Volume-activated chloride channels in HL-60 cells: Potent inhibition by an oxonol dye
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When swollen in hypotonic media, HL-60 cells exhibit a regulatory volume decrease (RVD) response as a result of net losses of K%2b 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 (P(X)/P(Cl)) calculated from the shifts of the reversal potentials, was SCN- > I- ≃ NO3/- > Br- > Cl- >> gluconate. 4-Acetamido-4%27-isothiocyanostilbene-2,2%27-disulfonic acid (SITS; 0.5 mM) inhibits HL-60 Cl- channels in a voltage-dependent manner, with ~10-fold increased affinity at potentials greater than %2b40 mV. Voltage-dependent blockade by SITS indicates that the binding site is located near the outside, where it senses 20%25 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 μM at room temperature. A similar apparent IC50 value (1.2 μM) was observed for net 36Cl- efflux into a Cl- free hypotonic medium at 21°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 %27mini-Cl-%27 channels described in lymphocytes and neutrophils and are strongly inhibited by low concentrations of diBA-(5)-C4.
When swollen in hypotonic media, HL-60 cells exhibit a regulatory volume decrease (RVD) response as a result of net losses of K%2b 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 (P(X)/P(Cl)) calculated from the shifts of the reversal potentials, was SCN- > I- ≃ 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 ~10-fold increased affinity at potentials greater than %2b40 mV. Voltage-dependent blockade by SITS indicates that the binding site is located near the outside, where it senses 20%25 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 μM at room temperature. A similar apparent IC50 value (1.2 μM) was observed for net 36Cl- efflux into a Cl- free hypotonic medium at 21°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.
