Non Channel Function of Kv2.1 to Facilitate Exocytosis; Underlying Molecular Mechanism

Abstract

WPB membrane composition and the mobilization of membrane and soluble cargo molecules retained within the post-fusion collapsed organelle. Exogenously applied (vibrant-DiI) or expressed plasma membrane markers (EGFP-Rab35) entered post-fusion WPB membranes. WPB membrane proteins showed differential changes in mobility; P-selectin-EGFP, immobile in the resting WPB membrane, became mobile, EGFP-CD63 mobility remained unchanged, and evidence was obtained for a slowing of EGFP-Rab27a mobility. The major core protein Von Willebrand factor (VWF) remained immobile, as well as in most cases the soluble VWF-propolypeptide. Using ECs expressing EGFP-CD63, it was found that post-fusion structures could support further cumulative fusion and exocytosis of WPBs. Together these data show that transient fusion of WPBs results in membrane mixing, a change in membrane identity, differential changes in cargo protein mobility and the formation of sites capable of supporting cumulative WPB exocytosis. At the first auditory synapse, the ribbon synapse of inner hair cell, neurotransmitter release is triggered by Ca 2þ influx through clusters of Ca v 1.3 channels. Previous work in our lab has shown marked heterogeneity in the size of Ca 2þchannel cluster (Meyer et al., 2009), as well as the amplitude and voltage dependence of microdomain Ca 2þ (Frank et al. 2009) at active zones within single inner hair cells. Such heterogeneity of presynaptic Ca 2þ signaling may contribute to the divergent sound coding properties of the postsynaptic spiral ganglion neurons that connect to the same hair cell. While potentially relevant for explaining large dynamic range sound encoding, its underlying mechanisms and the link to transmitter release are not well understood. Here we aimed at estimating the number of functional Ca 2þ channels at the single synapses by means of an optical fluctuation analysis approach. The fluctuation analysis approach is compared to whole-cell Ca 2þ current measurements in which single-synapse resolution is achieved by local pharmacological manipulation. In order to more systematically address synaptic heterogeneity we combined hair cell patch-clamp with spinning-disk confocal microscopy for simultaneous fast confocal Ca 2þ imaging at multiple active zones. This method also allows us to analyze microdomain properties as a function of location in 3D-reconstructed hair cells. In addition, measurement of exocytosis at individual synapse is essential in linking Ca 2þ signal amplitude to transmitter release. We are currently working on using optical reporters to monitor vesicle fusion at confocal resolution. Preliminary work demonstrates localized changes in VGLUT1-pHluorin fluorescence in response to voltage-gated Ca 2þ influx. Astrocytes are the most abundant cells of the central nervous system (CNS). In the CNS, astrocytes form part of the blood brain barrier, supply neurons and oligodendrocytes with substrates for energy metabolism, control homeostasis, regulate neurotransmitter release and modulate the immune response. Evid...