Zhao Wang

Synaptotagmin (syt) 7 is one of three syt isoforms found in all metazoans; it is ubiquitously expressed, yet its function in neurons remains obscure. Here, we resolved Ca²⁺-dependent and Ca²⁺-independent synaptic vesicle (SV) replenishment pathways, and found that syt 7 plays a selective and critical role in the Ca²⁺-dependent pathway. Mutations that disrupt Ca²⁺-binding to syt 7 abolish this function, suggesting that syt 7 functions as a Ca²⁺-sensor for replenishment. The Ca²⁺-binding protein calmodulin (CaM) has also been implicated in SV replenishment, and we found that loss of syt 7 was phenocopied by a CaM antagonist. Moreover, we discovered that syt 7 binds to CaM in a highly specific and Ca²⁺-dependent manner; this interaction requires intact Ca²⁺-binding sites within syt 7. Together, these data indicate that a complex of two conserved Ca²⁺-binding proteins, syt 7 and CaM, serve as a key regulator of SV replenishment in presynaptic nerve terminals.

Synaptotagmin is the major calcium sensor for fast synaptic transmission that requires the synchronous fusion of synaptic vesicles. Synaptotagmin contains two calcium-binding domains: C₂A and C₂B. Mutation of a positively charged residue (R233Q in rat) showed that Ca²⁺-dependent interactions between the C₂A domain and membranes play a role in the electrostatic switch that initiates fusion. Surprisingly, aspartate-to-asparagine mutations in C₂A that inhibit Ca²⁺binding support efficient synaptic transmission, suggesting that Ca²⁺binding by C₂A is not required for triggering synchronous fusion. Based on a structural analysis, we generated a novel mutation of a single Ca²⁺-binding residue in C₂A (D229E inDrosophila) that inhibited Ca²⁺binding but maintained the negative charge of the pocket. This C₂A aspartate-to-glutamate mutation resulted in ∼80% decrease in synchronous transmitter release and a decrease in the apparent Ca²⁺affinity of release. Previous aspartate-to-asparagine mutations in C₂A partially mimicked Ca²⁺binding by decreasing the negative charge of the pocket. We now show that the major function of Ca²⁺binding to C₂A is to neutralize the negative charge of the pocket, thereby unleashing the fusion-stimulating activity of synaptotagmin. Our results demonstrate that Ca²⁺binding by C₂A is a critical component of the electrostatic switch that triggers synchronous fusion. Thus, Ca²⁺binding by C₂B is necessary and sufficient to regulate the precise timing required for coupling vesicle fusion to Ca²⁺influx, but Ca²⁺binding by both C₂domains is required to flip the electrostatic switch that triggers efficient synchronous synaptic transmission.

The synaptic vesicle protein synaptotagmin I (syt) promotes exocytosis via its ability to penetrate membranes in response to binding Ca2+ and through direct interactions with SNARE proteins. However, studies using full-length (FL) membrane-embedded syt in reconstituted fusion assays have yielded conflicting results, including a lack of effect, or even inhibition of fusion, by Ca2+. In this paper, we show that reconstituted FL syt promoted rapid docking of vesicles (<1 min) followed by a priming step (3–9 min) that was required for subsequent Ca2+-triggered fusion between v- and t-SNARE liposomes. Moreover, fusion occurred only when phosphatidylinositol 4,5-bisphosphate was included in the target membrane. This system also recapitulates some of the effects of syt mutations that alter synaptic transmission in neurons. Finally, we demonstrate that the cytoplasmic domain of syt exhibited mixed agonist/antagonist activity during regulated membrane fusion in vitro and in cells. Together, these findings reveal further convergence of reconstituted and cell-based systems.

Many cells release multiple substances in different proportions according to the specific character of a stimulus. PC12 cells, a model neuroendocrine cell line, express multiple isoforms of the exocytotic Ca²⁺sensor synaptotagmin. We show that these isoforms sort to populations of dense-core vesicles that differ in size. These synaptotagmins differ in their Ca²⁺sensitivities, their preference for full fusion or kiss-and-run, and their sensitivity to inhibition by synaptotagmin IV. In PC12 cells, vesicles that harbor these different synaptotagmin isoforms can be preferentially triggered to fuse by different forms of stimulation. The mode of fusion is specified by the synaptotagmin isoform activated, and because kiss-and-run exocytosis can filter small molecules through a size-limiting fusion pore, the activation of isoforms that favor kiss-and-run will select smaller molecules over larger molecules packaged in the same vesicle. Thus synaptotagmin isoforms can provide multiple levels of control in the release of different molecules from the same cell.

Synaptotagmin-1 aggregates membranes in response to Ca2+. Using a variety of approaches, it was discovered that synaptotagmin-mediated membrane aggregation occurs through trans interactions between synaptotagmin molecules bound to different membranes. Synaptotagmin-regulated, SNARE-catalyzed fusion further requires synaptotagmin to drive the assembly of nonfusogenic syntaxin and SNAP25 into fusion-competent t-SNARE heterodimers. Synaptotagmin-1 is a Ca2+ sensor that triggers synchronous neurotransmitter release. The first documented biochemical property of synaptotagmin-1 was its ability to aggregate membranes in response to Ca2+. However, the mechanism and function of this process were poorly understood. Here we show that synaptotagmin-1–mediated vesicle aggregation is driven by trans interactions between synaptotagmin-1 molecules bound to different membranes. We found a strong correlation between the ability of Ca2+-bound synaptotagmin-1 to aggregate vesicles and to stimulate SNARE-mediated membrane fusion. Moreover, artificial aggregation of membranes—using non-synaptotagmin proteins—also efficiently promoted fusion of SNARE-bearing liposomes. Finally, using a modified fusion assay, we observed that synaptotagmin-1 drove the assembly of otherwise non-fusogenic individual t-SNARE proteins into fusion-competent heterodimers, independently of aggregation. Thus, membrane aggregation and t-SNARE assembly appear to be two key aspects of fusion reactions that are regulated by Ca2+-bound synaptotagmin-1 and catalyzed by SNAREs.

