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Am J Physiol. 1995 Oct;269(4 Pt 1):C1063-72.

Volume-activated chloride channels in HL-60 cells: potent inhibition by an oxonol dye.

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1
Department of Dental Research, University of Rochester, New York 14642, USA.

Abstract

When swollen in hypotonic media, HL-60 cells exhibit a regulatory volume decrease (RVD) response as a result of net losses of K+ and Cl-. This is primarily caused by a dramatic increase in Cl- permeability, which may reflect the opening of volume-sensitive channels (11). To test this hypothesis, we measured volume-activated Cl- currents in HL-60 cells using the patch-clamp technique. The whole cell Cl- conductance (in nS/pF at 100 mV) increased from 0.09 +/- 0.06 to 1.15 +/- 0.19 to 1.64 +/- 0.40 as the tonicity (in mosmol/kgH2O) of the external medium was decreased from 334 to 263 to 164, respectively. Cl- currents showed no significant inactivation during 800-ms pulses. Current-voltage curves exhibited outward rectification and were identical at holding potentials of 0 or -50 mV, suggesting that the gating of the channels is voltage independent. The selectivity sequence, based on permeability ratios (PX/PCl) calculated from the shifts of the reversal potentials, was SCN- > I- approximately NO3- > Br- > Cl- >> gluconate. 4-Acetamido-4'- isothiocyanostilbene-2,2'-disulfonic acid (SITS; 0.5 mM) inhibits HL-60 Cl- channels in a voltage-dependent manner, with approximately 10-fold increased affinity at potentials greater than +40 mV. Voltage-dependent blockade by SITS indicates that the binding site is located near the outside, where it senses 20% of the membrane potential. These Cl- channels were also inhibited in a voltage-independent manner by the oxonol dye bis-(1,3-dibutylbarbituric acid)pentamethine oxonol [diBA-(5)-C4] with a concentration that gives half inhibition (IC50) of 1.8 microM at room temperature. A similar apparent IC50 value (1.2 microM) was observed for net 36Cl- efflux into a Cl(-)-free hypotonic medium at 21 degrees C. It seems likely, therefore, that the volume-activated Cl- channels are responsible for the net Cl- efflux during RVD. These Cl- channels have properties similar to the "mini-Cl-" channels described in lymphocytes and neutrophils and are strongly inhibited by low concentrations of diBA-(5)-C4.

[Indexed for MEDLINE]

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