CHARACTERIZATION OF CA2+ DEPENDENT INWARDLY RECTIFYING K+ CURRENTS IN HELA-CELLS

The whole-cell configuration of the patch-clamp technique was used to examine K+ currents in HeLa cells. Under quasi-physiological ionic gradients, using an intracellular solution containing 10(-7) mol/l free Ca2+, mainly outward currents were observed. Large inwardly rectifying currents were elicited in symmetrical 145 mmol/l KCl. Replacement of all extracellular K+ by isomolar Na+, greatly decreased inward currents and shifted the reversal potential as expected for Kf selectivity. The inwardly rectifying Ki currents exhibited little or no apparent voltage dependence within the range of from -120 mV to 120 mV. A square-root relationship between chord conductance and [K+](o) at negative potentials could be established. The inwardly rectifying nature of the currents was unaltered after removal of intracellular Mg2+ and chelation with ATP and ethylenediaminetetraacetic acid (EDTA). Permeability ratios for other monovalent cations relative to K+ were: K+ (1.0) > Rb+ (0.86) > Cs+ (0.12) > Li+ (0.08) > Na+ (0.03). Slope conductance ratios measured at -100 mV were: Rb+ (1.66) > K+ (1.0) > Na+ (0.09) > Li+ (0.08) > Cs+ (0.06). K+ conductance was highly sensitive to intracellular free Ca2+ concentration. The relationship between conductance at 0 mV and Ca2+ concentration was well described by a Hill expression with a dissociation constant, K-D, of 70 nmol/l and a Hill coefficient, n, of 1.81. Extracellular Ba2+ blocked the currents in a concentration- and voltage-dependent manner. The dependence of the K-D for the blockade was analysed using a Woodhull-type treatment, locating the ion interaction site at 19% of the distance across the electrical field of the membrane and a K-D (0 mV) of 7 mmol/l, Tetraethylammonium and 4-aminopyridine were without effect whilst quinine and quinidine blocked the currents with concentrations for half-maximum effects equal to 7 mu mol/l and 3.5 mu mol/l, respectively, The unfractionated venom of the scorpion Leiurus quinquestriatus (LQV) blocked the K+ currents of HeLa cells. The toxins apamin and scyllatoxin had no detectable effect whilst charybdotoxin, a component of LQV, blocked in a voltage-dependent manner with half-maximal concentrations of 40 nmol/l at -120 mV and 189 nmol/l at 60 mV; blockade by charybdotoxin accounts for the effect of LQV. Application of ionomycin (5-10 mu mol/l), histamine (1 mmol/l) or bradykinin (1-10 mu mol/l) to cells dialysed with low-buffered intracellular solutions induced K+ currents showing inward rectification and a lack of voltage dependence.