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Am J Physiol Cell Physiol (March 28, 2007). doi:10.1152/ajpcell.00596.2006
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Submitted on November 30, 2006
Accepted on March 20, 2007

Relocalization of the V-ATPase B2 subunit to the apical membrane of epididymal clear cells of mice deficient in the B1 subunit

Nicolas Da Silva1*, Winnie WC Shum1, Jaafar El-Annan2, Teodor G Paunescu2, Mary McKee2, Peter JS Smith3, Dennis Brown1, and Sylvie Breton1

1 Nephrology Division - Program in Membrane Biology, Massachusetts General Hospital - Harvard Medical School, Boston, Massachusetts, United States
2 Boston, Massachusetts, United States; Nephrology Division - Program in Membrane Biology, Massachusetts General Hospital - Harvard Medical School, Boston, Massachusetts, United States
3 Molecular Physiology Program, MBL, BioCurrents Research Center, Woods Hole, Massachusetts, United States

* To whom correspondence should be addressed. E-mail: ndasilva{at}partners.org.

An acidic luminal pH in the epididymis contributes to maintaining sperm quiescent during their maturation and storage. The vacuolar H+ATPase, located in narrow and clear cells, is a major contributor to luminal acidification. Mutations in one of the V-ATPase subunits, ATP6V1B1, cause distal renal tubular acidosis in humans but surprisingly, B1 knockout mice do not develop metabolic acidosis and are fertile. While B1 is located in the apical membrane of narrow and clear cells, B2 localizes to sub-apical vesicles in wild type mouse, rat and human epididymis. However, a marked increase (84%) in the mean pixel intensity of B2-staining was observed in the apical pole of clear cells by immunofluorescence, and relocalization into their apical membrane was detected by confocal microscopy in B1-/- mice compared to B1+/+. Immunogold electron microscopy showed abundant B2 in the apical microvilli of clear cells in knockout mice. B2 mRNA expression, determined by real time RT-PCR using laser-microdissected epithelial cells, was identical in both groups. Semi-quantitative western blots from whole epididymis and cauda epididymidis showed no variation of B2 expression. Finally, the luminal pH of the cauda epididymidis was the same in B1-/- mice as in B1+/+ (6.7). These data indicate that, whereas overall expression of B2 is not affected in B1-/- mice, significant redistribution of B2-containing complexes occurs from intracellular compartments into the apical membrane of clear cells in B1-/- mice. This relocation compensates for the absence of functional B1 and maintains the luminal pH in an acidic range that is compatible with fertility.




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