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Exocytosis and Synaptic Vesicle Function

Ok‐Ho Shin

10.1002/cphy.c130021

Source: Volume 4, Issue 1, January 2014

Published online: January 2014

Full Article on Wiley Online Library



Abstract

Synaptic vesicles release their vesicular contents to the extracellular space by Ca2+‐triggered exocytosis. The Ca2+‐triggered exocytotic process is regulated by synaptotagmin (Syt), a vesicular Ca2+‐binding C2 domain protein. Synaptotagmin 1 (Syt1), the most studied major isoform among 16 Syt isoforms, mediates Ca2+‐triggered synaptic vesicle exocytosis by interacting with the target membranes and SNARE/complexin complex. In synapses of the central nervous system, synaptobrevin 2, a major vesicular SNARE protein, forms a ternary SNARE complex with the plasma membrane SNARE proteins, syntaxin 1 and SNAP25. The affinities of Ca2+‐dependent interactions between Syt1 and its targets (i.e., SNARE complexes and membranes) are well correlated with the efficacies of the corresponding exocytotic processes. Therefore, different SNARE protein isoforms and membrane lipids, which interact with Syt1 with various affinities, are capable of regulating the efficacy of Syt1‐mediated exocytosis. Otoferlin, another type of vesicular C2 domain protein that binds to the membrane in a Ca2+‐dependent manner, is also involved in the Ca2+‐triggered synaptic vesicle exocytosis in auditory hair cells. However, the functions of otoferlin in the exocytotic process are not well understood. In addition, at least five different types of synaptic vesicle proteins such as synaptic vesicle protein 2, cysteine string protein α, rab3, synapsin, and a group of proteins containing four transmembrane regions, which includes synaptophysin, synaptogyrin, and secretory carrier membrane protein, are involved in modulating the exocytotic process by regulating the formation and trafficking of synaptic vesicles. © 2014 American Physiological Society. Compr Physiol 4:149‐175, 2014.

