The transient receptor potential (TRP) 2 channel proteins comprise six transmembrane domains and multimerize to form ion channel complexes. Among the known families of ion channels, TRPs are unique in displaying an impressive diversity of cation selectivities, activation mechanisms, and functions (1). Specifically, members of the "canonical" TRP (TRPC) subfamily are non-selective cation channels that cause eventually Ca 2ϩ entry and collapse of the cell membrane potential (2). TRPCs are readily activated after stimulation of receptor-tyrosine kinases or G-protein-coupled receptors that activate the phospholipase C signaling pathway (3). It is believed that diacylglycerol, a product of the phospholipase C signaling pathway, activates ion channels formed by TRPC3, TRPC6, and TRPC7 (4), although the activation mechanisms of TRPC1, TRPC4, and TRPC5 appear to be more complex (5, 6).TRPC5 is enriched in the brain, where it is believed to control neurite extension and growth cone morphology (7,8). TRPC5-deficient mice exhibit diminished innate fear levels, suggesting an essential role for TRPC5 in the function of the amygdala (9). Furthermore, TRPC5 has been implicated in endothelial and mast cell function as well as in rheumatoid arthritis (2). In growth cones TRPC5 forms homomeric channels, but it forms heteromultimers with TRPC1 to build TRPC1/TRPC5 ion channels in the soma of neurones (8). Numerous stimuli are apparently able to control the activity of TRPC5 channels. By interacting with extracellularly located binding sites, thioredoxin, protons, and lanthanides enhance TRPC5 channel currents (10 -13). Acting intracellularly, nitric oxide enhances TRPC5 channel currents as well (14). Lysophospholipids and hypoosmotic-and pressure-induced membrane stretch act also as activators of TRPC5 channels (15,16). Under some circumstances, the externalization of TRPC5 contributes substantially to the overall enhancement of TRPC5 channel currents (17,18). All in all, TRPC5 are apparently target molecules of both extracellular and intracellular signals (5). As for other TRPC channels (e.g. Ref. 19), however, a central question is whether TRPC5 participates in store-operated Ca 2ϩ entry. Previous studies have shown that maneuvers that activate store-operated Ca 2ϩ entry also activate TRPC5 channels (20,21). In mast cells the Ca 2ϩ entry is apparently dependent on the presence of TRPC5 as well as on STIM1 and ORAI1, the key components of storeoperated Ca 2ϩ entry (22). Our understanding of the role of TRPC5 in store-operated Ca 2ϩ entry was advanced by the finding that STIM1 binds to TRPC5 and is obligatory for TRPC5 channel activation via membrane receptor stimulation (23). Thus, it is likely that TRPC5 is part of the protein complex responsible for store-operated Ca 2ϩ entry. However, it has
Neurotransmitter release is initiated by the influx of Ca 2+ via voltage-gated calcium channels. The accessory β-subunit (Ca V β) of these channels shapes synaptic transmission by associating with the pore-forming subunit (Ca V α 1 ) and up-regulating presynaptic calcium currents. Besides Ca V α 1, Ca V β interacts with several partners including actin filaments (F-actin). These filaments are known to associate with synaptic vesicles (SVs) at the presynaptic terminals and support their translocation within different pools, but the role of Ca V β/F-actin association on synaptic transmission has not yet been explored. We here study how Ca V β 4 , the major calcium channel β isoform in mamalian brain, modifies synaptic transmission in concert with F-actin in cultured hippocampal neurons. We analyzed the effect of exogenous Ca V β 4 before and after pharmacological disruption of the actin cytoskeleton and dissected calcium channel-dependent and -independent functions by comparing the effects of the wild-type subunit with the one bearing a double mutation that impairs binding to Ca V α 1 . We found that exogenously expressed wild-type Ca V β 4 enhances spontaneous and depolarization-evoked excitatory postsynaptic currents (EPSCs) without altering synaptogenesis. Ca V β 4 increases the size of the readily releasable pool (RRP) of SVs at resting conditions and accelerates their recovery after depletion. The enhanced neurotransmitter release induced by Ca V β 4 is abolished upon disruption of the actin cytoskeleton. The Ca V α 1 association-deficient Ca V β 4 mutant associates with actin filaments, but neither alters postsynaptic responses nor the time course of the RRP recovery. Furthermore, this mutant protein preserves the ability to increase the RRP size. These results indicate that the interplay between Ca V β 4 and F-actin also support the recruitment of SVs to the RRP in a Ca V α 1 -independent manner. Our studies show an emerging role of Ca V β in determining SV maturation toward the priming state and its replenishment after release. We envision that this subunit plays a role in coupling exocytosis to endocytosis during the vesicle cycle.
