, a subset of inhibitory syts down-regulated the ability of syt I to activate fusion, demonstrating that syt isoforms can modulate the function of each other.     

Ca2+-dependent synaptotagmin binding to SNAP-25 is essential for Ca2+-triggered exocytosis

Synaptotagmin is a proposed Ca2+ sensor on the vesicle for regulated exocytosis and exhibits Ca2+-dependent binding to phospholipids, syntaxin, and SNAP-25 in vitro, but the mechanism by which Ca2+ triggers membrane fusion is uncertain. Previous studies suggested that SNAP-25 plays a role in the Ca2+ regulation of secretion. We found that synaptotagmins I and IX associate with SNAP-25 during Ca2+-dependent exocytosis in PC12 cells, and we identified C-terminal amino acids in SNAP-25 (Asp179, Asp186, Asp193) that are required for Ca2+-dependent synaptotagmin binding. Replacement of SNAP-25 in PC12 cells with SNAP-25 containing C-terminal Asp mutations led to a loss-of-function in regulated exocytosis at the Ca2+-dependent fusion step. These results indicate that the Ca2+-dependent interaction of synaptotagmin with SNAP-25 is essential for the Ca2+-dependent triggering of membrane fusion.     

Synaptotagmin I and IX function redundantly in controlling fusion pore of large dense core vesicles

Synaptotagmins (Syts) constitute a large family of at least 16 members and individual Syt isoforms exhibit distinct Ca²⁺-binding properties and subcellular localization. It remains to be demonstrated whether multiple Syt isoforms can function independently or cooperatively on certain type of vesicle. In the current study, we have developed NPY-pHluorin to specifically assess exocytosis of large dense core vesicles (LDCVs) and studied the requirement of Syt I and Syt IX for LDCV exocytosis in PC12 cells. We found that down-regulation of both Syt I and Syt IX resulted in a significant loss of Ca²⁺-dependent LDCV exocytosis. Moreover, our results suggest Syt I and Syt IX play redundant role in controlling the choice of fusion modes. Down-regulation of both Syt I and Syt IX renders more fusion in the kiss-and-run mode. We conclude that Syt I and Syt IX function redundantly in Ca²⁺-sensing and fusion pore dilation on LDCVs in PC12 cells.     

CAPS acts at a prefusion step in dense-core vesicle exocytosis as a PIP2 binding protein

CAPS-1 is required for Ca2+-triggered fusion of dense-core vesicles with the plasma membrane, but its site of action and mechanism are unknown. We analyzed the kinetics of Ca2+-triggered exocytosis reconstituted in permeable PC12 cells. CAPS-1 increased the initial rate of Ca2+-triggered vesicle exocytosis by acting at a rate-limiting, Ca2+-dependent prefusion step. CAPS-1 activity depended upon prior ATP-dependent priming during which PIP2 synthesis occurs. CAPS-1 activity and binding to the plasma membrane depended upon PIP2. Ca2+ was ineffective in triggering vesicle fusion in the absence of CAPS-1 but instead promoted desensitization to CAPS-1 resulting from decreased plasma membrane PIP2. We conclude that CAPS-1 functions following ATP-dependent priming as a PIP2 binding protein to enhance Ca2+-dependent DCV exocytosis. Essential prefusion steps in dense-core vesicle exocytosis involve sequential ATP-dependent synthesis of PIP2 and the subsequent PIP2-dependent action of CAPS-1. Regulation of PIP2 levels and CAPS-1 activity would control the secretion of neuropeptides and monoaminergic transmitters.     

The tandem C2 domains of synaptotagmin contain redundant Ca2+ binding sites that cooperate to engage t-SNAREs and trigger exocytosis

Real-time voltammetry measurements from cracked PC12 cells were used to analyze the role of synaptotagmin-SNARE interactions during Ca2+-triggered exocytosis. The isolated C2A domain of synaptotagmin I neither binds SNAREs nor inhibits norepinephrine secretion. In contrast, two C2 domains in tandem (either C2A-C2B or C2A-C2A) bind strongly to SNAREs, displace native synaptotagmin from SNARE complexes, and rapidly inhibit exocytosis. The tandem C2 domains of synaptotagmin cooperate via a novel mechanism in which the disruptive effects of Ca2+ ligand mutations in one C2 domain can be partially alleviated by the presence of an adjacent C2 domain. Complete disruption of Ca2+-triggered membrane and target membrane SNARE interactions required simultaneous neutralization of Ca2+ ligands in both C2 domains of the protein. We conclude that synaptotagmin-SNARE interactions regulate membrane fusion and that cooperation between synaptotagmin's C2 domains is crucial to its function.     