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Figure 1. Figure 1. Synaptic vesicle proteins that are involved in regulating Ca2+‐triggered exocytotic processes. The domain structures of synaptic vesicle proteins, which are involved in Ca2+‐triggered exocytotic processes, are illustrated with a brief description of their major functions. The number in parentheses is the average copy number of protein per synaptic vesicle (235). Syt1, synaptotagmin 1; SV2, synaptic vesicle protein 2; SCAMP, secretory carrier membrane protein; Syb2, synaptobrevin 2 (also known as VAMP2); CSPα, cysteine string protein α; CaMKs, Ca2+/calmodulin‐dependent protein kinases; GAP, GTPase‐activating protein; GEF, guanine nucleotide exchange factor; PS, phosphatidylserine; PI, phosphatidylinositol; PIP2, phosphatidylinositol 4,5‐bisphosphate. Synapsin interacts with the acidic synaptic vesicle membrane in a phosphorylation/dephosphorylation‐dependent manner (98), while rab3a is associated with synaptic vesicles in a GTP‐dependent manner (72). The transmembrane region of Syb2 is associated with synaptophysin in the presence of cholesterol (54,145,167,267).
Figure 2. Figure 2. Ca2+‐tiggered exocytosis regulated by synaptotagmin 1 (Syt1). (A) Synaptotagmin 1 (Syt1) interacts with the target membrane by electrostatic and van der Waals interactions in a Ca2+‐dependent manner to mediate exocytosis (25,42,70,122,158,212). The R233 residue of the Syt1C2A domain and the K326, K327, R398, and R399 residues of the Syt1C2B domain are identified as critical residues for electrostatic interaction with membranes for the Syt1 function (3,70,123,262). The M173, F234 residues of the Syt1C2A domain and the V304 and I367 residues of the Syt1C2B domain penetrate into the membrane and form van der Waals interaction with hydrophobic lipids of the membrane (26,179,208). Syt1 also interacts with assembled SNARE complex composed of synaptobrevin 2 (Syb2), syntaxin 1 (Synt1), and SNAP25 (158). The Ca2+‐dependent interaction among Syt1, SNARE complex, and membranes are important to mediate the synaptic vesicle exocytosis (70,158), but the molecular mechanism of the interaction is not fully appreciated. (B) The domain structures of Syt1, Syb2, syntaxin 1A (Synt1A), SNAP25, and ferlins. SNAP25 is located in the plasma membrane, without transmembrane region, via palmitoylated C85, C88, C90, and C92 residues between the N‐terminal and C‐terminal SNARE motifs (207). The N‐terminal Habc domain (65) of Synt1A is capable of interacting with the SNARE motif of Synt1A, which hinders the efficient formation of ternary SNARE complex composed of Synt1A, Syb2, and SNAP25. Otoferlin is involved in regulating the Ca2+‐triggered synaptic vesicle exocytosis at synapses of auditory hair cells, where presynaptic SNARE proteins such as Synt1, SNAP25, and Syb2 are not expressed (153,177). However, unlike Syt1, the functions of otoferlin are not clearly demonstrated. Dysferlin, an isoform of otoferlin, regulates Ca2+‐dependent sarcolemma resealing which is mediated by an exocytotic process (5). Both otoferlin and dysferlin are probably involved in mediating Ca2+‐triggered exocytotic process by a similar mechanism, which is different from that of Syt1.
Figure 3. Figure 3. Mechanisms of the Ca2+‐dependent synaptotagmin 1 (Syt1) C2 domain binding to the membrane. (A) The Ca2+‐binding loops of the Syt1C2A and Syt1C2B domains. Aspartate (D) residues that directly interact with Ca2+ are indicated by red color, R233 residue that interacts with acidic head groups of phospholipids in the membrane is indicated by white color, and hydrophobic residues that penetrate into the phospholipid bilayer are indicated by blue color. (B) The hydrophobic residues of the synaptotagmin (Syt) C2A and C2B domains, which directly penetrate into the membrane and form van der Waals interaction with hydrophobic lipids. The Ca2+ affinities of the isolated C2 domains were measured in the presence of acidic‐liposomes reconstituted with 75% phosphatidylcholine (PC) and 25% phosphatidylserine (PS) (208,210,231). (C) The Ca2+ affinities and total amount of the wild‐type and mutant forms of Syt1C2A/B domain binding to synaptic‐liposomes (41% phosphatidylcholine, 32% phosphatidylethanolamine, 12% phosphatidylserine, 5% phosphatidylinositol, and 10% cholesterol) containing 0.25% phosphatidylinositol phosphate (PIP) and 0.05% phosphatidylinositol 4,5‐bisphosphate (PIP2) (123,179,212). The number in parentheses is an EC50 value of Ca2+ concentration for the Syt1 cytosolic fraction binding to synaptic‐liposomes containing 0.5% PIP and 0.1% PIP2 (158).
Figure 4. Figure 4. Regulation of the interaction between synaptotagmin 1 (Syt1) and the membrane by acidic‐phospholipids. (A) Acidic phospholipids such as phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP), and phosphatidylinositol 4,5‐bisphosphate (PIP2) are essential for the efficient Ca2+ binding to the C2 domains of synaptotagmin 1 (Syt1). The R233 residue of the Syt1C2A domain, and the K326 and K327 residues of the Syt1C2B domain are important for the efficient Syt1 binding to the membrane in a Ca2+‐dependent manner (70,123). In addition, the R398 and R399 residues of the Syt1C2B domain are essential for the Syt1 function that mediates Ca2+‐triggered exocytosis (262). These basic residues interact with acidic head groups of phospholipids in the membrane. Neurons increase PIP, PIP2, and inositol hexakisphosphate (IP6) concentrations by multiple action potential‐stimulations. IP6, a PIP2 metabolite inhibits the function of Syt1 by interacting with the Ca2+‐binding loops of the Syt1C2B domain (264). DAG, diacylglycerol. (B) The hydrophilic head groups of PI, PS, and PIP2. The Ca2+ affinity of Syt1 for the membrane binding depends on the acidity of the membrane (205,208,270). Therefore, PIP2 is much better than PI and PS for the Ca2+‐dependent Syt1 binding to the membrane. Even though the net acidities are the same between PS and PI, PS is better than PI for the Ca2+‐dependent Syt1 binding to the membrane (208).
Figure 5. Figure 5. The mechanisms of synaptic vesicle docking, which are mediated by synaptobrevin 2 (Syb2) and synaptotagmin 1 (Syt1). Synaptobrevin 2 (Syb2) is capable of interacting with syntaxin 1 (Synt1)/SNAP25 duplex (189), and the basic residues of the Syt1C2B domain are also capable of interacting with either PIP2 in the plasma membrane or Synt1/SNAP25 duplex (3,123,180). These Ca2+‐independent interactions lead to the synaptic vesicle docking and priming to the target plasma membrane. The primed synaptic vesicles are ready for Ca2+‐triggered exocytosis by extracellular Ca2+ influx after action potential‐stimulations. Neurons lacking Syb2 showed about tenfold decrease in the size of readily releasable pool (RRP), which is affected by the synaptic vesicle priming and docking (200). However, neurons expressing Syt1K326A,K327A did not alter the RRP size compared to that of neurons expressing wild‐type Syt1 (123). Therefore, the role of the Syt1C2B domain in mediating synaptic vesicle docking and priming is not universally accepted.
