MOLECULAR IDENTIFICATION AND FUNCTIONAL ANALYSIS OF Cl^- CHANNELS CONTRIBUTING OSTEOCLASTIC BONE RESORPTION.

破骨细胞中表达的ClC-7 Cl^-通道对骨吸收很重要，因为其基因在小鼠体内的破坏会导致严重的骨质疏松表型。然而，除ClC-7外，Cl^-通道在破骨细胞中的功能作用和表达尚不清楚。在这项研究中，我们鉴定了表达小鼠破骨细胞的Cl^-通道的分子类型，并研究了它们在骨吸收中的功能作用。全细胞膜片钳记录显示，从破骨细胞记录的Cl^-电流具有向外整流、快速激活和时间依赖性失活、阴离子渗透性（1 >Cl^-）和DIDS阻断的特征。Cl^-电流的这些性质类似于ClC-3而不是ClC-7。通过RT-PCR检测破骨细胞中ClC-3和ClC-7的转录本。免疫细胞化学分析也显示破骨细胞中存在ClC-3蛋白。通过特异性反义寡核苷酸和小干扰RNA （siRNA）抑制ClC-3在破骨细胞中的表达，显示出体外骨吸收活性的显著降低。在体外，与野生型（WT）小鼠相比，来自ClC-3缺陷（ClC-3 KO）小鼠的破骨细胞的骨吸收活性降低。来自ClC-3 KO和WT小鼠的破骨细胞中的Cl^-电流在电流动力学（快速激活和时间依赖性失活）、密度、精流和阴离子渗透性方面相似。此外，用抗clc -3抗体对破骨细胞进行细胞内透析，并未引起可检测到的向外整流Cl^-电流振幅的变化。这些结果排除了ClC-3是小鼠破骨细胞中负责向外整流Cl^-电流的通道。使用ph敏感染料吖啶橙，我们可视化了活破骨细胞的细胞内酸性区室（细胞器）。对ClC-3进行反义处理后，具有酸性细胞器的破骨细胞数量明显减少。siRNA对ClC-3也有类似的作用。此外，我们使用LysoSensor DND-160检测到ClC-3 KO破骨细胞细胞器pH值升高，该细胞器优先划分为酸性细胞器。这些结果表明，小鼠破骨细胞表达ClC-3，并且ClC-3和ClC-7有助于体外维持骨吸收活性。ClC-3可能作为细胞内的Cl^-通道，并有助于细胞器的酸化，从而导致有效的骨吸收。少

ClC-7 Cl^- channel expressing in osteoclasts is important for bone resorption since disruption of its gene in mice leads to a severe osteopetrotic phenotype. However, the functional roles and expression of Cl^- channels other than ClC-7 in osteoclasts are still obscure. In this study, we identified the molecular types of Cl^- channels expressing mouse osteoclasts and examined their functional role in bone resorption.1.Whole-cell patch-clamp recordings showed that Cl^- current recorded from osteoclasts was characterized by outward rectification, rapid activation and time-dependent inactivation, anion permeability (I>Cl^-), and block by DIDS. These properties of the Cl^- current resembled those of ClC-3 rather than ClC-7. Transcripts for ClC-3 as well as ClC-7 were detected in osteoclasts by RT-PCR. Immunocytochemical analysis also showed the presence of ClC-3 protein in osteoclasts.2.Reduction of ClC-3 expression in osteoclasts by specific antisense oligonucleotide and small interfering … More RNA (siRNA) against ClC-3 demonstrated a significant reduction in bone resorption activity in vitro. In an in vitro, bone resorption activity of osteoclasts derived from ClC-3-deficient (ClC-3 KO) mice was reduced compared with those from wild type (WT) mice.3.The Cl^- currents in osteoclasts from ClC-3 KO and WT mice were similar in terms of current kinetics (rapid activation and time-dependent inactivation), densities, rectification, and anion permeability. Furthermore, intracellular dialysis of osteoclasts with anti-ClC-3 antibody did not cause detectable change in the amplitude of outwardly rectifying Cl^- current. These results exclude ClC-3 as the channel responsible for the outwardly rectifying Cl^- current in mouse osteoclasts.4.Using a pH-sensitive dye, acridine orange, we visualized intracellular acidic compartments (organelles) in living osteoclasts. After treatment with antisense to ClC-3, the number of osteoclasts possessing acidic organelles was significantly decreased. Similar effects were also seen with siRNA against ClC-3. Furthermore, we detected an elevation in the organelle pH of ClC-3 KO osteoclasts, using LysoSensor DND-160, which preferentially partitions into acidic organelles.These results suggested that mouse osteoclasts expressed ClC-3 and that ClC-3 as well as ClC-7 contributes to maintaining bone resorption activity in vitro. ClC-3 may act as an intracellular Cl^- channel and contribute to acidification of organelles, which lead to the efficient bone resorption. Less