The vesicle protein synaptotagmin I is the Ca²⁺sensor that triggers fast, synchronous release of neurotransmitter. Specifically, Ca²⁺binding by the C₂B domain of synaptotagmin is required at intact synapses, yet the mechanism whereby Ca²⁺binding results in vesicle fusion remains controversial. Ca²⁺-dependent interactions between synaptotagmin and SNARE (solubleN-ethylmaleimide-sensitive fusion protein attachment receptor) complexes and/or anionic membranes are possible effector interactions. However, no effector-interaction mutations to date impact synaptic transmission as severely as mutation of the C₂B Ca²⁺-binding motif, suggesting that these interactions are facilitatory rather than essential. Here we useDrosophilato show the functional role of a highly conserved, hydrophobic residue located at the tip of each of the two Ca²⁺-binding pockets of synaptotagmin. Mutation of this residue in the C₂A domain (F286) resulted in a ∼50% decrease in evoked transmitter release at an intact synapse, again indicative of a facilitatory role. Mutation of this hydrophobic residue in the C₂B domain (I420), on the other hand, blocked all locomotion, was embryonic lethal even in syt I heterozygotes, and resulted in less evoked transmitter release than that insyt^nullmutants, which is more severe than the phenotype of C₂B Ca²⁺-binding mutants. Thus, mutation of a single, C₂B hydrophobic residue required for Ca²⁺-dependent penetration of anionic membranes results in the most severe disruption of synaptotagmin functionin vivoto date. Our results provide direct support for the hypothesis that plasma membrane penetration, specifically by the C₂B domain of synaptotagmin, is the critical effector interaction for coupling Ca²⁺binding with vesicle fusion.

Aqueous humor is formed by fluid transfer from the ciliary stroma sequentially across the pigmented ciliary epithelial (PE) cells, gap junctions, and nonpigmented ciliary epithelial (NPE) cells. Which connexins (Cx) contribute to PE-NPE gap junctional formation appears species specific. We tested whether small interfering RNA (siRNA) against Cx43 (siCx43) affects bovine PE-NPE communication and whether cAMP affects communication. Native bovine ciliary epithelial cells were studied by dual-cell patch clamping, Lucifer Yellow (LY) transfer, quantitative polymerase chain reaction with reverse transcription (qRT-PCR), and Western immunoblot. qRT-PCR revealed at least 100-fold greater expression for Cx43 than Cx40. siCx43 knocked down target mRNA expression by 55 ± 7% after 24 h, compared with nontargeting control siRNA (NTC1) transfection. After 48 h, siCx43 reduced Cx43 protein expression and LY transfer. The ratio of fluorescence intensity (R_f) in recipient to donor cell was 0.47 ± 0.09 ( n = 11) 10 min after whole cell patch formation in couplets transfected with NTC1. siCx43 decreased R_fby ∼60% to 0.20 ± 0.07 ( n = 13, P < 0.02). Dibutyryl-cAMP (500 μM) also reduced LY dye transfer by ∼60%, reducing R_ffrom 0.41 ± 0.05 ( n = 15) to 0.17 ± 0.05 ( n = 20) after 10 min. Junctional currents were lowered by ∼50% ( n = 6) after 10-min perfusion with 500 μM dibutyryl-cAMP ( n = 6); thereafter, heptanol abolished the currents ( n = 5). Preincubation with the PKA inhibitor H-89 (2 μM) prevented cAMP-triggered current reduction ( n = 6). We conclude that 1) Cx43, but not Cx40, is a major functional component of bovine PE-NPE gap junctions; and 2) under certain conditions, cAMP may act through PKA to inhibit bovine PE-NPE gap junctional communication.

Purpose. Conditional inactivation of connexin43 (Cx43) in the pigmented epithelium of the mouse eye results in a reduction in aqueous humor production and complete loss of the vitreous chamber. It was proposed that gap junctions between pigmented and nonpigmented epithelia of the ciliary body are critical for the production of the aqueous humor. To form such junctions, Cx43 in the pigmented epithelium must interact with connexin(s) present in the adjacent cells of the nonpigmented epithelium. The importance of Cx43 expression in the nonpigmented epithelium for the establishment of gap junctions and the regulation of intraocular pressure was tested. methods. To inactivate Cx43 in the nonpigmented epithelium of the mouse eye, a mouse line was crossed with a floxed Cx43 locus (Cx43^flox/flox) and a transgenic mouse line expressing cre recombinase under the control of the Pax6α promoter. General eye structure was evaluated by light microscopy, gap junctions were analyzed by electron microscopy, and intraocular pressure was directly assessed with micropipettes. results. In Pax6α-cre/Cx43^flox/flox mice, Cx43 was partially inactivated in the nonpigmented epithelium of the ciliary body and iris. Animals developed dilatations between the pigmented and nonpigmented epithelia and displayed a significant reduction in intraocular pressure. However, gap junctions between the ciliary epithelial layers were decreased but not eliminated. conclusions. Cx43 expression in the nonpigmented epithelium of the ciliary body contributes to the formation of gap junctions with the cells of the pigmented epithelium. These gap junctions play a critical role in maintaining the physical integrity of the ciliary body epithelium. Although the partial loss of Cx43 from the nonpigmented epithelium was correlated with a measurable drop in intraocular pressure, possible changes in Cx43 in the aqueous outflow pathway may provide an additional contribution to the observed phenotype.