Characterisation of Ca(2+)-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 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.
Díaz
M
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AFRC Babraham Institute, Babraham, Cambridge, UK.
Sepúlveda
F V
FV
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001464:Barium / Q000494:pharmacology; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002414:Cations, Monovalent; D002463:Cell Membrane Permeability; D018999:Charybdotoxin; D004492:Edetic Acid / Q000494:pharmacology; D004553:Electric Conductivity; D006367:HeLa Cells; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism* / Q000494:pharmacology; D015221:Potassium Channels / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying; D011802:Quinidine / Q000494:pharmacology; D011803:Quinine / Q000494:pharmacology; D012604:Scorpion Venoms / Q000494:pharmacology; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds / Q000494:pharmacology
D002414:Cations, Monovalent; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D012604:Scorpion Venoms; D013757:Tetraethylammonium Compounds; D018999:Charybdotoxin; D001464:Barium; D019789:Tetraethylammonium; D006632:Histamine; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D011803:Quinine; D008274:Magnesium; D011802:Quinidine; D011188:Potassium; D001920:Bradykinin; D002118:Calcium