Figure 6. Figure 6. Different modes of neurotransmitter release at presynaptic terminals. Three different modes of neurotransmitter release are illustrated (160). Action potentials trigger extracellular Ca2+ influx into the presynaptic terminals, and neurotransmitters are released by synchronous or asynchronous modes. Neurons also spontaneously release neurotransmitters without action potentials, but mainly in a Ca2+‐dependent manner (260). Ca2+ from either intracellular storages or local Ca2+ channel opening triggers spontaneous mini release. PIP2, phosphatidylinositol 4,5‐bisphosphate; DAG, diacylglycerol; IP3, inositol 1,4,5‐triphosphate; Syt, synaptotagmin; Doc2, double C2 protein; Syb, synaptobrevin (also known as vesicle‐associated membrane protein, VAMP); Vti1a, Vps10p‐tail‐interactor‐1a.
Figure 7. Figure 7. Comparison of amino acid sequences of the synaptotagmin 7 C2A/B (Syt7C2A/B) domains from zebrafish, mouse, rat, and human. Amino acid sequences of the Syt7C2A/B domains from zebrafish (genebank accession # NM_001245957), mouse (genebank accession # NM_173068), rat (genebank accession # NM_021659), and human (genebank accession # NM_004200). The residues that are not conserved in zebrafish are indicated by yellow color. Conserved residues among different species are indicated by grey boxes, and identical residues are not shown. Aspartate residues that directly interact with Ca2+ are indicated by red color.
Figure 8. Figure 8. Domain structures of the synaptic vesicle proteins that have four transmembrane regions. (A) Synaptophysin, synaptogyrin, and secretory carrier membrane protein (SCAMP) have four transmembrane regions. Synaptophysin has four isoforms—synaptophysin 1, synaptophysin 2 (also known as synaptoporin), pantophysin, and mitsugumin 29. Synaptogyrin has three isoforms (synaptogyrin 1‐3), and SCAMP has five isoforms (SCAMP 1‐5). NGly, N‐glycosylation site; Syb2, synaptobrevin 2; Synt1, syntaxin 1; PIP2, phosphatidylinositol 4,5‐bisphosphate; Syt, synaptotagmin; [P]n, multiple phosphorylation sites. (B) Synaptophysin 1 is capable of interacting with either dynamin or Syb2 (38,86,140), while SCAMP1 is capable of interacting with intersectin1 and γ‐synergin (69). Intersectin 1 and γ‐synergin are involved in the endocytotic process in the plasma membrane and vesicle formation in the trans‐Golgi complex, respectively. Therefore, the main function of these proteins in exocytotic processes is probably the generation of releasable synaptic vesicles, which is important for the synaptic facilitation and long‐term potentiation (LTP).
Figure 9. Figure 9. Domain structure of synapsin and its proposed mechanism of synaptic vesicle mobilization. (A) Conserved domains among synapsin isoforms. Phosphorylation sites of synapsin 1a are indicated by “P” in the circle. In particular, the phosphorylation sites by Ca2+/calmodulin‐dependent protein kinases (CaMKs) are indicated by red circles. The “A” domain is capable of interacting with the acidic synaptic vesicle membrane, but phosphorylation of serine9 residue in the “A” domain triggers the dissociation of synapsin from synaptic vesicles. The “C” domain is capable of interacting with the synaptic vesicle membrane and actin filaments, while the “D” domain can interact with SH3‐proteins. Both “C” and “E” domains are involved in synapsin oligomerization. (B) The proposed mechanism of synaptic vesicle mobilization by synapsin 1a and CaMKs (98,232). Repeated stimulations of neurons increase the intracellular Ca2+ concentration, and subsequently activate CaMKs in presynaptic terminals. Activated CaMKs phosphorylate synapsin 1a, and phosphorylated synapsins are dissociated from the cluster composed of synaptic vesicles, synapsin, and actin filaments. This mobilizes synaptic vesicles for exocytosis from the synapsin/CaMK‐responsive synaptic vesicle pools.
Figure 10. Figure 10. Domain structure of cysteine string protein (CSP) and its function in synaptic vesicle trafficking. (A) The domain structure of CSP isoforms. CSPα is capable of interacting with various types of proteins such as synaptotagmin 1 (Syt1), the C2A domain of synaptotagmin 9 (Syt9C2A), SNAP25, syntaxin (Synt), synaptobrevin 2 (Syb2), small glutamine‐rich TPR protein (SGT), GTP‐binding protein (Gαβγ), and N‐type Ca2+ channel. PKA, protein kinase A; PKB, protein kinase B; HIP14, huntingtin‐interacting protein 14. (B) The major function of CSPα, as a component of the chaperone complex composed of Hsc70 and SGT, is the maintenance of the expression levels of SNAP25 and dynamin 1 (191,271). Both SNAP25 and dynamin 1 play critical roles in synaptic vesicle exocytosis and endocytosis.
Figure 11. Figure 11. Mechanism of the rab3‐mediated synaptic vesicle priming to the active zone in the plasma membrane. Rab3a, rab3b, rab3c, and rab27b are expressed in synaptic vesicles (235). GTP‐bound form of rab3/rab27b interacts with both RIM and Munc13 proteins (51). RIM protein is a component of the presynaptic active zone protein complex (228). Munc13 is located in presynaptic active zone and binds to plasma membrane in a Ca2+/diacylglycerol (DAG)‐dependent manner (178,213). Solid line between proteins represents an interaction. Munc13 also stimulates the SNARE complex formation that mediates exocytosis. RIM‐BP, RIM‐binding protein; DAG, diacylglycerol; PIP2, phosphatidylinositol 4,5‐bisphosphate.
Figure 12. Figure 12. The effects of cholesterol and phosphatidylethanolamine (PE) on the Ca2+‐dependent synaptotagmin 1 (Syt1)‐binding to the membrane. Phosphatidylethanolamine (PE) and cholesterol enhanced the Ca2+‐dependent Syt1C2A/B domain binding to the membrane (212) probably because: (i) both PE and cholesterol have smaller hydrophilic head groups than that of phosphatidylcholine (PC) and (ii) cholesterol has a much bulkier hydrophobic structure than that of PC. The small size of hydrophilic head group of phospholipids probably helps the penetration of the hydrophobic residues (M173, F234, V304, and I367) in the Ca2+‐binding loops of the Syt1C2A/B domain into the phospholipid bilayer upon Ca2+‐dependent binding.
Figure 13. Figure 13. Regulation of the SNARE complex formation by lipids. (A) Sphingosine exposes the SNARE motif of synaptobrevin 2 (Syb2) from the synaptic vesicular membrane (41), while arachidonic acid releases the Habc domain from the SNARE motif of syntaxin 1A (Synt1A) (39,65). Both sphingosine and arachidonic acid are capable of promoting the formation of SNARE complex, and subsequent synaptic vesicle exocytosis. (B) Chemical structures of sphingosine and arachidonic acid. Both lipids have chemical properties of detergents.


