Molecular identification and functional analysis of Na^+/Ca^<2+> exchanger (NCX) associated with osteoclastic bone resorption

质膜Na~(++)/Ca~(2+)&gt；交换器(NCX)是一种双向转运体，它依赖于电化学梯度来调节Na~+与Ca~(2+)的交换。哺乳动物NCXs是一个由NCX1、NCX2和NCX3亚型组成的多基因家族。虽然已知大鼠破骨细胞中的NCX1与Na~+/H~+交换器偶联，以调节细胞内钙离子浓度([Ca^&lt；2+&gt；]_i)，但目前还不清楚NCX1的哪种变体在小鼠破骨细胞中表达，以及另外两种NCX亚型是否也存在于小鼠破骨细胞中。为了阐明NCXs在骨吸收中的作用，我们研究了NCXs在小鼠破骨细胞中的表达、NCXs的离子转运以及NCXs抑制剂对骨吸收活性的影响。通过RT-PCR、免疫细胞化学和Western印迹等方法检测到NCX1和NCX3的三种剪接变异体，即NCX1.3、NCX1.41和NCX3.2。其中，NCX1.41是一个新发现的剪接变异体。低钠([Na+]o)可通过NCX转运蛋白增加Fura-2负载破骨细胞内钙离子浓度。[Na~(++)]_o溶液引起的[Ca~(2+)]_i升高可被苄氧基苯基NCX抑制剂抑制。用膜片钳方法记录小鼠破骨细胞的双向NCX电流，NCX抑制剂可抑制该电流。NCX抑制剂还以剂量依赖的方式减少破骨细胞周围的吸收凹陷面积。此外，针对NCX1.3、1.41和NCX3.2的siRNA在小鼠破骨细胞中表达，抑制破骨细胞凹陷的形成。这些结果表明，三种NCX变异体在小鼠破骨细胞中都有表达，并且在破骨细胞性骨吸收过程中对钙的运输和调节起着重要作用。

The plasma membrane Na^+/Ca^<2+> exchanger (NCX) is a bi-directional 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, siRNAs targeted against NCX1.3, 1.41 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.