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Structure of membrane-associated neuronal SNARE complex: implication in neurotransmitter release.

Cho WJ, Shin L, Ren G, Jena BP - J. Cell. Mol. Med. (2009)

Bottom Line: Studies demonstrate the presence of SNAREs at the porosome base.Atomic force microscopy (AFM), electron microscopy (EM), and electron density measurement studies demonstrate that at the porosome base, where synaptic vesicles dock and transiently fuse, proteins, possibly comprised of t-SNAREs, are found assembled in a ring conformation.Our results demonstrate formation of 6-7 nm membrane-directed self-assembled t-/v-SNARE ring complexes, similar to, but twice as large as the ring structures present at the base of neuronal porosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201, USA.

ABSTRACT
To enable fusion between biological membranes, t-SNAREs and v-SNARE present in opposing bilayers, interact and assemble in a circular configuration forming ring-complexes, which establish continuity between the opposing membranes, in presence of calcium ions. The size of a t-/v-SNARE ring complex is dictated by the curvature of the opposing membrane. Hence smaller vesicles form small SNARE-ring complexes, as opposed to large vesicles. Neuronal communication depends on the fusion of 40-50 nm in diameter membrane-bound synaptic vesicles containing neurotransmitters at the nerve terminal. At the presynaptic membrane, 12-17 nm in diameter cup-shaped neuronal porosomes are present where synaptic vesicles transiently dock and fuse. Studies demonstrate the presence of SNAREs at the porosome base. Atomic force microscopy (AFM), electron microscopy (EM), and electron density measurement studies demonstrate that at the porosome base, where synaptic vesicles dock and transiently fuse, proteins, possibly comprised of t-SNAREs, are found assembled in a ring conformation. To further determine the structure and arrangement of the neuronal t-/v-SNARE complex, 50 nm t-and v-SNARE proteoliposomes were mixed, allowing t-SNARE-vesicles to interact with v-SNARE vesicles, followed by detergent solubilization and imaging of the resultant t-/v-SNARE complexes formed using both AFM and EM. Our results demonstrate formation of 6-7 nm membrane-directed self-assembled t-/v-SNARE ring complexes, similar to, but twice as large as the ring structures present at the base of neuronal porosomes. The smaller SNARE ring at the porosome base may reflect the 3-4 nm base diameter, where 40-50 nm in diameter v-SNARE-associated synaptic vesicle transiently dock and fuse to release neurotransmitters.

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Opposing bilayers containing t- and v-SNAREs, respectively, interact in a circular array to form conducting channels in presence of calcium. (A) Schematic diagram of the bilayer-electrophysiology setup (EPC9). (B) Lipid vesicle containing nystatin channels (red) and membrane bilayer with SNAREs demonstrate significant changes in capacitance and conductance. When t-SNARE vesicles were added to a v-SNARE membrane support, the SNAREs in opposing bilayers arranged in a ring pattern, forming pores (as seen in the AFM micrograph on the extreme right) and there were seen stepwise increases in capacitance and conductance (–60 mV holding potential). Docking and fusion of the vesicle at the bilayer membrane open vesicle-associated nystatin channels and SNARE-induced pore formation, allowing conductance of ions from cis to the trans side of the bilayer membrane. Then further addition of KCl to induce gradient-driven fusion resulted in little or no further increase in conductance and capacitance, demonstrating that docked vesicles have already fused [6]. (C) t-/v-SNARE ring complex at low and high resolution (D) is shown. Bar = 100 nm. (E–G) The size of the t-/v-SNARE complex is directly proportional to the size of the SNARE-reconstituted vesicles [8]. (E) Schematic diagram depicting the interaction of t-SNARE-reconstituted and v-SNARE-reconstituted liposomes [8]. (F) AFM images of vesicle before and after their removal using the AFM cantilever tip, exposing the t-/v-SNARE-ring complex at the centre. (G) Note the high correlation coefficient between vesicle diameter and size of the SNARE complex [8]. (H) Schematic diagram depicting the possible molecular mechanism of SNARE ring complex formation, when t-SNARE-vesicles and V-SNARE-vesicles meet. The process may occur due to a progressive recruitment of t-/v-SNARE pairs as the opposing vesicles are pulled toward each other, until a complete ring is established, preventing any further recruitment of t-/v-SNARE pairs to the complex. The top panel is a side view of two vesicles (one t-SNARE-reconstituted, and the other v-SNARE reconstituted) interacting to form a single t-/v-SNARE complex, leading progressively (from left to right) to the formation of the ring complex. The lower panel is a top view of the two interacting vesicles [16].
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fig01: Opposing bilayers containing t- and v-SNAREs, respectively, interact in a circular array to form conducting channels in presence of calcium. (A) Schematic diagram of the bilayer-electrophysiology setup (EPC9). (B) Lipid vesicle containing nystatin channels (red) and membrane bilayer with SNAREs demonstrate significant changes in capacitance and conductance. When t-SNARE vesicles were added to a v-SNARE membrane support, the SNAREs in opposing bilayers arranged in a ring pattern, forming pores (as seen in the AFM micrograph on the extreme right) and there were seen stepwise increases in capacitance and conductance (–60 mV holding potential). Docking and fusion of the vesicle at the bilayer membrane open vesicle-associated nystatin channels and SNARE-induced pore formation, allowing conductance of ions from cis to the trans side of the bilayer membrane. Then further addition of KCl to induce gradient-driven fusion resulted in little or no further increase in conductance and capacitance, demonstrating that docked vesicles have already fused [6]. (C) t-/v-SNARE ring complex at low and high resolution (D) is shown. Bar = 100 nm. (E–G) The size of the t-/v-SNARE complex is directly proportional to the size of the SNARE-reconstituted vesicles [8]. (E) Schematic diagram depicting the interaction of t-SNARE-reconstituted and v-SNARE-reconstituted liposomes [8]. (F) AFM images of vesicle before and after their removal using the AFM cantilever tip, exposing the t-/v-SNARE-ring complex at the centre. (G) Note the high correlation coefficient between vesicle diameter and size of the SNARE complex [8]. (H) Schematic diagram depicting the possible molecular mechanism of SNARE ring complex formation, when t-SNARE-vesicles and V-SNARE-vesicles meet. The process may occur due to a progressive recruitment of t-/v-SNARE pairs as the opposing vesicles are pulled toward each other, until a complete ring is established, preventing any further recruitment of t-/v-SNARE pairs to the complex. The top panel is a side view of two vesicles (one t-SNARE-reconstituted, and the other v-SNARE reconstituted) interacting to form a single t-/v-SNARE complex, leading progressively (from left to right) to the formation of the ring complex. The lower panel is a top view of the two interacting vesicles [16].

