Volume-activated chloride channels in hl-60 cells: potent inhibition by an oxonol dye. Arreola, Jorge, Kenneth R. Hallows, and Philip A. Knauf. Departments of Dental Research, Physiology, and Biophysics, University of Rochester, Rochester NY 14642
APStracts 2:0163C, 1995.
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 mosm/kg) 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- @ NO3- > Br - > Cl- >> gluconate. SITS (0.5 mM) inhibits HL-60 Cl- channels in a voltage-dependent manner, with 10-fold increased affinity at potentials >+40 mV. Voltage-dependent blockade by SITS indicates that the binding site is located near the outside, where it senses 20% of Vm. These Cl- channels were also inhibited in a voltage-independent manner by the oxonol dye diBA-(5) -C4 (bis-(1,3-dibutylbarbituric acid)pentamethine oxonol) with an IC50 value (concentration which gives half inhibition) of 1.8 mM at room temperature. A similar apparent IC50 value (1.2 mM) was observed for net 36Cl- efflux into a Cl--free hypotonic medium at 21oC. 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. 

Received 2 August 1994; accepted in final form 4 April 1995.
APS Manuscript Number C455-4.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 19 April 1995.