application): Agonist activation of arterial smooth muscle produces sustained Ca2+ influx and membrane depolarization that contribute to tonic force maintenance. The PI has shown that Ca2+ influx modulates whole cell K+ currents (Ik) with characteristics that suggest the inhibition of voltage-gated K+ channels (Kv) by L-type Ca2+ channel (CaL) influx. Also, this mechanism appears to be larger in SHR than in WKY myocytes providing an explanation for many previous observations, and also providing an opportunity to investigate the molecular mechanisms involved. This research project is based upon the hypothesis that: (a) Ca2+ influx through CaL inhibits Kv channels, (b) by a direct action on the channel, providing a mechanism for sustained membrane depolarization during agonist activation, and (d) which is more effective in myocytes from SHR versus WKY. The following specific aims will test these hypotheses using myocytes, RNA and membranes isolated from mesenteric arteries (MA) of WKY and SHR: 1) To demonstrate that CaL influx inhibits Ik when maxi K channels are blocked by iberiotoxin; 2) To demonstrate that Ca2+- dependent signal transduction mechanisms (PKC and CaM kinase II) are not involved in this effect; 3) To determine Kv isoform expression in MA from WKY and SHR; 4) To determine the (cytosolic) Ca2+ sensitivity (inhibition) of these Kv isoforms expressed in Xenopus oocytes; 5) To determine the Ca2+-sensitivity of Kv in native arterial myocytes; and 6) to determine the role of Kv inhibition by Ca2+ influx on sustained depolarization with agonist activation. Ik will be measured by perforated patch variant of the whole cell patch clamp method. Ca2+ influx will be inhibited by Cd2+, nisoldipine, or lowering external Ca2+, and increased by Bay K 8644 and ionomycin. PKC and CaM kinase II will be inhibited by calphostin C and KT5926 and inhibitory peptides. Kv expression will be determined by RT-PCR and by Southern and Western Blotting. The effects of Ca2+ on Kv expressed in oocytes and native myocytes will be determined by whole cell and single channel methods. The PI suggests that inhibition of Kv by Ca2+ influx represents a positive feedback mechanism which sustains tonic contractions during agonist activation through membrane depolarization and sustained CaL activation. These studies will provide the first detailed investigation of Kv expression and functional properties in arterial myocytes. In addition, these studies will provide a new molecular target for genetic linkage studies in hypertension as well as new potential therapeutic targets.
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