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

P-type ATPases require both alpha- and beta-subunits for functional activit y. Although an alpha-subunit for colonic apical membrane H-K-ATPase (HKc al pha) has been identified and studied, its beta-subunit has not been identif ied. 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 a nd sequence homology to other rat beta-subunits. Northern blot analysis dem onstrates 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 dista l colon but not proximal colon, testis, or lung of K-depleted rats. In cont rast, 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 d istal colon, does not increase HKc beta mRNA abundance. Western blot analys es using a polyclonal antibody raised to a glutathione S-transferase-HKc be ta fusion protein established expression of a 45-kDa HKc beta protein in bo th apical and basolateral membranes of rat distal colon, but K depletion in creased HKc beta protein expression only in apical membranes. Physical asso ciation between HKc beta and HKc alpha proteins was demonstrated by Western blot analysis performed with HKc beta antibody on immunoprecipitate of api cal membranes of rat distal colon and HKc alpha antibody. Tissue-specific u pregulation of this beta-subunit mRNA in response to K depletion, localizat ion of its protein, its upregulation by K depletion in apical membranes of distal colon, and its physical association with HKc alpha protein provide c ompelling evidence that HKc beta is the putative beta-subunit of colonic H- K-ATPase.