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Compensatory membrane expression of the V-ATPase B2 subunit isoform in renal medullary intercalated cells of B1-deficient mice

Teodor G. Punescu,¹ Leileata M. Russo,¹ Nicolas Da Silva,¹ Jana Kovacikova,² Nilufar Mohebbi,² Alfred N. Van Hoek,¹ Mary McKee,¹ Carsten A. Wagner,² Sylvie Breton,¹ and Dennis Brown¹

¹Center for Systems Biology, Program in Membrane Biology, and Division of Nephrology, Massachusetts General Hospital, and Harvard Medical School, Boston, Massachusetts; and ²Institute of Physiology and Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Zurich, Switzerland

Submitted 5 April 2007 ; accepted in final form 22 September 2007

Mice deficient in the ATP6V1B1 ("B1") subunit of the vacuolar proton-pumping ATPase (V-ATPase) maintain body acid-base homeostasis under normal conditions, but not when exposed to an acid load. Here, compensatory mechanisms involving the alternate ATP6V1B2 ("B2") isoform were examined to explain the persistence of baseline pH regulation in these animals. By immunocytochemistry, the mean pixel intensity of apical B2 immunostaining in medullary A intercalated cells (A-ICs) was twofold greater in B1–/– mice than in B1+/+ animals, and B2 was colocalized with other V-ATPase subunits. No significant upregulation of B2 mRNA or protein expression was detected in B1–/– mice compared with wild-type controls. We conclude that increased apical B2 staining is due to relocalization of B2-containing V-ATPase complexes from the cytosol to the plasma membrane. Recycling of B2-containing holoenzymes between these domains was confirmed by the intracellular accumulation of B1-deficient V-ATPases in response to the microtubule-disrupting drug colchicine. V-ATPase membrane expression is further supported by the presence of "rod-shaped" intramembranous particles seen by freeze fracture microscopy in apical membranes of normal and B1-deficient A-ICs. Intracellular pH recovery assays show that significant (28–40% of normal) V-ATPase function is preserved in medullary ICs from B1–/– mice. We conclude that the activity of apical B2-containing V-ATPase holoenzymes in A-ICs is sufficient to maintain baseline acid-base homeostasis in B1-deficient mice. However, our results show no increase in cell surface V-ATPase activity in response to metabolic acidosis in ICs from these animals, consistent with their inability to appropriately acidify their urine under these conditions.

proton pump; immunofluorescence; pH homeostasis; urinary acidification; Atp6v1b1–/– mice

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