ClC-3, ClC-4, and ClC-5 are electrogenic chloride/proton exchangers that can be found in endosomal compartments of mammalian cells. Although the association with genetic diseases and the severe phenotype of knockout animals illustrate their physiological importance, the cellular functions of these proteins have remained insufficiently understood. We here study the role of two Clcn3 splice variants, ClC-3b and ClC-3c, in granular exocytosis and catecholamine accumulation of adrenal chromaffin cells using a combination of high-resolution capacitance measurements, amperometry, protein expression/gene knock-out/down, rescue experiments, and confocal microscopy. We demonstrate that ClC-3c resides in immature as well as in mature secretory granules, where it regulates catecholamine accumulation and contributes to the establishment of the readily releasable pool of secretory vesicles. The lysosomal splice variant ClC-3b contributes to vesicle priming only with low efficiency and leaves the vesicular catecholamine content unaltered. The related Cl -/H + antiporter ClC-5 undergoes age-dependent downregulation in wild-type conditions. Its upregulation in Clcn3 -/cells partially rescues the exocytotic mutant defect. Our study demonstrates how different CLC transporters with similar transport functions, but distinct localizations can contribute to vesicle functions in the regulated secretory pathway of granule secretion in chromaffin cells. Significance StatementCl -/H + exchangers are expressed along the endosomal/lysosomal system of mammalian cells, however, their exact subcellular functions have remained insufficiently understood. We used chromaffin cells, a system extensively used to understand presynaptic mechanisms of synaptic transmission, to define the role of CLC exchangers in neurosecretion. Disruption of ClC-3 impairs catecholamine accumulation and secretory vesicle priming. There are multiple ClC-3 splice variants, and only expression of one, ClC-3c, in double Cl -/H + exchanger-deficient cells fully rescues the WT phenotype. Another splice variant, ClC-3b, is present in lysosomes and is not necessary for catecholamine secretion. The distinct functions of 4 ClC-3c and ClC-3b illustrate the impact of expressing multiple CLC transporters with similar transport functions and separate localizations in different endosomal compartments.
Membrane depolarization activates the multisubunit CaV1.2 L‐type calcium channel initiating various excitation coupling responses. Intracellular trafficking into and out of the plasma membrane regulates the channel's surface expression and stability, and thus, the strength of CaV1.2‐mediated Ca2+ signals. The mechanisms regulating the residency time of the channel at the cell membrane are unclear. Here, we coexpressed the channel core complex CaV1.2α1 pore‐forming and auxiliary CaVβ subunits and analyzed their trafficking dynamics from single‐particle‐tracking trajectories. Speed histograms obtained for each subunit were best fitted to a sum of diffusive and directed motion terms. The same mean speed for the highest‐mobility state underlying directed motion was found for all subunits. The frequency of this component increased by covalent linkage of CaVβ to CaV1.2α1 suggesting that high‐speed transport occurs in association with CaVβ. Selective tracking of CaV1.2α1 along the postendocytic pathway failed to show the highly mobile state, implying CaVβ‐independent retrograde transport. Retrograde speeds of CaV1.2α1 are compatible with myosin VI‐mediated backward transport. Moreover, residency time at the cell surface was significantly prolonged when CaV1.2α1 was covalently linked to CaVβ. Thus, CaVβ promotes fast transport speed along anterograde trafficking and acts as a molecular switch controlling the endocytic turnover of L‐type calcium channels.
In mouse chromaffin cells expressing green synaptopHluorin in the granules, cell membranes were stained from the outside with red lipophilic dyes that rapidly redistribute by flip-flop between both leaflets. Fusion of secretory granules was monitored by evanescent wave microscopy. Exocytosis was triggered by superfusion with high Kþ solution, and double images were taken at 491 and 560 nm excitation, respectively. Fluorescence signals of the membrane probes recorded in the red channel were spatially and temporally aligned with respect to fusion events in the green channel to yield average movies with high signal-to-noise ratio. We found the membrane fluorescent signals to be slightly increased in diffraction-limited spots at locations of docked granules for up to a second prior to fusion. The fluorescent signals, however, rapidly decreased to background levels upon fusion of the granules at the respective sites, with the fluorescence dissipating from the center to the periphery. Our results are best explained by mixing of lipids prior to fusion in a hemifused state.
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