Mouse RPE Cells Have an Unusually High Conductance and Permeability to Thiocyanate

Abstract

Purpose : Anion channels mediate the movement of Cl^- across the RPE basolateral membrane in the final step of active Cl^- absorption and also influence the transport of other ions by helping set the membrane potentials. Multiple types of regulated Cl^- channels have been found in the RPE, but the channel underlying the resting Cl^- conductance has yet to be identified. The purpose of this study was to determine the permeation properties of mouse RPE cells with respect to thiocyanate (SCN^-), nitrate (NO₃^-), and other anions as a first step toward functionally identifying the predominant anion conductance in the RPE under resting conditions.

Methods : RPE cells were enzymatically isolated from the eyes of wild-type C57/B6 mice. Freshly isolated RPE cells were placed in a constantly superfused recording chamber and whole-cell currents were recorded using the patch-clamp technique. The pipette solution contained 140 mM NMDG-Cl (nominally 30 nM free Ca²⁺, pH 7.2) and the bath contained 140 mM NaX, where X = Cl^-, NO₃^-, I^-, Br^-, SCN^-, or gluconate^-. Slope conductance was calculated in the voltage range +30 to +60 mV and the permeability ratio was estimated from the change in reversal potential resulting from the replacement of Cl^- with the test anion.

Results : Isolated mouse RPE cells had a membrane capacitance of 114.6 ± 20.2 pF (mean ± SEM, n = 16). When bathed with 140 mM NaCl solution, whole-cell currents in response to 1-sec voltage steps were essentially time independent and exhibited mild inward rectification. The relative permeabilities under bi-ionic conditions were: SCN^- >> NO₃^- >> I^- > Br^- > Cl^- >> gluconate^-, with P_SCN/P_Cl = 23.1 ± 7.2 (mean ± SEM, n = 7). The conductance sequence was SCN^- >> NO₃^- > I^- > Br^- > Cl^- >> gluconate, with G_SCN = 617.0 ± 114.9 pS/pF (n = 7) and G_Cl = 21.2 ± 1.6 pS/pF (n = 16).

Conclusions : Under resting conditions, freshly isolated mouse RPE cells exhibit a remarkably high conductance and permeability to SCN^-. This novel finding suggests that the RPE plasma membrane contains an anion channel or transporter that has not been described previously. Further characterization of this membrane mechanism may lead to new insight into RPE transport function.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.