MOLECULAR STUDIES OF VOLTAGE DEPENDENT POTASSIUM CHANNELS

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Colonic muscles display a heterogeneous collection of K+ conductances which contribute to motility through excitation-contraction coupling. While electrophysiological studies in dispersed smooth muscle cells have generated a considerable body of information concerning the properties of these currents, the molecular basis for colonic motility has yet to be investigated. Specifically, K+ channel gene products responsible for the K+ currents demonstrated in GI smooth muscle have not been elucidated nor have their expression patterns in these diverse tissues been studied. The experiments proposed in this application will test the hypotheses that: (1) the diverse electrical behavior of the colon is determined by several distinct K+ channel genes expressed in colonic smooth muscle, (2) the differential genetic expression of the genes encoding these channels contributes to the regional pattern of electrical activity demonstrated in the GI tract. In addition, by identifying and isolating the molecular counterparts for the K+ currents present in the colon, it will be possible to characterize the electrophysiological and pharmacological properties of these channels in the absence of contaminating currents. To address hypothesis (1), full-length cDNAs encoding K+ channel proteins will be isolated from colonic smooth muscle cDNA libraries, including libraries specific for longitudinal muscle and cultured interstitial cells. By injecting in vitro transcribed RNA made from these cDNA clones into Xenopus oocytes, the electrophysiological characteristics of these ion channels will be determined and related to data collected by Project 2, which has been obtained from dissociated colonic myocytes. Biophysical studies will be conducted to determine the mechanism and structural relationships of 4-AP block. Hypothesis (2) will be addressed by determining the molecular distribution of K+ channel expression in the colon and other regions of the GI tract. The possibility of colonic K+ channel regulation will be explored with experiments which will determine the effects of kinase activity, temperature and divalent cations on cloned colonic K+ channel functions.