This application proposes continuation of physiologic and molecular studies of potassium absorption in the rat distal colon. The PI has identified an active K absorptive process in the rat distal colon associated with an apical K/H exchange energized by a novel H,K-ATPase. The PI has also shown that this process is upregulated by both aldosterone and dietary K depletion. The PI's recent studies have focused on correlating the H,K-ATPase activity with the upregulation of K absorption both at the molecular and physiologic levels of study. The PI has reported that H,K-ATPase of rat distal colon is partially ouabain sensitive, pointing to the possible presence of two different potassium absorptive processes and H,K-ATPase isoforms. His group was recently successful in cloning the HCKalpha1 cDNA. The expression of this cDNA in SF9 cells showed ouabain insensitivity in contrast to studies from other groups using the oocyte expression system. In this proposal he, therefore, plans to determine the physiological function of the transporter cDNAs after transfection in a mammalian cell expression system. Studies are also proposed to determine whether the recently cloned rat colon-specific beta subunit is the elusive and putative beta subunit for optimal functional expression. The PI has identified an ouabain-sensitive K-dependent pHi recovery process in the colonic crypt. The PI hypothesizes that this pHi regulation will be modified by dietary K depletion and will, therefore, represent the second H,K-ATPase function. To identify the second colonic H,K-ATPase, the PI will use a cloning strategy based on its probable upregulation by dietary potassium depletion, its identity to the H-3 domain region rather than to the 5' end of HCKalpha1 cDNA and its exclusive localization in colonic crypts. It is also hypothesized that dietary potassium depletion might regulate the second colonic H,K-ATPase (HCKa2 and/or HCKab) at the apical membrane and/or electroneutral KCl cotransport at the basolateral membrane. The PI plans to employ cell and molecular biology studies to accomplish his four specific aims aimed at gaining more understanding of colonic potassium absorption. This proposal certainly has relevance to clinical medicine and would enhance our knowledge of potassium transport.
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