Colonic H-K-ATPase β-subunit:: identification in apical membranes and regulation by dietary K depletion

P-type ATPases require both alpha- and beta-subunits for functional activity. Although an alpha-subunit for colonic apical membrane H-K-ATPase (HKc alpha) has been identified and studied, its beta-subunit has not been identified. We cloned putative beta-subunit rat colonic H-K-ATPase (HKc beta) cDNA that encodes a 279-amino-acid protein with a single transmembrane domain and sequence homology to other rat beta-subunits. Northern blot analysis demonstrates that this HKc beta is expressed in several rat tissues, including distal and proximal colon, and is highly expressed in testis and lung. HKc beta mRNA abundance is upregulated threefold compared with normal in distal colon but not proximal colon, testis, or lung of K-depleted rats. In contrast, Na-K-ATPase beta(1) mRNA abundance is unaltered in distal colon of K-depleted rats. Na depletion, which also stimulates active K absorption in distal colon, does not increase HKc beta mRNA abundance. Western blot analyses using a polyclonal antibody raised to a glutathione S-transferase-HKc beta fusion protein established expression of a 45-kDa HKc beta protein in both apical and basolateral membranes of rat distal colon, but K depletion increased HKc beta protein expression only in apical membranes. Physical association between HKc beta and HKc alpha proteins was demonstrated by Western blot analysis performed with HKc beta antibody on immunoprecipitate of apical membranes of rat distal colon and HKc alpha antibody. Tissue-specific upregulation of this beta-subunit mRNA in response to K depletion, localization of its protein, its upregulation by K depletion in apical membranes of distal colon, and its physical association with HKc alpha protein provide compelling evidence that HKc beta is the putative beta-subunit of colonic H-K-ATPase.