title: Volume regulation in a toad epithelial cell line: role of coactivation of K+ and Cl- channels.
abstract: 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.
authors.affiliation: KU Leuven, Laboratorium voor Fysiologie, Belgium.
authors.forename: B
authors.initials: B
authors.lastname: Nilius
authors.forename: J
authors.initials: J
authors.lastname: Sehrer
authors.forename: P
authors.initials: P
authors.lastname: De Smet
authors.forename: W
authors.initials: W
authors.lastname: Van Driessche
authors.forename: G
authors.initials: G
authors.lastname: Droogmans
mesh_terms: D000818:Animals; D001001:Anura; D002460:Cell Line; D018118:Chloride Channels / Q000378:metabolism*; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D007038:Hypotonic Solutions; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism*; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D007686:Kidney Tubules, Distal / Q000166:cytology / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D009579:Nitrobenzoates / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000378:metabolism*; D011803:Quinine / Q000494:pharmacology
chemical_list: D018118:Chloride Channels; D007038:Hypotonic Solutions; D009579:Nitrobenzoates; D015221:Potassium Channels; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D011803:Quinine