ARF6 regulates a plasma membrane pool of phosphatidylinositol(4,5)bisphosphate required for regulated exocytosis

ADP-ribosylation factor (ARF) 6 regulates endosomal plasma membrane trafficking in many cell types, but is also suggested to play a role in Ca2+-dependent dense-core vesicle (DCV) exocytosis in neuroendocrine cells. In the present work, expression of the constitutively active GTPase-defective ARF6Q67L mutant in PC12 cells was found to inhibit Ca2+-dependent DCV exocytosis. The inhibition of exocytosis was accompanied by accumulation of ARFQ67L, phosphatidylinositol 4,5-bisphosphate (PIP2), and the phosphatidylinositol 4-phosphate 5-kinase type I (PIP5KI) on endosomal membranes with their corresponding depletion from the plasma membrane. That the depletion of PIP2 and PIP5K from the plasma membrane caused the inhibition of DCV exocytosis was demonstrated directly in permeable cell reconstitution studies in which overexpression or addition of PIP5KIgamma restored Ca2+-dependent exocytosis. The restoration of exocytosis in ARF6Q67L-expressing permeable cells unexpectedly exhibited a Ca2+ dependence, which was attributed to the dephosphorylation and activation of PIP5K. Increased Ca2+ and dephosphorylation stimulated the association of PIP5KIgamma with ARF6. The results reveal a mechanism by which Ca2+ influx promotes increased ARF6-dependent synthesis of PIP2. We conclude that ARF6 plays a role in Ca2+-dependent DCV exocytosis by regulating the activity of PIP5K for the synthesis of an essential plasma membrane pool of PIP2.     

Synaptotagmin VII is targeted to secretory organelles in PC12 cells, where it functions as a high-affinity calcium sensor

Synaptotagmin (syt) I is thought to act as a Ca2+ sensor that regulates neuronal exocytosis. Fifteen additional isoforms of syt have been identified, but their functions are less well understood. Here, we used PC12 cells to test the idea that different isoforms of syt impart cells with distinct metal (i.e., Ca2+, Ba2+, and Sr2+) requirements for secretion. These cells express syt's I and IX (syt IX sometimes referred to as syt V), which have low apparent metal affinities, at much higher levels than syt VII, which we show has a relatively high apparent affinity for metals. We found that syt I and VII partially colocalize on large dense core vesicles and that upregulation of syt VII produces a concomitant increase in the divalent cation sensitivity of catecholamine release from PC12 cells. Furthermore, RNA interference-mediated knockdown of endogenous syt VII reduced the metal sensitivity of release. These data support the hypothesis that the complement of syt's expressed by a cell, in conjunction with their metal affinity, determines the divalent cation sensitivity of exocytosis.     

Synaptotagmin VII is targeted to dense-core vesicles and regulates their Ca2+ -dependent exocytosis in PC12 cells

