Membrane potential and ionic currents were studied in cultured rabbit retinal pigment epithelial (RPE) cells using whole-cell patch clamp and perforated-patch recording techniques. RPE cells exhibited both outward and inward voltage-dependent currents and had a mean membrane capacitance of 26 +/- 12 pF (SD, n = 92). The resting membrane potential averaged -31 +/- 15 mV (n = 37), but it was as high as -60 mV in some cells. When K+ was the principal cation in the recording electrode, depolarization-activated outward currents were apparent in 91% of cells studied. Tail current analysis revealed that the outward currents were primarily K+ selective. The most frequently observed outward K+ current was a voltage- and time-dependent outward current (IK) which resembled the delayed rectifier K+ current described in other cells. IK was blocked by tetraethylammonium ions (TEA) and barium (Ba2+) and reduced by 4-aminopyridine (4-AP). In a few cells (3-4%), depolarization to -50 mV or more negative potentials evoked an outwardly rectifying K+ current (IKt) which showed more rapid inactivation at depolarized potentials. Inwardly rectifying K+ current (IKI) was also present in 41% of cells. IKI was blocked by extracellular Ba2+ or Cs+ and exhibited time-dependent decay, due to Na+ blockade, at negative potentials. We conclude that cultured rabbit RPE cells exhibit at least three voltage-dependent K+ currents. The K+ conductances reported here may provide conductive pathways important in maintaining ion and fluid homeostasis in the subretinal space.
Calcium-activated potassium current was studied in cultured rabbit retinal pigment epithelial (RPE) cells using whole-cell and single channel patch-clamp recording techniques. When K+ was the principal cation in the electrode, depolarizing voltage steps from a holding potential of -60 mV activated outwardly rectifying current. Outward K+ current was increased by the Ca2+ ionophore ionomycin and reduced when the extracellular Ca2+ concentration was decreased from 2.5 mM to 100 nM in the presence of ionomycin. Outward K+ current recorded in the presence of ionomycin was blocked by iberiotoxin and by charybdotoxin. Single channel recording from cell-attached and excised membrane patches revealed a large conductance Ca2+-activated K+ (K(Ca)) channel. Identification of K(Ca) channels was based on: 1) the voltage-dependence of channel opening; 2) the large unitary conductance (> 200 pS with symmetrical 130 mM K+); 3) the dependence of the reversal potential on the K+ gradient; and 4) increased channel opening after exposure of the cytosolic surface of excised membrane patches to elevated Ca2+. These results demonstrate that Ca2+-activated K+ channels are present in rabbit RPE cells and may play an essential role in the regulation of membrane potential and ion transport.
1. The effects of adrenergic agonists on K¤ currents were studied in cultured rabbit pigmented ciliary epithelial (PCE) cells.
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