Abstract
Purpose :
Thiocyanate (SCN-), a biologically-active anion known to have both positive and negative effects in the body outside the eye, is actively transported by a variety of epithelia. Plasma levels of SCN- typically range from 25 µM to 100 µM but can exceed 150 µM in smokers. Our recent electrophysiological study of isolated mouse RPE cells demonstrated that the basolateral membrane contains an anion conductance that is highly selective for SCN- and that in the presence of 10 external SCN-, voltage steps produce large, transient whole-cell currents whose time-dependent decay most likely results from the accumulation or depletion of intracellular SCN-. In the present study, we investigated the effects of more physiologically relevant concentrations of SCN- on RPE electrophysiological properties.
Methods :
Whole-cell currents of freshly isolated C57B/6 mouse RPE cells were recorded using the patch-clamp technique. Currents were evoked by voltage steps in the presence of external SCN- at concentrations ranging from 50 to 500 µM or by the rapid application of 500 µM SCN- by pressure ejection via a second pipette. The voltage dependence of SCN- fluxes into and out of voltage-clamped BALB/c mouse RPE cells was determined by monitoring changes in the fluorescence of the anion-sensitive dye MQAE loaded into the cytoplasm via the patch pipette.
Results :
RPE cells exhibited transient currents at all SCN- concentrations tested, with the amplitude of the peak current increasing as the SCN- concentration was increased. Transient currents were also produced when the membrane potential was held constant and the external SCN- concentration was increased rapidly by the pressure ejection of 500 µM SCN-. Finally, the flux of SCN- into or out of BALB/c RPE cells exposed to 500 µM SCN-, measured by the rate of intracellular MQAE fluorescence quenching, was strongly voltage-dependent.
Conclusions :
Our results indicate that isolated mouse RPE cells have an unexpectedly large SCN- conductance in the presence of physiologically-relevant concentrations of SCN-. Fluxes of SCN- via this conductance are sufficiently large that SCN- rapidly approaches electrochemical equilibrium in the cytoplasm when the membrane potential is altered. The specific role that this unusual anion conductance plays in RPE transport physiology remains to be determined.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.