RESUMO
Osteoclasts acidify bone resorption lacunae through proton translocation by plasma membrane V-ATPase (vacuolar-type ATPase) which has an a3 isoform, one of the four isoforms of the trans-membrane a subunit (Toyomura et al., J. Biol. Chem., 278, 22023-22030, 2003). d2, a kidney- and epididymis-specific isoform of the d subunit, was also induced in osteoclast-like cells derived from the RAW264.7 line, and formed V-ATPase with a3. The amount of d2 in osteoclasts was 4-fold higher than that of d1, a ubiquitous isoform. These results indicate that V-ATPase with d2/a3 is a major osteoclast proton pump. Essentially the same results were obtained with osteoclasts derived from mouse spleen macrophages. Macrophages from a3-knock-out mice could differentiate into multi-nuclear cells with osteoclast-specific enzymes. In these cells, the d2 isoform was also induced and assembled in V-ATPase with the a1 or a2 isoform. However, they did not absorb calcium phosphate, indicating that V-ATPase with d2/a1 or d2/a2 could not perform the function of that with d2/a3.