Membrane fusion is a process that intimately involves both proteins and lipids. Although the SNARE proteins, which ultimately overcome the energy barrier for fusion, have been extensively studied, regulation of the energy barrier itself, determined by specific membrane lipids, has been largely overlooked. Our findings reveal a novel function for SNARE proteins in reducing the energy barrier for fusion, by directly binding and sequestering fusogenic lipids to sites of fusion. We demonstrate a specific interaction between Syntaxin1A and the fusogenic lipid phosphatidic acid, in addition to multiple polyphosphoinositide lipids, and define a polybasic juxtamembrane region within Syntaxin1A as its lipidbinding domain. In PC-12 cells, Syntaxin1A mutations that progressively reduced lipid binding resulted in a progressive reduction in evoked secretion. Moreover, amperometric analysis of fusion events driven by a lipid-binding-deficient Syntaxin1A mutant (5RK/A) demonstrated alterations in fusion pore dynamics, suggestive of an energetic defect in secretion. Overexpression of the phosphatidic acid-generating enzyme, phospholipase D1, completely rescued the secretory defect seen with the 5RK/A mutant. Moreover, knockdown of phospholipase D1 activity drastically reduced control secretion, while leaving 5RK/A-mediated secretion relatively unaffected. Altogether, these data suggest that Syntaxin1A-lipid interactions are a critical determinant of the energetics of SNARE-catalyzed fusion events. INTRODUCTIONMembrane fusion is a process that underlies compartmentalization within all eukaryotic cells, and allows for the many critical and diverse physiological functions in higher organisms. Despite the essential and ubiquitous nature of this process, a considerable energetic expenditure is required to overcome the electrostatic repulsion between opposing lipid bilayers and to deform and ultimately rupture these bilayers (Chernomordik and Kozlov, 2003;Cohen and Melikyan, 2004). As a result, substantial effort has been placed on defining the molecular machinery that overcomes this energetic barrier to accomplish regulated and rapid membrane fusion. SNARE (soluble n-ethylmaleimide-sensitive fusion factor attachment protein receptor) proteins have now been identified as the minimal protein machinery required for membrane fusion (Jahn and Scheller, 2006). Their critical role is supported by multiple lines of evidence, including that SNARE proteins are sufficient to drive membrane fusion when reconstituted into liposomes in vitro (Weber et al., 1998) and that cleavage of SNARE proteins by clostridial toxins (Schiavo et al., 1992; Blasi et al., 1993a,b), as well as genetic mutations resulting in loss of SNARE protein function (Broadie et al., 1995;Littleton et al., 1998;Saifee et al., 1998), strongly inhibit neurotransmitter release. Currently, the role of SNARE proteins in membrane fusion is believed to be predominantly mechanical. During neurotransmitter release, nucleation and zippering of a highly stable SNARE core complex formed f...

Voltage‐gated Na+ current is reduced by acid solution. Protons reduce peak Na+ conductance by lowering single channel conductance and shift the voltage range of gating by neutralizing surface charges. Structure‐function studies identify six carboxyls and a lysine in the channel's outer vestibule. We examined the roles of the superficial ring of carboxyls in acid block of Nav1.4 (the rat skeletal muscle Na+ channel isoform) by measuring the effects of their neutralization or their substitution by lysine on sensitivity to acid solutions, using the two‐micropipette voltage clamp in Xenopus oocytes. Alteration of the outer ring of carboxylates had little effect on the voltage for half‐activation of Na+ current, as if they are distant from the channels' voltage sensors. The mutations did not abolish proton block; rather, they all shifted the pKa (‐log of the dissociation constant) in the acid direction. Effects of neutralization on pKa were not identical for different mutations, with E758Q > D1241A > D1532N > E403Q. E758K showed double the effect of E758Q, and the other lysine mutations all produced larger effects than the neutralizing mutations. Calculation of the electrostatic potential produced by these carboxylates using a pore model showed that the pKa values of carboxylates of Glu‐403, Glu‐758, and Asp‐1532 are shifted to values similar to the experimentally measured pKa. Calculations also predict the experimentally observed changes in pKa that result from mutational neutralization or introduction of a positive charge. We propose that proton block results from partial protonation of these outer ring carboxylates and that all of the carboxylates contribute to a composite Na+ site.

