Three Na+/Ca2+Exchanger (NCX) Variants Are Expressed in Mouse Osteoclasts and Mediate Calcium Transport during Bone Resorption

The plasma membrane Na(+)/Ca(2+) exchanger (NCX) is a bidirectional transporter that mediates the exchange of Na(+) for Ca(2+) depending on the electrochemical gradients. Mammalian NCXs form a multigene family comprising NCX1, NCX2, and NCX3 isoforms. Although it has been known that NCX1 in rat osteoclasts is coupled with the Na(+)/ H(+) exchanger for regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)), it is unclear what kind of NCX1 variants are expressed and whether the other two NCX isoforms are also present in mouse osteoclasts. To clarify the role of NCXs during bone resorption, we investigated the expression of NCXs, the ion transport via NCXs, and the effects of NCX inhibitors on bone-resorbing activity in mouse osteoclasts. Using RT-PCR, immunocytochemical, and Western blot methods, we detected three splice variants of NCX1 and NCX3, namely NCX1.3, NCX1.41, and NCX3.2. Of these, NCX1.41 is a newly identified splice variant. Low extracellular sodium ([Na(+)](o)) solution increases the intracellular Ca(2+) concentration via NCX transporter in fura-2-loaded osteoclasts. The [Na(+)](o)-free solution-induced [Ca(2+)](i) increase was suppressed by benzyloxyphenyl NCX inhibitors. Bidirectional NCX currents in mouse osteoclasts were recorded using the patch clamp method and could be suppressed with NCX inhibitors. NCX inhibitors also decreased the resorption pit area surrounding osteoclasts in a dose-dependent manner. Furthermore, small interference RNAs targeted against NCX1.3, NCX1.41, and NCX3.2 expressed in mouse osteoclasts suppressed osteoclastic pit formation. These results show that three NCX variants are expressed in mouse osteoclasts and play an important role for Ca(2+) transport and regulation during osteoclastic bone resorption.