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 the 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.
Rugolo
M
M
Dipartimento di Biologia E.S., Università di Bologna, Italy.
Mastrocola
T
T
De Luca
M
M
Romeo
G
G
Galietta
L J
LJ
D001692:Biological Transport; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D005576:Colforsin / Q000031:analogs & derivatives / Q000494:pharmacology; D005942:Gluconates / Q000494:pharmacology; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D008564:Membrane Potentials; D009994:Osmolar Concentration
D002712:Chlorides; D005942:Gluconates; D005576:Colforsin; C047983:1,9-dideoxyforskolin; C030691:gluconic acid