Tomosyn is a 130-kDa cytosolic R-SNARE protein that associates with Q-SNAREs and reduces exocytotic activity. Two paralogous genes, tomosyn-1 and -2, occur in mammals and produce seven different isoforms via alternative splicing. Here, we map the structural differences between the yeast homologue of m-tomosyn-1, Sro7, and tomosyn genes/isoforms to identify domains critical to the regulation of exocytotic activity to tomosyn that are outside the soluble N-ethylmaleimide-sensitive attachment receptor motif. Homology modeling of m-tomosyn-1 based on the known structure of yeast Sro7 revealed a highly conserved functional conformation but with tomosyn containing three additional loop domains that emanate from a ␤-propeller core. Notably, deletion of loops 1 and 3 eliminates tomosyn inhibitory activity on secretion without altering its soluble N-ethylmaleimide-sensitive attachment receptor pairing with syntaxin1A. By comparison, deletion of loop 2, which contains the hypervariable splice region, did not reduce the ability of tomosyn to inhibit regulated secretion. However, exon variation within the hypervariable splice region resulted in significant differences in protein accumulation of tomosyn-2 isoforms. Functional analysis of s-tomosyn-1, m-tomosyn-1, m-tomosyn-2, and xb-tomosyn-2 demonstrated that they exert similar inhibitory effects on elevated K ؉ -induced secretion in PC12 cells, although m-tomosyn-2 was novel in strongly augmenting basal secretion. Finally, we report that m-tomosyn-1 is a target substrate for SUMO 2/3 conjugation and that mutation of this small ubiquitin-related modifier target site (Lys-730) enhances m-tomosyn-1 inhibition of secretion without altering interaction with syntaxin1A. Together these results suggest that multiple domains outside the R-SNARE of tomosyn are critical to the efficacy of inhibition by tomosyn on exocytotic secretion.Synaptic vesicle fusion and the subsequent release of neurotransmitter require the formation of heterotrimeric SNARE 2 complexes formed from plasma membrane proteins syntaxin1A and SNAP-25 (Q-SNAREs) with the synaptic vesicle membrane protein VAMP/synaptobrevin (R-SNARE) (1-3). Present on opposing membranes, these SNAREs combine and engage in thermodynamically stable coiled-coil interactions that bridge the two membranes and catalyze their fusion (4). The formation of SNARE complexes is spatially and temporally controlled by accessory components that lend additional specificity to SNARE pairing, arrest SNARE complex intermediates, and/or lower the energy required for fusion (4 -6). Ultimately, it is the functional activity of these regulators on SNARE complex assembly that determines the dynamics of the exocytotic event.Tomosyn is an important regulator of SNARE complex formation whose mechanism of action remains unclear. Initially identified in neurons (7-8), tomosyn, a soluble R-SNARE protein, was considered to be a negative effector of fusogenic SNARE complex assembly through interactions with syntaxin1A and SNAP-25 that preclude the binding of VAMP2,...

Tomosyn, a soluble R-SNARE protein identified as a binding partner of the Q-SNARE syntaxin 1A, is thought to be critical in setting the level of fusion-competent SNARE complexes for neurosecretion. To date, there has been no direct evaluation of the dynamics in which tomosyn transits through tomosyn-SNARE complexes or of the extent to which tomosyn-SNARE complexes are regulated by secretory demand. Here, we employed biochemical and optical approaches to characterize the dynamic properties of tomosyn-syntaxin 1A complexes in live adrenal chromaffin cells. We demonstrate that secretagogue stimulation results in the rapid translocation of tomosyn from the cytosol to plasma membrane regions and that this translocation is associated with an increase in the tomosyn-syntaxin 1A interaction, including increased cycling of tomosyn into tomosyn-SNARE complexes. The secretagogue-induced interaction was strongly reduced by pharmacological inhibition of the Rho-associated coiled-coil forming kinase, a result consistent with findings demonstrating secretagogue-induced activation of RhoA. Stimulation of chromaffin cells with lysophosphatidic acid, a nonsecretory stimulus that strongly activates RhoA, resulted in effects on tomosyn similar to that of application of the secretagogue. In PC-12 cells overexpressing tomosyn, secretagogue stimulation in the presence of lysophosphatidic acid resulted in reduced evoked secretory responses, an effect that was eliminated upon inhibition of Rho-associated coiled-coil forming kinase. Moreover, this effect required an intact interaction between tomosyn and syntaxin 1A. Thus, modulation of the tomosyn-syntaxin 1A interaction in response to secretagogue activation is an important mechanism allowing for dynamic regulation of the secretory response.Regulated neurotransmitter release requires the well orchestrated spatial and temporal actions of many presynaptic proteins (1). Although the primary molecular entities in the release pathway have been identified, the exact mechanics of synaptic vesicle fusion and its precise regulation are still not established. Central to the fusion process is the transient formation of SNARE 4 core complexes that include the target membrane SNARE proteins syntaxin 1A and SNAP25 and the vesicle SNARE protein synaptobrevin/VAMP (2-4). A SNARE core complex is a highly stable, four-␣-helix parallel bundle consisting of one SNARE motif from each of syntaxin 1A and synaptobrevin/VAMP, and two SNARE motifs from SNAP25 (5, 6). Although these proteins alone are sufficient to induce a slow fusion when reconstituted into liposomes (7), additional proteins are necessary to establish the properties that describe fast, Ca 2ϩ -dependent neurotransmitter release (8). For example, assembly of SNARE core complexes is subject to temporal and spatial regulation by a variety of protein families, including Rab-GTPases (9 -13), Sec/Munc18s (14 -16), exocyst tethering complexes (17-20), and Munc13s (21-24). In addition, recent evidence suggests that the temporal and spatial availability...