It has recently been proposed that synaptotagmin (Syt) VII functions as a plasma membrane Ca2+ sensor for dense-core vesicle exocytosis in PC12 cells based on the results of transient overexpression studies using green fluorescent protein (GFP)-tagged Syt VII; however, the precise subcellular localization of Syt VII is still a matter of controversy (plasma membrane versus secretory granules). In this study we established a PC12 cell line "stably expressing" the Syt VII-GFP molecule and demonstrated by immunocytochemical and immunoelectron microscopic analyses that the Syt VII-GFP protein is localized on dense-core vesicles as well as in other intracellular membranous structures, such as the trans-Golgi network and lysosomes. Syt VII-GFP forms a complex with endogenous Syts I and IX, but not with Syt IV, and it colocalize well with Syts I and IX in the cellular processes (where dense-core vesicles are accumulated) in the PC12 cell line. We further demonstrated by an N-terminal antibody-uptake experiment that Syt VII-GFP-containing dense-core vesicles undergo Ca2+ -dependent exocytosis, the same as endogenous Syt IX-containing vesicles. Moreover, silencing of Syt VII-GFP with specific small interfering RNA dramatically reduced high KCl-dependent neuropeptide Y secretion from the stable PC12 cell line (approximately 60% of the control cells), whereas the same small interfering RNA had little effect on neuropeptide Y secretion from the wild-type PC12 cells (approximately 85-90% of the control cells), indicating that the level of endogenous expression of Syt VII molecules must be low. Our results indicate that the targeting of Syt VII-GFP molecules to specific membrane compartment(s) is affected by the transfection method (transient expression versus stable expression) and suggested that Syt VII molecule on dense-core vesicles functions as a vesicular Ca2+ sensor for exocytosis in endocrine cells.     

Synaptotagmin isoforms couple distinct ranges of Ca2+, Ba2+, and Sr2+ concentration to SNARE-mediated membrane fusion

Ca2+-triggered exocytosis of synaptic vesicles is controlled by the Ca2+-binding protein synaptotagmin (syt) I. Fifteen additional isoforms of syt have been identified. Here, we compared the abilities of three syt isoforms (I, VII, and IX) to regulate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion in vitro in response to divalent cations. We found that different isoforms of syt couple distinct ranges of Ca2+, Ba2+, and Sr2+ to membrane fusion; syt VII was approximately 400-fold more sensitive to Ca2+ than was syt I. Omission of phosphatidylserine (PS) from both populations of liposomes completely abrogated the ability of all three isoforms of syt to stimulate fusion. Mutations that selectively inhibit syt.target-SNARE (t-SNARE) interactions reduced syt stimulation of fusion. Using Sr2+ and Ba2+, we found that binding of syt to PS and t-SNAREs can be dissociated from activation of fusion, uncovering posteffector-binding functions for syt. Our data demonstrate that different syt isoforms are specialized to sense different ranges of divalent cations and that PS is an essential effector of Ca2+.syt action.     

Mutations in the effector binding loops in the C2A and C2B domains of synaptotagmin I disrupt exocytosis in a nonadditive manner

The secretory vesicle protein synaptotagmin I (syt) plays a critical role in Ca2+-triggered exocytosis. Its cytoplasmic domain is composed of tandem C2 domains, C2A and C2B; each C2 domain binds Ca2+. Upon binding Ca2+, positively charged residues within the Ca2+-binding loops are thought to interact with negatively charged phospholipids in the target membrane to mediate docking of the cytoplasmic domain of syt onto lipid bilayers. The C2 domains of syt also interact with syntaxin and SNAP-25, two components of a conserved membrane fusion complex. Here, we have neutralized single positively charged residues at the membrane-binding interface of C2A (R233Q) and C2B (K366Q). Either of these mutations shifted the Ca2+ requirements for syt-liposome interactions from approximately 20 to approximately 40 microm Ca2+. Kinetic analysis revealed that the reduction in Ca2+-sensing activity was associated with a decrease in affinity for membranes. These mutations did not affect sytsyntaxin interactions but resulted in an approximately 50% loss in SNAP-25 binding activity, suggesting that these residues lie at an interface between membranes and SNAP-25. Expression of full-length versions of syt that harbored these mutations reduced the rate of exocytosis in PC12 cells. In both biochemical and functional assays, effects of the R233Q and K366Q mutations were not additive, indicating that mutations in one domain affect the activity of the adjacent domain. These findings indicate that the tandem C2 domains of syt cooperate with one another to trigger release via loop-mediated electrostatic interactions with effector molecules.     

