Characterization of a volume-sensitive chloride current in rat osteoblast-like (ROS 17/2.8) cells

1. During osmotic swelling, cultured osteoblastic cells (ROS 17/2.8) exhibited activation of large amplitude Cl^- currents in the whole-cell configuration of the patch-clamp technique. Effects of hypotonic shock on cell volume and membrane conductance were rapidly reversed on return to isotonic conditions. 2. Voltage command pulses in the range -80 to +50 mV produce instantaneous activation of Cl^- currents. At potentials more positive than +50 mV the current exhibited time-dependent inactivation. The instantaneous current-voltage relationship was outwardly rectifying. 3. The anion permeability sequence of the induced current was SCN^- (2.2) > I^- (1.9) > Br^- (1.5) > Cl^- (1.0) > F^- (0.8) > gluconate^- (0.2). This corresponds to Eisenman's sequence I. 4. The volume-sensitive Cl^- current was effectively inhibited by the Cl^- channel blockers 4,4'-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). Outward currents were more effectively suppressed by DIDS than inward currents. The concentrations for 50% inhibition (IC₅₀) of outward and inward currents were 81 and 298 μM, respectively. NPPB was equally effective at inhibiting outward and inward currents (IC₅₀ of 64 μM). The current was relatively insensitive to diphenylamine-2-carboxylate (DPC), 500 μM producing only 22.5 ± 4.0% inhibition. 5. Inhibitors of protein kinase A (H-89, 1 μM) and tyrosine kinase (tyrphostin A25, 200 μM) were without effect upon activation of Cl^- currents in response to hypotonic shock. Under isotonic conditions, elevation of intracellular Ca²⁺ by ionomycin (1 μM) or activation of protein kinase C by 12-O-tetradecanoylphorbol 13-acetate (TPA, 0.1 μM) failed to evoke increases in basal Cl^- conductance levels. 6. It is concluded that an outwardly rectifying Cl^- conductance is activated upon osmotic swelling and may be involved in cell volume regulation of ROS 17/2.8 cells.