Figure 1. Synaptic vesicle proteins that are involved in regulating Ca2+‐triggered exocytotic processes. The domain structures of synaptic vesicle proteins, which are involved in Ca2+‐triggered exocytotic processes, are illustrated with a brief description of their major functions. The number in parentheses is the average copy number of protein per synaptic vesicle (235). Syt1, synaptotagmin 1; SV2, synaptic vesicle protein 2; SCAMP, secretory carrier membrane protein; Syb2, synaptobrevin 2 (also known as VAMP2); CSPα, cysteine string protein α; CaMKs, Ca2+/calmodulin‐dependent protein kinases; GAP, GTPase‐activating protein; GEF, guanine nucleotide exchange factor; PS, phosphatidylserine; PI, phosphatidylinositol; PIP2, phosphatidylinositol 4,5‐bisphosphate. Synapsin interacts with the acidic synaptic vesicle membrane in a phosphorylation/dephosphorylation‐dependent manner (98), while rab3a is associated with synaptic vesicles in a GTP‐dependent manner (72). The transmembrane region of Syb2 is associated with synaptophysin in the presence of cholesterol (54,145,167,267).


Figure 2. Ca2+‐tiggered exocytosis regulated by synaptotagmin 1 (Syt1). (A) Synaptotagmin 1 (Syt1) interacts with the target membrane by electrostatic and van der Waals interactions in a Ca2+‐dependent manner to mediate exocytosis (25,42,70,122,158,212). The R233 residue of the Syt1C2A domain and the K326, K327, R398, and R399 residues of the Syt1C2B domain are identified as critical residues for electrostatic interaction with membranes for the Syt1 function (3,70,123,262). The M173, F234 residues of the Syt1C2A domain and the V304 and I367 residues of the Syt1C2B domain penetrate into the membrane and form van der Waals interaction with hydrophobic lipids of the membrane (26,179,208). Syt1 also interacts with assembled SNARE complex composed of synaptobrevin 2 (Syb2), syntaxin 1 (Synt1), and SNAP25 (158). The Ca2+‐dependent interaction among Syt1, SNARE complex, and membranes are important to mediate the synaptic vesicle exocytosis (70,158), but the molecular mechanism of the interaction is not fully appreciated. (B) The domain structures of Syt1, Syb2, syntaxin 1A (Synt1A), SNAP25, and ferlins. SNAP25 is located in the plasma membrane, without transmembrane region, via palmitoylated C85, C88, C90, and C92 residues between the N‐terminal and C‐terminal SNARE motifs (207). The N‐terminal Habc domain (65) of Synt1A is capable of interacting with the SNARE motif of Synt1A, which hinders the efficient formation of ternary SNARE complex composed of Synt1A, Syb2, and SNAP25. Otoferlin is involved in regulating the Ca2+‐triggered synaptic vesicle exocytosis at synapses of auditory hair cells, where presynaptic SNARE proteins such as Synt1, SNAP25, and Syb2 are not expressed (153,177). However, unlike Syt1, the functions of otoferlin are not clearly demonstrated. Dysferlin, an isoform of otoferlin, regulates Ca2+‐dependent sarcolemma resealing which is mediated by an exocytotic process (5). Both otoferlin and dysferlin are probably involved in mediating Ca2+‐triggered exocytotic process by a similar mechanism, which is different from that of Syt1.


