Volume regulation in a toad epithelial cell line: role of coactivation of K+ and Cl‐ channels.

1. We have measured changes in cell volume, membrane potential and ionic currents in distal nephron A6 cells following a challenge with hypotonic solutions (HTS). 2. The volume increase induced by HTS is compensated by a regulatory volume decrease (RVD), which is inhibited by both 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate (NPPB) and quinine. Quinine (500 microM) completely blocked RVD, whereas 100 microM NPPB delayed and attenuated RVD. 3. The resting potential in A6 cells was ‐52.3 +/‐ 4.8 mV (n = 53), and shifted to ‐35.1 +/‐ 2.2 mV (n = 33) during HTS. 4. Resting membrane current in A6 cells was 0.35 +/‐ 0.12 pA pF‐1 at ‐80 mV and 0.51 +/‐ 0.16 pA pF‐1 at +80 mV (n = 5). During cell swelling these values increased to 11.5 +/‐ 1.1 and 29.3 +/‐ 2.8 pA pF‐1 (n = 29), respectively. 5. Quinine (500 microM) completely blocked the HTS‐activated current at ‐15 mV, the reversal potential for Cl‐ currents, but exerted only a small block at ‐100 mV (K+ equilibrium potential). NPPB (100 microM) inhibited the current at both potentials almost to the same extent. The HTS‐induced net current reversed at ‐41 +/‐ 2.5 mV (n = 15), which is close to the measured resting potential during HTS. 6. The quinine‐insensitive current reversed near the Cl‐ equilibrium potential. The quinine‐sensitive current reversed near the K+ equilibrium potential. The respective conductances activated by HTS at the zero‐current potential were 2.1 +/‐ 0.7 nS for K+ and 5.2 +/‐ 1.3 nS for Cl‐ (n = 15). 7. Single channel analysis unveiled activation of at least two different channels during HTS. A 36 pS channel reversing at the Cl‐ equilibrium potential showed increased open probability at depolarized potentials. HTS also activated a K+ channel with a 29 pS conductance in high‐K+ extracellular solutions (130 mM) or 12 pS in 2.5 mM K+. 8. This coactivation of K+ and Cl‐ channels shifts the membrane potential towards a value between EK and ECl (the reversal potentials for K+ and Cl‐), where a net efflux of Cl‐ (Cl‐ inward current) and K+ (K+ outward current) under zero‐current conditions occurs. Block of either the K+ or the Cl‐ conductance will shift the zero‐current potential towards the equilibrium potential of the unblocked channel, preventing net efflux of osmolytes and RVD. This coactivation of K+ and Cl‐ currents causes a shift of osmolytes out of the cells, which almost completely accounts for the observed RVD.