Mentions: A general understanding of membrane fusion in cells has been made possible following discovery of an N-ethylmaleimide-sensitive factor (NSF) [1] and SNARE proteins [2–4], and the mechanism of their participation [5–11]. Target membrane proteins at the cell plasma membrane SNAP-25 and syntaxin termed t-SNAREs, and secretory vesicle-associated membrane protein VAMP or v-SNARE, are part of the conserved protein complex involved in fusion of opposing cellular membranes. VAMP and syntaxin are both integral membrane proteins, whereas the soluble SNAP-25 protein associates with syntaxin. Therefore, the key to our understanding of SNARE-induced membrane fusion requires determination of the atomic arrangement and interaction between membrane-associated v- and t-SNAREs. Ideally, the atomic coordinates of membrane-associated SNARE complex using x-ray crystallography would help elucidate the chemistry of SNARE-induced membrane fusion in cells. So far such structural details at the atomic level of membrane-associated t-/v-SNARE complex have not been realized, primarily due to solubility problems of membrane-associated SNAREs and due to the fact that v-SNARE and t-SNAREs need to reside in opposing membranes when they meet to be able to assemble in a physiologically relevant conformation [6, 8, 9]. The remaining option has been the use of nuclear magnetic resonance spectroscopy (NMR), which too has been unsuccessful due to the size of the t-/v-SNARE complex being larger than current NMR capabilities. Regardless of these setbacks, atomic force microscopy (AFM) force spectroscopy has provided, at nm resolution, an understanding of the structure, assembly, and disassembly of membrane-associated t-/v-SNARE complex, in physiological buffered solution [6, 8, 9, 11]. The structure and arrangement of SNARE-associated with lipid bilayer were first determined using AFM (Fig. 1) [6]. A bilayer electrophysiological setup allowed measurements of membrane conductance and capacitance during fusion of v-SNARE-reconstituted liposomes with t-SNARE-reconstituted membrane, and vice-versa. Results from these studies demonstrated that t-SNAREs and v-SNARE when present in opposing membranes interact and assemble in a circular array (ring complexes), and form conducting channels in presence of calcium [6]. The interaction between t-SNAREs and v-SNARE proteins to form such conducting channel is strictly dependent on the association of SNAREs in opposing bilayers. In the absence of membrane, either v-SNARE or t-SNAREs, fail to appropriately interact and form the ring complex, and to establish continuity between the opposing bilayers [6]. The size of the t-/v-SNARE complex is dictated by the curvature of the opposing membranes hence smaller vesicles form smaller SNARE-ring complexes (Fig. 1) [8].