Figure 3. Mechanisms of the Ca2+‐dependent synaptotagmin 1 (Syt1) C2 domain binding to the membrane. (A) The Ca2+‐binding loops of the Syt1C2A and Syt1C2B domains. Aspartate (D) residues that directly interact with Ca2+ are indicated by red color, R233 residue that interacts with acidic head groups of phospholipids in the membrane is indicated by white color, and hydrophobic residues that penetrate into the phospholipid bilayer are indicated by blue color. (B) The hydrophobic residues of the synaptotagmin (Syt) C2A and C2B domains, which directly penetrate into the membrane and form van der Waals interaction with hydrophobic lipids. The Ca2+ affinities of the isolated C2 domains were measured in the presence of acidic‐liposomes reconstituted with 75% phosphatidylcholine (PC) and 25% phosphatidylserine (PS) (208,210,231). (C) The Ca2+ affinities and total amount of the wild‐type and mutant forms of Syt1C2A/B domain binding to synaptic‐liposomes (41% phosphatidylcholine, 32% phosphatidylethanolamine, 12% phosphatidylserine, 5% phosphatidylinositol, and 10% cholesterol) containing 0.25% phosphatidylinositol phosphate (PIP) and 0.05% phosphatidylinositol 4,5‐bisphosphate (PIP2) (123,179,212). The number in parentheses is an EC50 value of Ca2+ concentration for the Syt1 cytosolic fraction binding to synaptic‐liposomes containing 0.5% PIP and 0.1% PIP2 (158).


