Characterisation of Ca(2+)-dependent inwardly rectifying K+ currents in HeLa cells

Abstract

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 K+ selectivity. The inwardly rectifying K+ 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+] 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, KD, 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 KD 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 KD (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 mumol/l and 3.5 mumol/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 mumol/l), histamine (1 mmol/l) or bradykinin (1-10 mumol/l) to cells dialysed with low-buffered intracellular solutions induced K+ currents showing inward rectification and a lack of voltage dependence.