Structure of membrane-associated neuronal SNARE complex: implication in neurotransmitter release.

Cho WJ, Shin L, Ren G, Jena BP - J. Cell. Mol. Med. (2009)

Opposing bilayers containing t- and v-SNAREs, respectively, interact in a circular array to form conducting channels in presence of calcium. (A) Schematic diagram of the bilayer-electrophysiology setup (EPC9). (B) Lipid vesicle containing nystatin channels (red) and membrane bilayer with SNAREs demonstrate significant changes in capacitance and conductance. When t-SNARE vesicles were added to a v-SNARE membrane support, the SNAREs in opposing bilayers arranged in a ring pattern, forming pores (as seen in the AFM micrograph on the extreme right) and there were seen stepwise increases in capacitance and conductance (–60 mV holding potential). Docking and fusion of the vesicle at the bilayer membrane open vesicle-associated nystatin channels and SNARE-induced pore formation, allowing conductance of ions from cis to the trans side of the bilayer membrane. Then further addition of KCl to induce gradient-driven fusion resulted in little or no further increase in conductance and capacitance, demonstrating that docked vesicles have already fused [6]. (C) t-/v-SNARE ring complex at low and high resolution (D) is shown. Bar = 100 nm. (E–G) The size of the t-/v-SNARE complex is directly proportional to the size of the SNARE-reconstituted vesicles [8]. (E) Schematic diagram depicting the interaction of t-SNARE-reconstituted and v-SNARE-reconstituted liposomes [8]. (F) AFM images of vesicle before and after their removal using the AFM cantilever tip, exposing the t-/v-SNARE-ring complex at the centre. (G) Note the high correlation coefficient between vesicle diameter and size of the SNARE complex [8]. (H) Schematic diagram depicting the possible molecular mechanism of SNARE ring complex formation, when t-SNARE-vesicles and V-SNARE-vesicles meet. The process may occur due to a progressive recruitment of t-/v-SNARE pairs as the opposing vesicles are pulled toward each other, until a complete ring is established, preventing any further recruitment of t-/v-SNARE pairs to the complex. The top panel is a side view of two vesicles (one t-SNARE-reconstituted, and the other v-SNARE reconstituted) interacting to form a single t-/v-SNARE complex, leading progressively (from left to right) to the formation of the ring complex. The lower panel is a top view of the two interacting vesicles [16].
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4496122&req=5

