Kv channels inhibit release indirectly by hyperpolarizing membrane potential, but the significance of Kv channel interaction with the secretory apparatus is not known. The Kv2.1 channel is commonly expressed in the soma and dendrites of neurons, where it could influence the release of neuropeptides and neurotrophins, and in neuroendocrine cells, where it could influence hormone release. Here we show that Kv2.1 channels increase dense-core vesicle (DCV)-mediated release after elevation of cytoplasmic Ca 2ϩ . This facilitation occurs even after disruption of pore function and cannot be explained by changes in membrane potential and cytoplasmic Ca 2ϩ . However, triggering release increases channel binding to syntaxin, a secretory apparatus protein. Disrupting this interaction with competing peptides or by deleting the syntaxin association domain of the channel at the C terminus blocks facilitation of release. Thus, direct association of Kv2.1 with syntaxin promotes exocytosis. The dual functioning of the Kv channel to influence release, through its pore to hyperpolarize the membrane potential and through its C-terminal association with syntaxin to directly facilitate release, reinforces the requirements for repetitive firing for exocytosis of DCVs in neuroendocrine cells and in dendrites.

Interdomain interactions between intracellular N and C termini have been described for various K ؉ channels, including the voltage-gated Kv2.1, and suggested to affect channel gating. However, no channel regulatory protein directly affecting N/C interactions has been demonstrated. Most Kv2.1 channel interactions with regulatory factors occur at its C terminus. The vesicular SNARE that is also present at a high concentration in the neuronal plasma membrane, VAMP2, is the only protein documented to affect Kv2.1 gating by binding to its N terminus. As its binding target has been mapped near a site implicated in Kv2.1 N/C interactions, we hypothesized that VAMP2 binding to the N terminus requires concomitant conformational changes in the C terminus, which wraps around the N terminus from the outside, to give VAMP2 access. Here, we first determined that the Kv2.1 N terminus, although crucial, is not sufficient to convey functional interaction with VAMP2, and that, concomitant to its binding to the "docking loop" at the Kv2.1 N terminus, VAMP2 binds to the proximal part of the Kv2.1 C terminus, C1a. Next, using computational biology approaches (ab initio modeling, docking, and molecular dynamics simulations) supported by molecular biology, biochemical, electrophysiological, and fluorescence resonance energy transfer analyses, we mapped the interaction sites on both VAMP2 and Kv2.1 and found that this interaction is accompanied by rearrangements in the relative orientation of Kv2.1 cytoplasmic domains. We propose that VAMP2 modulates Kv2.1 inactivation by interfering with the interaction between the docking loop and C1a, a mechanism for gating regulation that may pertain also to other Kv channels.Interdomain interactions between intracellularly located N and C termini have been described for various K ϩ channels, including inwardly rectifying Kir2.3 and Kir6.2 (1, 2), small conductance Ca 2ϩ -activated (hSK3) (3), and voltage-gated Kv2.1 (4) and Kv4.1 (5) channels. In the case of Kv2.1, two modes of interaction have been proposed: an association of the distal part of Kv2.1 C terminus (termed CTA domain; amino acids (aa) 741-853) 4 with aa 67 and 75 of the Kv2.1 N terminus (4); or an association between the proximal part of the Kv2.1 C terminus (aa 444 -477) and the predicted loop structure (aa 55-71) in the N-terminal T1 domain (6). In addition, involvement of the S4-S5 linker in this interaction has been suggested (7). Although these studies propose two different C-terminal sites, they indicate a specific loop in the N terminus of Kv2.1 (6, 8), which could be functionally related to the Shaker and Shal docking loops in the lateral part of their T1 domains (9, 10). These latter loops are responsible for the subfamily-specific association with ␤-subunits (Kv␤ and KChIP, respectively). Further, the interaction between the N and C cytoplasmic termini (N/C interaction) of Kv2.1 has been shown to be dynamic and voltage-dependent and to involve structural rearrangements between these domains, which could affect both activati...

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...