A volume-sensitive chloride conductance revealed in cultured human keratinocytes by 36Cl- efflux and whole-cell patch clamp recording

The Cl^- transport mechanism responsible for the stimulation of ³⁶Cl^- efflux after exposure to hypotonic medium (210 mosmol/kg) was investigated in human keratinocytes. The involvement of the anion exchanger and of the Cl^-/cation cotransporters was ruled out by the finding that replacement of extracellular Cl^- by the poorly permeant anion gluconate, and the addition of bumetanide and furosemide, inhibitors of Na⁺/K⁺/Cl^- and K⁺/Cl^- cotransporters, respectively, failed to significantly reduce the activation of Cl^- efflux by hypotonic medium. 'Whole cell' configuration of the patch clamp technique directly revealed the presence of a macroscopic Cl^- current, which was evoked by incubation with hypotonic medium and was reversed by elevation of the extracellular osmolality. Volume-sensitive current showed outward rectification of the current-voltage relationship and time-dependent inactivation at depolarizing voltages. This current was Cl^- selective, because the zero-current reversal potential approached the Cl^- equilibrium potential, when extracellular Cl^- was replaced by gluconate. 0.1 mM, 1,9-dideoxyforskolin significantly reduced either ³⁶Cl^- efflux and the Cl^- current, suggesting that the Cl^- efflux and the macroscopic current activated after exposure to hypotonic medium are mediated by the same pathway. Electronic cell sizing showed that in keratinocytes hypotonic swelling was not followed by a significant regulatory volume decrease response.