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Lisa Ebihara, Jun-Jie Tong; Calcium-activated chloride currents in peripheral mouse fiber cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2476.
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© ARVO (1962-2015); The Authors (2016-present)
Chloride channels play a critical role in regulating lens volume and maintaining optical transparency. Unfortunately, little information is available about how these channels operate in lens fiber cells. Recently, our laboratory developed a new technique for isolating mouse fiber cells utilizing transgenic mouse lenses. The purpose of this study was to characterize the biophysical properties of chloride conductances in mouse fiber cells using this new technique.
Differentiating fiber cells were isolated from the lenses of double knockout mice that lack both Cx50 and Cx46 using collagenase. Membrane currents were recorded in fiber cells using the whole cell patch clamp technique. RT-PCR was used to determine whether TMEM16A or B were expressed in the lens. The distribution of TMEM16A in the lens was analyzed by immunofluorescence microscopy.
We studied fiber cells from double knockout lenses ranging between 50 and 600 microns in length using the whole cell patch clamp technique. These experiments indicated that sodium conductances were absent or small in fiber cells lacking connexins. Rather, our work suggested that the membrane conductance was dominated by potassium conductances. In addition, there was a voltage-dependent chloride current that could be activated by raising the free intracellular calcium to concentrations greater than 50-100 nM. The main biophysical characteristics of this current were: (1) It slowly activated on depolarization and deactivated upon repolarization; (2) It showed steady state outward rectification at low and intermediate calcium concentrations, but exhibited a linear I-V relationship at high calcium concentrations; (3) It’s permeability sequence for anions was Cl >> Glu; (4) It was blocked by standard calcium-activated chloride channel blockers such as niflumic acid and tannic acid. These properties resembled those of the TMEM16A and B calcium-activated chloride currents. RT-PCR demonstrated the presence of TMEM16A and possibly TMEM16B transcripts in wild-type mouse lenses. Immunohistochemical staining revealed the presence of TMEM16A protein in peripheral fiber cells.
Our results strongly suggest that peripheral mouse fiber cells functionally express calcium-activated chloride channels which can be at least in part attributed to TMEM16A.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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