The Basal Membrane of Mouse RPE Contains a Unique Anion Channel with a High Permeability to Thiocyanate

Abstract

Purpose : The anion conductance of the RPE basal membrane plays a key role in the active transport of Cl^- between the subretinal space and choroid. Previously, we reported that exposure of isolated mouse RPE cells to thiocyanate (SCN^-) evokes a large whole-cell current, suggesting that the RPE may contain a previously unknown anion channel. The purpose of this study was to characterize the biophysical and pharmacological properties of this channel in excised membrane patches as the next step towards determining its identity.

Methods : RPE cells were freshly isolated from the eyecups of C57/B6 mice and placed in a constantly perfused recording chamber. Currents from excised outside-out patches of basal or apical membrane were recorded using the patch-clamp technique. The pipette solution contained 135 mM NMDG-Cl and the bath solution contained 140 mM NaCl or 140 mM NaSCN. The relative permeability (P_SCN/P_Cl) was calculated using the GHK equation.

Results : Replacement of external Cl^- with SCN^- increased the amplitude of the current in both basal and apical membrane patches with no obvious increase in noise, but basal membrane patches were significantly more responsive. Replacement of external Cl^- with SCN^- produced nearly a 6-fold increase in conductance in basal membrane patches (from 276 ± 47 pS/pF to 1,535 ± 168 pS/pF) compared to a 2-fold increase in apical membrane patches (from 410 ± 104 pS/pF to 834 ± 165 pS/pF). Moreover, replacement of external Cl^- with SCN^- shifted the reversal potential by -80.1± 4.6 mV (n=36) in basal membrane patches and by -43.1 ± 5.6 mV (n=14) in apical membrane patches, giving P_SCN/P_Cl values of 47.9 ± 12.6 and 7.0 ± 2.2, respectively. Exposure of basal membrane patches to the anion channel blockers tannic acid (100 µM, n=4), DIDS (500 µM, n=4), NPPB (200 µM, n=3), CFTR_inh-172 (3 µM, n=3) had minor effects on the SCN^--evoked current, but zinc (1 mM) reduced the current by 47.6 ± 10.6 % (n=4).

Conclusions : Our results demonstrate that the basal membrane of the mouse RPE expresses an anion conductance with high SCN^- permeability. The blocker sensitivity of this conductance does not correspond to that of any of the Cl^- channels thought to be expressed in the RPE. Further experiments will be needed to identify the specific channel that underlies the SCN^--selective conductance in the RPE basal membrane.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.