Figure 4. Regulation of the interaction between synaptotagmin 1 (Syt1) and the membrane by acidic‐phospholipids. (A) Acidic phospholipids such as phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP), and phosphatidylinositol 4,5‐bisphosphate (PIP2) are essential for the efficient Ca2+ binding to the C2 domains of synaptotagmin 1 (Syt1). The R233 residue of the Syt1C2A domain, and the K326 and K327 residues of the Syt1C2B domain are important for the efficient Syt1 binding to the membrane in a Ca2+‐dependent manner (70,123). In addition, the R398 and R399 residues of the Syt1C2B domain are essential for the Syt1 function that mediates Ca2+‐triggered exocytosis (262). These basic residues interact with acidic head groups of phospholipids in the membrane. Neurons increase PIP, PIP2, and inositol hexakisphosphate (IP6) concentrations by multiple action potential‐stimulations. IP6, a PIP2 metabolite inhibits the function of Syt1 by interacting with the Ca2+‐binding loops of the Syt1C2B domain (264). DAG, diacylglycerol. (B) The hydrophilic head groups of PI, PS, and PIP2. The Ca2+ affinity of Syt1 for the membrane binding depends on the acidity of the membrane (205,208,270). Therefore, PIP2 is much better than PI and PS for the Ca2+‐dependent Syt1 binding to the membrane. Even though the net acidities are the same between PS and PI, PS is better than PI for the Ca2+‐dependent Syt1 binding to the membrane (208).


Figure 5. The mechanisms of synaptic vesicle docking, which are mediated by synaptobrevin 2 (Syb2) and synaptotagmin 1 (Syt1). Synaptobrevin 2 (Syb2) is capable of interacting with syntaxin 1 (Synt1)/SNAP25 duplex (189), and the basic residues of the Syt1C2B domain are also capable of interacting with either PIP2 in the plasma membrane or Synt1/SNAP25 duplex (3,123,180). These Ca2+‐independent interactions lead to the synaptic vesicle docking and priming to the target plasma membrane. The primed synaptic vesicles are ready for Ca2+‐triggered exocytosis by extracellular Ca2+ influx after action potential‐stimulations. Neurons lacking Syb2 showed about tenfold decrease in the size of readily releasable pool (RRP), which is affected by the synaptic vesicle priming and docking (200). However, neurons expressing Syt1K326A,K327A did not alter the RRP size compared to that of neurons expressing wild‐type Syt1 (123). Therefore, the role of the Syt1C2B domain in mediating synaptic vesicle docking and priming is not universally accepted.


