No abstract

Although it is well established that exocytosis of neurotransmitters and hormones is highly regulated by numerous secretory proteins, such as SNARE proteins, there is an increasing appreciation of the importance of the chemophysical properties and organization of membrane lipids to various aspects of the exocytotic program. Based on amperometric recordings by carbon fiber microelectrodes, we show that deprivation of membrane cholesterol by methyl-beta-cyclodextrin not only inhibited the extent of membrane depolarization-induced exocytosis, it also adversely affected the kinetics and quantal size of vesicle fusion in neuroendocrine PC12 cells. In addition, total internal fluorescence microscopy studies revealed that cholesterol depletion impaired vesicle docking and trafficking, which are believed to correlate with the dynamics of exocytosis. Furthermore, we found that free cholesterol is able to directly trigger vesicle fusion, albeit with less potency and slower kinetics as compared to membrane depolarization stimulation. These results underscore the versatile roles of cholesterol in facilitating exocytosis.

The tandem C2-domains of synaptotagmin 1 (syt) function as Ca2+-binding modules that trigger exocytosis; in the absence of Ca2+, syt inhibits spontaneous release. Here, we used proline linkers to constrain and alter the relative orientation of these C2-domains. Short poly-proline helices have a period of three, so large changes in the relative disposition of the C2-domains result from changing the length of the poly-proline linker by a single residue. The length of the linker was varied one residue at a time, revealing a periodicity of three for the ability of the linker mutants to interact with anionic phospholipids and drive evoked synaptic transmission; syt efficiently drove exocytosis when its tandem C2-domains pointed in the same direction. Analysis of spontaneous release revealed a reciprocal relationship between the activation and clamping activities of the linker mutants. Hence, different structural states of syt underlie the control of distinct forms of synaptic transmission.

The Ca2+ sensor for rapid synaptic vesicle exocytosis, synaptotagmin I (syt), is largely composed of two Ca2+-sensing C2-domains, C2A and C2B. We have investigated the apparent synergy between the tandem C2 domains by altering the length and rigidity of the linker that connects them. The behavior of the linker mutants revealed a correlation between the ability of the C2-domains to penetrate membranes in response to Ca2+ and to drive evoked neurotransmitter release in cultured mouse neurons, uncovering a step in excitation-secretion coupling. Atomic force microscopy experiments indicate that the synergy between these C2-domains involves intra-molecular interactions between them. Thus, syt function is profoundly affected by changes in the physical nature of the linker that connects its tandem C2-domains. Moreover, the linker mutations uncoupled syt-mediated regulation of evoked and spontaneous release, revealing that syt also acts as a fusion clamp prior to the Ca2+ trigger.