Regulation of Exocytosis and Fusion Pores by Synaptotagmin-Effector Interactions

Synaptotagmin (syt) serves as a Ca²⁺ sensor in the release of neurotransmitters and hormones. This function depends on the ability of syt to interact with other molecules. Syt binds to phosphatidylserine (PS)-containing lipid bilayers as well as to soluble N-ethylmaleimide sensitive factor receptors (SNAREs) and promotes SNARE assembly. All these interactions are regulated by Ca²⁺, but their specific roles in distinct kinetic steps of exocytosis are not well understood. To explore these questions we used amperometry recording from PC12 cells to investigate the kinetics of exocytosis. Syt isoforms and syt I mutants were overexpressed to perturb syt-PS and syt-SNARE interactions to varying degrees and evaluate the effects on fusion event frequency and the rates of fusion pore transitions. Syt I produced more rapid dilation of fusion pores than syt VII or syt IX, consistent with its role in synchronous synaptic release. Stronger syt-PS interactions were accompanied by a higher frequency of fusion events and more stable fusion pores. By contrast, syt-SNARE interactions and syt-induced SNARE assembly were uncorrelated with rates of exocytosis. This associates the syt-PS interaction with two distinct kinetic steps in Ca²⁺ triggered exocytosis and supports a role for the syt-PS interaction in stabilizing open fusion pores.     

Synaptotagmin IX regulates Ca2+-dependent secretion in PC12 cells.

Synaptotagmin (Syt) I-deficient phaeochromocytoma (PC12) cell lines show normal Ca(2+)-dependent norepinephrine (NE) release (Shoji-Kasai, Y., Yoshida, A., Sato, K., Hoshino, T., Ogura, A., Kondo, S., Fujimoto, Y., Kuwahara, R., Kato, R., and Takahashi, M. (1992) Science 256, 1821-1823). To identify an alternative Ca(2+) sensor, we searched for other Syt isoforms in Syt I-deficient PC12 cells and identified Syt IX, an isoform closely related to Syt I, as an abundantly expressed dense-core vesicle protein. Here we show that Syt IX is required for the Ca(2+)-dependent release of NE from PC12 cells. Antibodies directed against the C2A domain of either Syt IX or Syt I inhibited Ca(2+)-dependent NE release in permeable PC12 cells indicating that both Syt proteins function in dense-core vesicle exocytosis. Our results support the idea that Syt family proteins that co-reside on secretory vesicles may function cooperatively and redundantly as potential Ca(2+) sensors for exocytosis.     

Fusion pore dynamics are regulated by synaptotagmin*t-SNARE interactions

Exocytosis involves the formation of a fusion pore that connects the lumen of secretory vesicles with the extracellular space. Exocytosis from neurons and neuroendocrine cells is tightly regulated by intracellular [Ca2+] and occurs rapidly, but the molecular events that mediate the opening and subsequent dilation of fusion pores remain to be determined. A putative Ca2+ sensor for release, synaptotagmin I (syt), binds directly to syntaxin and SNAP-25, which are components of a conserved membrane fusion complex. Here, we show that Ca2+-triggered syt*SNAP-25 interactions occur rapidly. The tandem C2 domains of syt cooperate to mediate binding to syntaxin/SNAP-25; lengthening the linker that connects C2A and C2B selectively disrupts this interaction. Expression of the linker mutants in PC12 cells results in graded reductions in the stability of fusion pores. Thus, the final step of Ca2+-triggered exocytosis is regulated, at least in part, by direct contacts between syt and SNAP-25/syntaxin.     

Synaptotagmin C2A loop 2 mediates Ca2+-dependent SNARE interactions essential for Ca2+-triggered vesicle exocytosis

Synaptotagmins contain tandem C2 domains and function as Ca(2+) sensors for vesicle exocytosis but the mechanism for coupling Ca(2+) rises to membrane fusion remains undefined. Synaptotagmins bind SNAREs, essential components of the membrane fusion machinery, but the role of these interactions in Ca(2+)-triggered vesicle exocytosis has not been directly assessed. We identified sites on synaptotagmin-1 that mediate Ca(2+)-dependent SNAP25 binding by zero-length cross-linking. Mutation of these sites in C2A and C2B eliminated Ca(2+)-dependent synaptotagmin-1 binding to SNAREs without affecting Ca(2+)-dependent membrane binding. The mutants failed to confer Ca(2+) regulation on SNARE-dependent liposome fusion and failed to restore Ca(2+)-triggered vesicle exocytosis in synaptotagmin-deficient PC12 cells. The results provide direct evidence that Ca(2+)-dependent SNARE binding by synaptotagmin is essential for Ca(2+)-triggered vesicle exocytosis and that Ca(2+)-dependent membrane binding by itself is insufficient to trigger fusion. A structure-based model of the SNARE-binding surface of C2A provided a new view of how Ca(2+)-dependent SNARE and membrane binding occur simultaneously.     

