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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Expression of the 56-kDa B2 subunit isoform of the vacuolar H⁺-ATPase in proton-secreting cells of the kidney and epididymis

Teodor G. Punescu,^* Nicolas Da Silva,^* Vladimir Marshansky, Mary McKee, Sylvie Breton, and Dennis Brown

Program in Membrane Biology and Renal Unit, Massachusetts General Hospital, Charlestown 02129; and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115

Submitted 12 February 2003 ; accepted in final form 12 February 2004

B1 and B2 are two highly homologous isoforms of the vacuolar H⁺-ATPase (V-ATPase) 56-kDa B subunit. We investigated whether the B2 subunit is expressed alongside B1 in proton-secreting cells of the rodent kidney collecting duct (intercalated cells, IC) and epididymis (clear cells) by using antibodies against distinct COOH-terminal peptides from the two B isoforms. B2 was detected not only in the kidney proximal tubule, thick ascending limb, distal convoluted tubule, and connecting segment but also in A- and B-type IC of collecting ducts (CD) in both rat and mouse. B2 had a predominant cytoplasmic localization in most IC but was clearly located in a tighter apical band together with the V-ATPase 31-kDa E subunit in some A-IC, especially in the medulla. Apical membrane staining was confirmed by immunogold electron microscopy. B2 was very weakly expressed on the basolateral membranes of B-IC in control kidney CD, but some connecting segment B-IC had more distinct basolateral staining. In response to chronic carbonic anhydrase inhibition by acetazolamide, many A-IC showed a strong apical membrane localization of B2, where it colocalized with E and B1. In rat and mouse epididymis, B2 isoform expression was detected in clear cells, where it was concentrated in subapical vesicles. Unlike B1, B2 did not colocalize with the E subunit in the apical microvilli. These findings indicate that in addition to its role in the acidification of intracellular organelles, the B2 isoform could also contribute to transepithelial proton secretion and the maintenance of acid-base homeostasis.

vacuolar H⁺-ATPase B subunit; intercalated cells; clear cells; urogenital tract; immunofluorescence

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