pH dependence of K+ conductances of rat cortical collecting duct principal cells

The K⁺ channels of the principal cells of rat cortical collecting duct (CCD) are pH sensitive in excised membranes. K⁺ secretion is decreased with increased H⁺ secretion during acidosis. We examined whether the pH sensitivity of these K⁺ channels is present also in the intact cell and thus could explain the coupling between K⁺ and H⁺ secretion. Membrane voltages (V_m), whole-cell conductances (g_c), and single-channel currents of K⁺ channels were recorded from freshly isolated CCD cells or isolated CCD segments with the patch-clamp method. Intracellular pH (pH_i) was measured using the pH-sensitive fluorescent dye 2-7-bis(carboxyethyl)-5-6-carboxyfluorescein (BCECF). Acetate (20 mmol/l) had no effect on V_m, g_c, or the activity of the K⁺ channels in these cells. Acetate, however, acidified pH_i slightly by 0.17±0.04 pH units (n=19). V_m depolarized by 12±3 mV (n=26) and by 23±2 mV (n=66) and g_c decreased by 26±5% (n=13) and by 55±5% (n=12) with 3–5 or 8–10% CO₂, respectively. The same CO₂ concentrations decreased pH_i by 0.49±0.07 (n=15) and 0.73±0.11 pH units (n=12), respectively. Open probability (P_o) of all four K⁺ channels in the intact rat CCD cells was reversibly inhibited by 8–10% CO₂. pH_i increased with the addition of 20 mmol/l NH₄⁺/NH₃ by a maximum of 0.64±0.08 pH units (n=33) and acidified transiently by 0.37±0.05 pH units (n=33) upon NH₄⁺/NH₃ removal. In the presence of NH₄⁺/NH₃V_m depolarized by 16±2 mV (n=66) and g_c decreased by 26±7% (n=16). The activity of all four K⁺ channels was also strongly inhibited in the presence of NH₄⁺/NH₃. The effect of NH₄⁺/NH₃ on V_m and g_c was markedly increased when the pH of the NH₄⁺/NH₃-containing solution was set to 8.5 or 9.2. From these data we conclude that cellular acidification in rat CCD principal cells down-regulates K⁺ conductances, thus reduces K⁺ secretion by direct inhibition of K⁺ channel activity. This pH dependence is present in all four K⁺ channels of the rat CCD. The inhibition of K⁺ channels by NH₄⁺/NH₃ is independent of changes in pH_i and rather involves an effect of NH₃.