CLCN7 POINT MUTANT INHIBITED Cl-EXTRUSION, RESUTLING IN OSTEOPETROSIS

ClC7 Cl^-通道对破骨细胞骨吸收至关重要，因为它们在酸分泌中起重要作用。ClC7基因（CLCN7）缺失的小鼠在骨吸收过程中破骨细胞Cl挤压受损，导致严重的骨质疏松。也有报道称，常染色体II型骨质疏松症（ADO II）是由CLCN7的杂合突变引起的。虽然已知细胞外酸化可在转染CLCN7的卵母细胞中诱导ClC7 Cl^-电流，但这种酸诱导的Cl^-电流的其他特征以及ADO II中突变体CLCN7对破骨细胞Cl^-挤压活性的影响尚不清楚。本研究的目的是表征从哺乳动物破骨细胞中克隆的ClC7 Cl^-电流，并阐明在ADO II患者中发现的点突变对Cl^-挤压活性的影响。细胞外酸化（pH 6.0）在小鼠破骨细胞中引起小的天然Cl^-电流。与模拟转染的细胞相比，野生型人CLC7在HEK293细胞中的瞬时表达可诱导显著的Cl^-电流。这些酸激活的Cl^-电流与细胞内酸化或[Ca^<2+>]i的增加无关。相比之下，HEK293细胞中的CLCN7突变基因表达不表现出这些酸激活的Cl电流。此外，与野生型CLCN7表达相比，G215R处CLCN7突变的RAW 264.7在破骨细胞分化潜能方面没有变化，但骨吸收活性显著降低。总之，我们的研究结果表明，与ADO II相关的G215R位点的CLCN7突变导致破骨细胞骨吸收过程中Cl挤压活性的抑制。

ClC7 Cl^- channels are crucial for osteoclastic bone resorption since they play an important role for acid secretion. Mice with a deficient ClC7 gene (CLCN7) have impaired osteoclastic Cl extrusion during bone resorption, resulting in severe osteopetrosis. It also has been reported that autosomal osteopetrosis type II (ADO II) results from heterozygous mutations in CLCN7. Although extracellular acidification is known to induce ClC7 Cl^- current in CLCN7 transfected oocytes, other characteristics of this acid-induced Cl^- current as well as effects of mutant CLCN7 in ADO II on osteoclastic Cl^- extrusion activity are unknown. The aim of present study was to characterize the ClC7 Cl^- currents cloned from mammalian osteoclasts and to clarify the effect of point mutations found in ADO II patients on Cl^- extrusion activity. Extracellular acidification (pH 6.0) evoked small native Cl^- currents in mouse osteoclasts. Transient expression of wild type human CLC7 in HEK293 cells induced significant Cl^- currents compared to mock-transfected cells. These acid-activated Cl^- currents were independent of intracellular acidification or [Ca^<2+>]i increase. In contrast, CLCN7 mutant gene expression in HEK293 cells did not display these acid-activated Cl currents. Furthermore, RAW 264.7 with the CLCN7 mutation at G215R demonstrated no change in osteoclast differentiation potential but a significant decrease in bone resorption activity compared to wild type CLCN7 expression. In summary, our results indicate that a CLCN7 mutation at the G215R locus associated with ADO II results in suppression of Cl extrusion activity during osteoclastic bone resorption.