Department of Physiology and Biophysics, Rosalind Franklin University of Medicine and Science, Chicago, Illinois, United States.
Invest Ophthalmol Vis Sci. 2019 Apr 1;60(5):1621-1629. doi: 10.1167/iovs.19-26626.
Chloride channels have been proposed to play an important role in the regulation of lens volume. Unfortunately, little information is available about the molecular identity of these channels or how they are regulated in the lens due to the difficulties in isolating mouse fiber cells. Recently, our laboratory has developed a new technique for isolating these cells by using transgenic mouse lenses that lack both Cx50 and Cx46. The purpose of this study was to test the hypothesis that newly differentiating mouse fiber cells express calcium-activated chloride channels (CaCCs) by using this technique.
Differentiating fiber cells were isolated from lenses of double knockout mice that lack both Cx50 and Cx46 by using collagenase. Membrane currents were studied using the whole-cell patch clamp technique. The molecular identity and distribution of CaCCs were investigated using RT-PCR and immunofluorescence.
Our electrophysiologic experiments suggest that peripheral fiber cells express a calcium-activated chloride current. The voltage gating properties, calcium sensitivity, and pharmacologic properties of this current resembled those of TMEM16 CaCCs. RT-PCR analysis demonstrated the presence of TMEM16A and TMEM16B transcripts in wild-type and double knockout mouse lenses. Both TMEM16A and TMEM16B proteins were detected in the differentiating epithelial cells and newly elongating fiber cells near the equator of the lens by immunohistochemistry.
Our results demonstrate that membrane conductance of peripheral fiber cells contain CaCCs that can be attributed to TMEM16A and TMEM16B. Given their critical role in volume regulation in other tissues, we speculate that these channels play a similar role in the lens.
氯离子通道被认为在调节晶状体体积中发挥重要作用。由于难以分离小鼠纤维细胞，因此有关这些通道的分子特性或其在晶状体中的调节方式的信息很少。最近，我们的实验室开发了一种使用缺乏 Cx50 和 Cx46 的转基因小鼠晶状体来分离这些细胞的新技术。本研究的目的是通过使用该技术来测试新分化的小鼠纤维细胞表达钙激活氯离子通道（CaCCs）的假设。
通过胶原酶从缺乏 Cx50 和 Cx46 的双敲除小鼠晶状体中分离出分化的纤维细胞。使用全细胞膜片钳技术研究膜电流。通过 RT-PCR 和免疫荧光研究 CaCC 的分子特性和分布。
我们的电生理实验表明，周围纤维细胞表达钙激活氯离子电流。该电流的电压门控特性、钙敏感性和药理学特性与 TMEM16 CaCC 相似。RT-PCR 分析表明，野生型和双敲除小鼠晶状体中存在 TMEM16A 和 TMEM16B 转录本。免疫组织化学显示，TMEM16A 和 TMEM16B 蛋白均存在于分化的上皮细胞和晶状体赤道附近新伸长的纤维细胞中。
我们的结果表明，周围纤维细胞的膜电导包含可归因于 TMEM16A 和 TMEM16B 的 CaCC。鉴于它们在其他组织中对体积调节的关键作用，我们推测这些通道在晶状体中发挥类似的作用。
