Cloning and functional analysis of the genes related to endothelium derived hyperpolarizing factor

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The purpose of this project was to clarify the molecular mechanisms of endothelium-derived hyperpolarizing factor(EDHF)-mediated arterial relaxation and membrane hyperpolarization in the rat mesenteric arteries. We tried to identify the ion channels and gap junctional channels involved in the EDHF action. RT-PCR experiment showed that mRNA of small-conductance Ca^<2+>-activated K^+(SK)3, intermediate-conductance Ca^<2+>-activated K^+(IK), large-conductance Ca^<2+>-activated K^+(BK) channels and connexin-37,-40,-43,-45 but not SK1 and SK2 were exist in the rat mesenteric artery. Genes of these ion channels and connexins were cloned by RT-PCR and cDNA library screening. New gene similar to SK3 channel was cloned. Functional analysis using patch clamp techniques showed that the new SK3-related channel has almost the same ion channel functions such as voltage sensitivity and toxin sensitivity. However the Ca^<2+> sensitivity of the channel was less sensitive compared with the reported SK3 channel. The new channel may relate to the arterial function. Immunohistochemical analysis showed that the SK3 channel was expressed in the endothelium. The CX37 and 43 were expressed in both the endothelial and smooth muscle cells, however CX40 was expressed in only the endothelial cells. To clarify the functional role of these genes in the native artery, adenoviruses expressing these genes were constructed. The functional analysis of these genes using adenovirus in the native artery was under estimation.