Figure 6. Different modes of neurotransmitter release at presynaptic terminals. Three different modes of neurotransmitter release are illustrated (160). Action potentials trigger extracellular Ca2+ influx into the presynaptic terminals, and neurotransmitters are released by synchronous or asynchronous modes. Neurons also spontaneously release neurotransmitters without action potentials, but mainly in a Ca2+‐dependent manner (260). Ca2+ from either intracellular storages or local Ca2+ channel opening triggers spontaneous mini release. PIP2, phosphatidylinositol 4,5‐bisphosphate; DAG, diacylglycerol; IP3, inositol 1,4,5‐triphosphate; Syt, synaptotagmin; Doc2, double C2 protein; Syb, synaptobrevin (also known as vesicle‐associated membrane protein, VAMP); Vti1a, Vps10p‐tail‐interactor‐1a.


Figure 7. Comparison of amino acid sequences of the synaptotagmin 7 C2A/B (Syt7C2A/B) domains from zebrafish, mouse, rat, and human. Amino acid sequences of the Syt7C2A/B domains from zebrafish (genebank accession # NM_001245957), mouse (genebank accession # NM_173068), rat (genebank accession # NM_021659), and human (genebank accession # NM_004200). The residues that are not conserved in zebrafish are indicated by yellow color. Conserved residues among different species are indicated by grey boxes, and identical residues are not shown. Aspartate residues that directly interact with Ca2+ are indicated by red color.


Figure 8. Domain structures of the synaptic vesicle proteins that have four transmembrane regions. (A) Synaptophysin, synaptogyrin, and secretory carrier membrane protein (SCAMP) have four transmembrane regions. Synaptophysin has four isoforms—synaptophysin 1, synaptophysin 2 (also known as synaptoporin), pantophysin, and mitsugumin 29. Synaptogyrin has three isoforms (synaptogyrin 1‐3), and SCAMP has five isoforms (SCAMP 1‐5). NGly, N‐glycosylation site; Syb2, synaptobrevin 2; Synt1, syntaxin 1; PIP2, phosphatidylinositol 4,5‐bisphosphate; Syt, synaptotagmin; [P]n, multiple phosphorylation sites. (B) Synaptophysin 1 is capable of interacting with either dynamin or Syb2 (38,86,140), while SCAMP1 is capable of interacting with intersectin1 and γ‐synergin (69). Intersectin 1 and γ‐synergin are involved in the endocytotic process in the plasma membrane and vesicle formation in the trans‐Golgi complex, respectively. Therefore, the main function of these proteins in exocytotic processes is probably the generation of releasable synaptic vesicles, which is important for the synaptic facilitation and long‐term potentiation (LTP).


Figure 9. Domain structure of synapsin and its proposed mechanism of synaptic vesicle mobilization. (A) Conserved domains among synapsin isoforms. Phosphorylation sites of synapsin 1a are indicated by “P” in the circle. In particular, the phosphorylation sites by Ca2+/calmodulin‐dependent protein kinases (CaMKs) are indicated by red circles. The “A” domain is capable of interacting with the acidic synaptic vesicle membrane, but phosphorylation of serine9 residue in the “A” domain triggers the dissociation of synapsin from synaptic vesicles. The “C” domain is capable of interacting with the synaptic vesicle membrane and actin filaments, while the “D” domain can interact with SH3‐proteins. Both “C” and “E” domains are involved in synapsin oligomerization. (B) The proposed mechanism of synaptic vesicle mobilization by synapsin 1a and CaMKs (98,232). Repeated stimulations of neurons increase the intracellular Ca2+ concentration, and subsequently activate CaMKs in presynaptic terminals. Activated CaMKs phosphorylate synapsin 1a, and phosphorylated synapsins are dissociated from the cluster composed of synaptic vesicles, synapsin, and actin filaments. This mobilizes synaptic vesicles for exocytosis from the synapsin/CaMK‐responsive synaptic vesicle pools.