Synaptotagmin VII as a plasma membrane Ca(2+) sensor in exocytosis

Synaptotagmins I and II are Ca(2+) binding proteins of synaptic vesicles essential for fast Ca(2+)-triggered neurotransmitter release. However, central synapses and neuroendocrine cells lacking these synaptotagmins still exhibit Ca(2+)-evoked exocytosis. We now propose that synaptotagmin VII functions as a plasma membrane Ca(2+) sensor in synaptic exocytosis complementary to vesicular synaptotagmins. We show that alternatively spliced forms of synaptotagmin VII are expressed in a developmentally regulated pattern in brain and are concentrated in presynaptic active zones of central synapses. In neuroendocrine PC12 cells, the C(2)A and C(2)B domains of synaptotagmin VII are potent inhibitors of Ca(2+)-dependent exocytosis, but only when they bind Ca(2+). Our data suggest that in synaptic vesicle exocytosis, distinct synaptotagmins function as independent Ca(2+) sensors on the two fusion partners, the plasma membrane (synaptotagmin VII) versus synaptic vesicles (synaptotagmins I and II).     

Calmodulin-dependent regulation of a lipid binding domain in the v-SNARE synaptobrevin and its role in vesicular fusion

Trans SNARE complex assembly is an essential step in Ca2+-dependent membrane fusion, although the SNARE proteins do not bind Ca2+ ions. Studies to evaluate how the Ca2+sensor protein calmodulin might regulate this process led to the identification of a consensus calmodulin binding motif in the v-SNARE VAMP2. This sequence (residues 77-90) is situated precisely C-terminal to the tetanus toxin (TeNT) and botulinum B toxin cleavage site (76Q-F77) close to the transmembrane anchor. The same domain also binds acidic phospholipids and Ca2+/calmodulin or lipid binding are mutually exclusive. Directed mutagenesis of basic or hydrophobic residues within this motif reduced interactions with both Ca2+/calmodulin and phospholipids to a similar extent. The effects of these mutations on Ca2+-dependent exocytosis was explored using an hGH release assay in permeabilized pheochromocytoma PC12 cells. Treatment of cells with tetanus toxin (TeNT), which cleaves endogenous VAMP, abolished secretion. Secretion could be re-established by transfecting TeNT-resistant VAMP with mutations (Q76V,F77W) in the cleavage site. However rescue of exocytosis was abolished when additional mutations (K83A,K87V or W89A,W90A) were introduced that inhibited calmodulin and phospholipid binding to VAMP. Thus calmodulin and/or phospholipid binding to the membrane proximal region of VAMP is required for Ca2+-dependent exocytosis. We speculate that interactions between cis phospholipids at the vesicle surface and the membrane proximal region of VAMP inhibits SNARE complex assembly. Displacement of these interactions by Ca2+/calmodulin may promote SNARE complex assembly and lead to trans interactions between the membrane proximal region of VAMP and phospholipids in the plasma membrane.     

