VOLUME REGULATION IN A TOAD EPITHELIAL-CELL LINE - ROLE OF COACTIVATION OF K- CHANNELS( AND CL)

1. We have measured changes in cell volume, membrane potential and ion ic currents in distal nephron A6 cells following a challenge with hypo tonic solutions (HTS). 2. The volume increase induced by HTS is compen sated by a regulatory volume decrease (RVD), which is inhibited by bot h 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and quinine. Quinine (500 mu M) completely blocked RVD, whereas 100 mu M 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 -8 0 mV and 0.51 +/- 0.16 pA pF(-1) at +80 mV (n = 5). During cell swelli ng these values increased to 11.5 +/- 1.1 and 29.3 +/- 2.8 pA pF(-1) ( n = 29), respectively. 5. Quinine (500 mu M) completely blocked the HT S-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 mu M) inhibited the current at both potentials almost to t he 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+ eqilibr ium potential. The respective conductances activated by HTS at the zer o-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 lea st two different channels during HTS. A 36 pS channel reversing at the Cl- equilibrium potential showed increased open probability at depola rized potentials. HTS also activated a K+ channel with a 29 pS conduct ance 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 pote ntial towards a value between E(K) and E(Cl) (the reversal potentials for K+ and Cl-), where a net efflux of Cl- (Cl- inward current) and Kf (Kf outward current) under zero-current conditions occurs. Block of e ither the K+ or the Cl- conductance will shift the zero-current potent ial towards the equilibrium potential of the unblocked channel, preven ting net efflux of osmolytes and RVD. This coactivation of K+ and Cl- currents causes a shift of osmolytes out of the cells, which almost co mpletely accounts for the observed RVD.