Calcium-Activated Chloride Channels in Newly Differentiating Mouse Lens Fiber Cells and Their Role in Volume Regulation.

Abstract

PurposeChloride 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.MethodsDifferentiating 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.ResultsOur 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.ConclusionsOur 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.