The long term goal of the present proposal is to define in coronary smooth muscle the biophysical and pharmacological properties of Ca2+ dependent K (KCa) channels, to determine their regulation mechanism(s) and, to establish a correlation between the structure and the functional properties of the channel. This project will be addressed using a combined approach which consists of a study using: a) KCa channels incorporated into bilayers, and b) molecular cloning of KCa channels. The central hypothesis proposes that plasma membrane KCa channels are regulated by hormones, thus playing a major role in the control of arterial tone. We plan to study the biophysical characteristics of KCa channels and their regulatory mechanism(s). To perform these studies we have recently isolated a plasma membrane enriched fraction from the coronary smooth muscle, and reconstituted in a reproducible manner functional KCa channels into artificial membranes. Our initial findings indicate that these channels are inhibited by the vasoconstrictor peptide angiotensin II, and by the vasoconstrictor lipid thromboxane A2 probably in a direct manner. These findings suggest that coronary smooth muscle KCa channels may possess recognition sites for circulating hormones. One important question is to define whether these regulatory sites are integral part of the channel or if they are receptors directly or indirectly (via G proteins) coupled to the channel. The physiological studies in conjunction with molecular biological studies will be performed to define the primary regions in the sequence of coronary smooth muscle KCa channels which will give important clues to the mechanism(s) of the channel regulation. To obtain the primary sequence through the cloning of its cDNA we have initiated the isolation of total coronary smooth muscle mRNA and established and electrophysiological assay to follow functional expression in mRNA microinjected Xenopus oocytes. Cloning of the cDNA encoding the KCa channel will be attempted following expression of the channel activity either in Xenopus oocytes microinjected with in vitro transcribed RNA from the transcription competent cDNA library or in COS cells transfected with the cDNA library made in a high expression eukaryotic expression vector. We plan to study the tissue specificity and recognition sites for hormones in the primary structure of channels using molecular biology techniques.
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