Figure 10. Domain structure of cysteine string protein (CSP) and its function in synaptic vesicle trafficking. (A) The domain structure of CSP isoforms. CSPα is capable of interacting with various types of proteins such as synaptotagmin 1 (Syt1), the C2A domain of synaptotagmin 9 (Syt9C2A), SNAP25, syntaxin (Synt), synaptobrevin 2 (Syb2), small glutamine‐rich TPR protein (SGT), GTP‐binding protein (Gαβγ), and N‐type Ca2+ channel. PKA, protein kinase A; PKB, protein kinase B; HIP14, huntingtin‐interacting protein 14. (B) The major function of CSPα, as a component of the chaperone complex composed of Hsc70 and SGT, is the maintenance of the expression levels of SNAP25 and dynamin 1 (191,271). Both SNAP25 and dynamin 1 play critical roles in synaptic vesicle exocytosis and endocytosis.


Figure 11. Mechanism of the rab3‐mediated synaptic vesicle priming to the active zone in the plasma membrane. Rab3a, rab3b, rab3c, and rab27b are expressed in synaptic vesicles (235). GTP‐bound form of rab3/rab27b interacts with both RIM and Munc13 proteins (51). RIM protein is a component of the presynaptic active zone protein complex (228). Munc13 is located in presynaptic active zone and binds to plasma membrane in a Ca2+/diacylglycerol (DAG)‐dependent manner (178,213). Solid line between proteins represents an interaction. Munc13 also stimulates the SNARE complex formation that mediates exocytosis. RIM‐BP, RIM‐binding protein; DAG, diacylglycerol; PIP2, phosphatidylinositol 4,5‐bisphosphate.


Figure 12. The effects of cholesterol and phosphatidylethanolamine (PE) on the Ca2+‐dependent synaptotagmin 1 (Syt1)‐binding to the membrane. Phosphatidylethanolamine (PE) and cholesterol enhanced the Ca2+‐dependent Syt1C2A/B domain binding to the membrane (212) probably because: (i) both PE and cholesterol have smaller hydrophilic head groups than that of phosphatidylcholine (PC) and (ii) cholesterol has a much bulkier hydrophobic structure than that of PC. The small size of hydrophilic head group of phospholipids probably helps the penetration of the hydrophobic residues (M173, F234, V304, and I367) in the Ca2+‐binding loops of the Syt1C2A/B domain into the phospholipid bilayer upon Ca2+‐dependent binding.


Figure 13. Regulation of the SNARE complex formation by lipids. (A) Sphingosine exposes the SNARE motif of synaptobrevin 2 (Syb2) from the synaptic vesicular membrane (41), while arachidonic acid releases the Habc domain from the SNARE motif of syntaxin 1A (Synt1A) (39,65). Both sphingosine and arachidonic acid are capable of promoting the formation of SNARE complex, and subsequent synaptic vesicle exocytosis. (B) Chemical structures of sphingosine and arachidonic acid. Both lipids have chemical properties of detergents.
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Further Reading
 1. Chapman ER. How does synaptotagmin trigger neurotransmitter release? Annu Rev Biochem 77: 615–641, 2008.
 2. Jahn R , Lang T , Südhof TC . Membrane fusion. Cell 112: 519–533, 2003.
 3. Martin TF. Role of PI(4,5)P2 in vesicle exocytosis and membrane fusion. Subcell Biochem 59: 111–130, 2012.
 4. Südhof TC , Rizo J . Synaptic vesicle exocytosis. Cold Spring Harb Perspect Biol 3: a005637, 2011.

Further Reading
  • Chapman ER. How does synaptotagmin trigger neurotransmitter release? Annu Rev Biochem 77: 615-641, 2008.
  • Jahn R, Lang T, Südhof TC. Membrane fusion. Cell 112: 519-533, 2003.
  • Martin TF. Role of PI(4,5)P2 in vesicle exocytosis and membrane fusion. Subcell Biochem 59: 111-130, 2012.
  • Südhof TC, Rizo J. Synaptic vesicle exocytosis. Cold Spring Harb Perspect Biol 3: a005637, 2011.

 


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Article Title: Exocytosis and Synaptic Vesicle Function
 

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Ok‐Ho Shin. Exocytosis and Synaptic Vesicle Function. Compr Physiol 2014, 4: 149-175. doi: 10.1002/cphy.c130021