Possible thiol group involvement in intracellular pH effect on low-conductance Ca(2+)-dependent K+ channels.
We have studied the effect of intracellular pH (pHi) shifts on the activity of Ca(2+)-dependent, inwardly rectifying K+ channels of HeLa cells. Recordings of macroscopic currents in symmetrical 145 mK K+ and internal pH of 7.4 gave moderate inward rectification of the current. At pH 6.4, inward rectification was more marked, whereas it switched to outward rectification at pH 8.2. In excised inside-out membrane patches, similar changes in pHi did not affect the single-channel conductance of the channels underlying the Ca(2+)-dependent K+ currents. At neutral pH, the open state probability (Po) was independent of voltage in the range from -70 to 70 mV. At alkaline pH, Po became voltage dependent, decreasing at negative potentials and increasing with depolarization compared with pH 7.4. These changes accounted for the pH-dependent changes in rectification of the macroscopic current. The possibility that voltage dependence might arise from the ionization of a thiol group was tested by using thiol-directed reagents. The decrease in Po produced by intracellular alkalinization at negative potential was reverted by treatment with N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic acid), and 2,2'-dithiodipyridine. The effect of intracellular alkalinization is speculated to occur through ionization of a cysteine group(s) within the field of the membrane affecting gating.
Riquelme
G
G
Departamento de Fisiología y Biofísica, Faculated de Medicina, Universidad de Chile, Santiago-7, Chile.
Díaz
M
M
Sepúlveda
F V
FV
D002118:Calcium / Q000494:pharmacology*; D003545:Cysteine; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004558:Electric Stimulation; D005033:Ethylmaleimide / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid / Q000502:physiology; D015640:Ion Channel Gating / Q000502:physiology; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying; D011336:Probability; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D013997:Time Factors
D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D013439:Sulfhydryl Reagents; D004228:Dithionitrobenzoic Acid; D003545:Cysteine; D005033:Ethylmaleimide; D002118:Calcium; D004229:Dithiothreitol