Voltage–Dependent Currents in Freshly Isolated Cells of the Mouse Retinal Pigment Epithelium

Abstract

Abstract: : Purpose: A loss of Cl channel function the retinal pigment epithelium (RPE) leads to different types of retinal degenerations. The purpose of the study is to investigate ion currents in freshly isolated mouse RPE cells to study mechanisms leading to retinal degeneration in transgenic or knock–out mouse models. Methods: The RPE from 2–3 months old mice was dissolved into single cell suspension by gentle digestion using accutase. After cell attachment to poly–lysine coated cover slips membrane currents were measured under intra–/ extracellular K⁺–free conditions. Results: Voltage–dependent currents with an overall density of 26.9 ± 6 pA/pF (n = 4) were activated from a holding potential of –40 mV (depolarisation: to +50 mV, voltage–steps of 50 ms duration, 10 mV increment; hyperpolarisation: to –130 mV in voltage–steps, –10 mV increment, 50 ms duration). All cells responded to hyperpolarisation to potentials more negative than – 32 ± 4.2 mV (n = 9) with inwardly rectifying, sometimes slowly activating, currents. In one third of all cells an additional outwardly rectifying current of 7.5 ± 2.4 pA/pF (n = 4) was observed. This current was increased to 14 ± 3.6 pA/pF (n = 7) when intracellular free Ca²⁺ was increased from 10 nM to 400 nM. Conclusions: Fresh mouse RPE cells show two types of voltage–dependent currents under K⁺–free conditions: a ClC–2 like inwardly–rectifying current and a current with properties of Ca²⁺ activated Cl channels. Thus, the method provides a tool to study retinal degeneration in knock–out or transgenic mouse models.

Keywords: ion channels • retinal pigment epithelium • electrophysiology: non-clinical