(Verbatim from the application): This proposal is aimed towards finding what distinction channels in vascular SMC are responsible for capacitative (store-operated) Ca2+ influx, and how they are regulated by the stores. Recently, we described capacitative Ca2+ entry (CCE) in SMC and human platelets and showed that nitric oxide (NO) inhibits it through SERCA-dependent refilling of the stores, but the nature of ion channels that are responsible for CCE in SMC, and the distinct mechanisms which link channel activation and inhibition to the depletion and refilling of the stores remains totally unknown. Our preliminary data showed that upon depletion of intracellular stores novel small (3 pS) nonselective cation channels are activated in SMC from rabbit and mouse aorta. We also found that the same channels could be activated by a putative calcium influx factor (CIF) that has been partially purified from yeast and platelets. The overall goal of my proposal is to characterize these novel nonselective cation channels, establish their functional role in capacitative Ca2+ influx in SMC, and to define the molecular mechanism of their store-dependent activation and inhibition. The main hypothesis of this proposal is that in vascular SMC capacitative Ca2+ entry is mediated by novel nonselective cation channels that are directly activated by a Ca2+ influx factor (CIF) that is produced by endoplasmic reticulum during depletion of Ca2+ stores and is delivered to the plasma membrane via exocytosis-like process. Inhibition of store-operated Ca2+ influx could result from impairment of CIF production, delivery, and/or CIF sensitivity of store-operated channels. This hypothesis will be tested at the level of single store-operated channels, whole-cell currents, cation influx, intracellular Ca2+ and CIF produced by SMC and platelets.
Specific aims of this proposal are:
Aim 1. To determine ion channels in SMC that are responsible for store-operated Ca2+ influx. We will characterize novel small (3 pS) nonselective cation channels and establish their regulation by the filling state of Ca2+ stores.
Aim 2. To define the molecular mechanisms of store-operated channel regulation in SMC. We will determine if the store-operated channels are activated directly by a putative CIF produced by endoplasmic reticulum of SMC and platelets, what other known substances could activate this channel, if specific substrates or functional connection with cytoskeleton is required for channel activation, if phosphatase inhibitors or GTPgammaS could affect channel sensitivity to CIF, if CIF needs to be delivered to plasma membrane to activate the channels via exocytosis-like process requiring anchoring proteins. We will also define the physiological mechanisms of inhibition of store-operated channels and CCE on the level of CIF production, delivery and CIF-sensitivity of single channels.
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