The calcium-binding loops of the tandem C2 domains of synaptotagmin VII cooperatively mediate calcium-dependent oligomerization

Synaptotagmin VII (Syt VII), a proposed regulator for Ca2+-dependent exocytosis, showed a robust Ca2+-dependent oligomerization property via its two C2 domains (Fukuda, M., and Mikoshiba, K. (2001) J. Biol. Chem. 276, 27670-27676), but little is known about its structure or the critical residues directly involved in the oligomerization interface. In this study, site-directed mutagenesis and chimeric analysis between Syt I and Syt VII showed that three Asp residues in Ca2+-binding loop 1 or 3 (Asp-172, Asp-303, and Asp-357) are crucial to robust Ca(2+)-dependent oligomerization. Unlike Syt I, however, the polybasic sequence in the beta4 strands of the C2 structures (so-called "C2 effector domain") is not involved in the Ca2+-dependent oligomerization of Syt VII. The results also showed that the Ca2+-binding loops of the two C2 domains cooperatively mediate Syt VII oligomerization (i.e. the presence of redundant Ca2+-binding site(s)) as well as the importance of Ca2+-dependent oligomerization of Syt VII in Ca2+-regulated secretion. Expression of wild-type tandem C2 domains of Syt VII in PC12 cells inhibited Ca2+-dependent neuropeptide Y release, whereas mutant fragments lacking Ca2+-dependent oligomerization activity had no effect. Finally, rotary-shadowing electron microscopy showed that the Ca2+-dependent oligomer of Syt VII is "a large linear structure," not an irregular aggregate. By contrast, in the absence of Ca2+ Syt VII molecules were observed to form a globular structure. Based on these results, we suggest that the linear Ca2+-dependent oligomer may be aligned at the fusion site between vesicles and plasma membrane and modulate Ca2+-regulated exocytosis by opening or dilating fusion pores.     

Silence of Synaptotagmin VII inhibits release of dense core vesicles in PC12 cells

Synaptotagmin VII (Syt VII), which has a higher Ca²⁺ affinity and slower disassembly kinetics with lipid than Syt I and Syt IX, was regarded as being uninvolved in synaptic vesicle (SV) exocytosis but instead possibly as a calcium sensor for the slower kinetic phase of dense core vesicles (DCVs) release. By using high temporal resolution capacitance and amperometry measurements, it was demonstrated that the knockdown of endogenous Syt VII attenuated the fusion of DCV with the plasma membrane, reduced the amplitude of the exocytotic burst of the Ca²⁺-triggered DCV release without affecting the slope of the sustained component, and blocked the fusion pore expansion. This suggests that Syt VII is the Ca²⁺ sensor of DCV fusion machinery and is an essential factor for the establishment and maintenance of the pool size of releasable DCVs in PC12 cells.     

Kinetic diversity in the fusion of exocytotic vesicles.

The speed at which secretory vesicles fuse with the plasma membrane is a key parameter for neuronal and endocrine functions. We determined the precise time courses for fusion of small clear and large dense-core vesicles in PC12 and chromaffin cells by simultaneously measuring both plasma membrane areas and release of vesicular contents. We found that instantaneous increases in cytosolic Ca2+ concentration evoked vesicle fusion, but with time constants that varied over four orders of magnitude among different types of vesicles and cells. This indicates that the molecular machinery for the final Ca2+-dependent fusion steps of exocytosis is highly variable and is as critical as Ca2+ signalling processes in determining the speed and amount of secretion of neurotransmitters and hormones. Our results suggest a new possibility that the molecules responsible for the final fusion reaction that leads to vesicle fusion are key determinants for neuronal plasticity and hormonal disorders.     

Axonal and dendritic synaptotagmin isoforms revealed by a pHluorin-syt functional screen

The synaptotagmins (syts) are a family of molecules that regulate membrane fusion. There are 17 mammalian syt isoforms, most of which are expressed in the brain. However, little is known regarding the subcellular location and function of the majority of these syts in neurons, largely due to a lack of isoform-specific antibodies. Here we generated pHluorin-syt constructs harboring a luminal domain pH sensor, which reports localization, pH of organelles to which syts are targeted, and the kinetics and sites of exocytosis and endocytosis. Of interest, only syt-1 and 2 are targeted to synaptic vesicles, whereas other isoforms selectively recycle in dendrites (syt-3 and 11), axons (syt-5, 7, 10, and 17), or both axons and dendrites (syt-4, 6, 9, and 12), where they undergo exocytosis and endocytosis with distinctive kinetics. Hence most syt isoforms localize to distinct secretory organelles in both axons and dendrites and may regulate neuropeptide/neurotrophin release to modulate neuronal function.