fig01: Opposing bilayers containing t- and v-SNAREs, respectively, interact in a circular array to form conducting channels in presence of calcium. (A) Schematic diagram of the bilayer-electrophysiology setup (EPC9). (B) Lipid vesicle containing nystatin channels (red) and membrane bilayer with SNAREs demonstrate significant changes in capacitance and conductance. When t-SNARE vesicles were added to a v-SNARE membrane support, the SNAREs in opposing bilayers arranged in a ring pattern, forming pores (as seen in the AFM micrograph on the extreme right) and there were seen stepwise increases in capacitance and conductance (–60 mV holding potential). Docking and fusion of the vesicle at the bilayer membrane open vesicle-associated nystatin channels and SNARE-induced pore formation, allowing conductance of ions from cis to the trans side of the bilayer membrane. Then further addition of KCl to induce gradient-driven fusion resulted in little or no further increase in conductance and capacitance, demonstrating that docked vesicles have already fused [6]. (C) t-/v-SNARE ring complex at low and high resolution (D) is shown. Bar = 100 nm. (E–G) The size of the t-/v-SNARE complex is directly proportional to the size of the SNARE-reconstituted vesicles [8]. (E) Schematic diagram depicting the interaction of t-SNARE-reconstituted and v-SNARE-reconstituted liposomes [8]. (F) AFM images of vesicle before and after their removal using the AFM cantilever tip, exposing the t-/v-SNARE-ring complex at the centre. (G) Note the high correlation coefficient between vesicle diameter and size of the SNARE complex [8]. (H) Schematic diagram depicting the possible molecular mechanism of SNARE ring complex formation, when t-SNARE-vesicles and V-SNARE-vesicles meet. The process may occur due to a progressive recruitment of t-/v-SNARE pairs as the opposing vesicles are pulled toward each other, until a complete ring is established, preventing any further recruitment of t-/v-SNARE pairs to the complex. The top panel is a side view of two vesicles (one t-SNARE-reconstituted, and the other v-SNARE reconstituted) interacting to form a single t-/v-SNARE complex, leading progressively (from left to right) to the formation of the ring complex. The lower panel is a top view of the two interacting vesicles [16].
Mentions: A general understanding of membrane fusion in cells has been made possible following discovery of an N-ethylmaleimide-sensitive factor (NSF) [1] and SNARE proteins [2–4], and the mechanism of their participation [5–11]. Target membrane proteins at the cell plasma membrane SNAP-25 and syntaxin termed t-SNAREs, and secretory vesicle-associated membrane protein VAMP or v-SNARE, are part of the conserved protein complex involved in fusion of opposing cellular membranes. VAMP and syntaxin are both integral membrane proteins, whereas the soluble SNAP-25 protein associates with syntaxin. Therefore, the key to our understanding of SNARE-induced membrane fusion requires determination of the atomic arrangement and interaction between membrane-associated v- and t-SNAREs. Ideally, the atomic coordinates of membrane-associated SNARE complex using x-ray crystallography would help elucidate the chemistry of SNARE-induced membrane fusion in cells. So far such structural details at the atomic level of membrane-associated t-/v-SNARE complex have not been realized, primarily due to solubility problems of membrane-associated SNAREs and due to the fact that v-SNARE and t-SNAREs need to reside in opposing membranes when they meet to be able to assemble in a physiologically relevant conformation [6, 8, 9]. The remaining option has been the use of nuclear magnetic resonance spectroscopy (NMR), which too has been unsuccessful due to the size of the t-/v-SNARE complex being larger than current NMR capabilities. Regardless of these setbacks, atomic force microscopy (AFM) force spectroscopy has provided, at nm resolution, an understanding of the structure, assembly, and disassembly of membrane-associated t-/v-SNARE complex, in physiological buffered solution [6, 8, 9, 11]. The structure and arrangement of SNARE-associated with lipid bilayer were first determined using AFM (Fig. 1) [6]. A bilayer electrophysiological setup allowed measurements of membrane conductance and capacitance during fusion of v-SNARE-reconstituted liposomes with t-SNARE-reconstituted membrane, and vice-versa. Results from these studies demonstrated that t-SNAREs and v-SNARE when present in opposing membranes interact and assemble in a circular array (ring complexes), and form conducting channels in presence of calcium [6]. The interaction between t-SNAREs and v-SNARE proteins to form such conducting channel is strictly dependent on the association of SNAREs in opposing bilayers. In the absence of membrane, either v-SNARE or t-SNAREs, fail to appropriately interact and form the ring complex, and to establish continuity between the opposing bilayers [6]. The size of the t-/v-SNARE complex is dictated by the curvature of the opposing membranes hence smaller vesicles form smaller SNARE-ring complexes (Fig. 1) [8].

Bottom Line: Studies demonstrate the presence of SNAREs at the porosome base.Atomic force microscopy (AFM), electron microscopy (EM), and electron density measurement studies demonstrate that at the porosome base, where synaptic vesicles dock and transiently fuse, proteins, possibly comprised of t-SNAREs, are found assembled in a ring conformation.Our results demonstrate formation of 6-7 nm membrane-directed self-assembled t-/v-SNARE ring complexes, similar to, but twice as large as the ring structures present at the base of neuronal porosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201, USA.

ABSTRACT
To enable fusion between biological membranes, t-SNAREs and v-SNARE present in opposing bilayers, interact and assemble in a circular configuration forming ring-complexes, which establish continuity between the opposing membranes, in presence of calcium ions. The size of a t-/v-SNARE ring complex is dictated by the curvature of the opposing membrane. Hence smaller vesicles form small SNARE-ring complexes, as opposed to large vesicles. Neuronal communication depends on the fusion of 40-50 nm in diameter membrane-bound synaptic vesicles containing neurotransmitters at the nerve terminal. At the presynaptic membrane, 12-17 nm in diameter cup-shaped neuronal porosomes are present where synaptic vesicles transiently dock and fuse. Studies demonstrate the presence of SNAREs at the porosome base. Atomic force microscopy (AFM), electron microscopy (EM), and electron density measurement studies demonstrate that at the porosome base, where synaptic vesicles dock and transiently fuse, proteins, possibly comprised of t-SNAREs, are found assembled in a ring conformation. To further determine the structure and arrangement of the neuronal t-/v-SNARE complex, 50 nm t-and v-SNARE proteoliposomes were mixed, allowing t-SNARE-vesicles to interact with v-SNARE vesicles, followed by detergent solubilization and imaging of the resultant t-/v-SNARE complexes formed using both AFM and EM. Our results demonstrate formation of 6-7 nm membrane-directed self-assembled t-/v-SNARE ring complexes, similar to, but twice as large as the ring structures present at the base of neuronal porosomes. The smaller SNARE ring at the porosome base may reflect the 3-4 nm base diameter, where 40-50 nm in diameter v-SNARE-associated synaptic vesicle transiently dock and fuse to release neurotransmitters.

Show MeSH
Related in: MedlinePlus