ROMK potassium (K+) channels are present in the apical membrane of principal cells of the kidney cortical collecting ducts (CCD) and are responsible for luminal K+ secretion in this nephron segment. K+ secretion in CCD is regulated by dietary K+ intake and the hormone vasopressin. It has been shown that dietary K+ intake and vasopressin alters K+ secretion, at least partly, by altering the number of active K+ channels in CCD. How this occurs is not fully understood, but may involve alteration of membrane trafficking of the channels and/or activation of silent channels. Our long-term objectives are to understand the molecular mechanism for membrane trafficking of renal K+ channels and its importance in physiological regulation of the channels. There are multiple pathways for endocytosis. Our preliminary results indicate that endocytosis of ROMK is mediated via clathrin-coated vesicles (CCVs).
The specific aims of the first part of the application are to examine the structural elements of ROMK and CCVs involved in the endocytosis of the channels and to examine whether low dietary K+ intake decreases K+ channels by increasing endocytosis and subsequent degradation of ROMK. Association of ROMK with CCVs will be studied by immunofluorescent colocalization, biochemical purification, and immunocoprecipitation. Functional significance of protein interaction will be examined using two-electrode voltage clamp and patch-clamp recording in Xenopus oocytes as well as CCD. We also found that some of the ROMK channels in the apical membrane of CCD are inhibited by syntaxin-lA. The second part of the application is to test the hypothesis that vasopressin increases density of active channels by activating pre-existing silent channels. Biochemical binding assay and patch-clamp recording will be performed to examine this hypothesis.
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