Characterization of Ca(2+)-activated 86Rb+ fluxes in rat C6 glioma cells: a system for identifying novel IKCa-channel toxins.
1. The pharmacological characteristics of a putative Ca2+ activated K+ channel (IKCa channel) in rat glioma C6 cells were studied in the presence of the Ca2+ ionophore, ionomycin and various K+ channel blockers, 86Rb+ being used as a radioisotopic tracer for K+. 2. The resting 86Rb+ influx into C6 cells was 318 +/- 20 pmol s-1. The threshold for ionomycin activation of 86Rb+ influx was approx. 100 nM. At ionomycin concentrations above the activation threshold, the initial rate of 86Rb+ influx was proportional to ionophore concentration. Ionomycin-activated 86Rb+ flux was saturable (EC50 = 0.62 +/- 0.03 microM) and was not inhibited by ouabain. 3. Intracellular Ca2+ increased within 30 s from a basal level of 42 +/- 2 nM to 233 +/- 17 nM, after addition of 2 microM ionomycin. During this period, intracellular pH fell from 7.03 +/- 0.04 to 6.87 +/- 0.03 and the cell hyperpolarized from -34 +/- 10 mV to -76 +/- 2 mV. 4. Single channel conductance measurements on inside-out patches in physiological K+ solutions identified a 14 +/- 3 pS CA(2+)-activated K+ current between -25 mV and +50 mV. In symmetrical (100 mM) K+, the single channel conductance was 26 pS. 5. Externally applied quinine (IC50 = 0.12 +/- 0.34 mM) and tetraethylammonium chloride (IC50 = 10 +/- 1.9 mM) inhibited 86Rb+ influx into C6 cells in a concentration-dependent manner. Charybdotoxin (IC50 = 0.5 +/- 0.02 nM) and iberiotoxin (IC50 = 800 +/- 150 nM), as well as the crude venoms from the scorpions Leiurus quinquestriatus and Mesobuthus tamulus, also inhibited 86Rb+ influx. In contrast, apamin and toxin I had no inhibitory effects on 86Rb+ flux. A screen of fractions from cation exchange h.p.l.c. of Mesob. tamulus venom revealed the presence of at least four charybdotoxin-like peptides. One of these was iberiotoxin; the other three are novel toxins. 6. The ionomycin-activated 86Rb+ influx into rat C6 glioma cells has proved to be a valuable pharmacological assay for the screening of toxins and crude venoms which modify intermediate conductance, Ca2+ activated K+ channel activity.
de-Allie
F A
FA
Department of Paediatrics and Neonatal Medicine, Royal Postgraduate Medical School, London.
Bolsover
S R
SR
Nowicky
A V
AV
Strong
P N
PN
D000818:Animals; D001932:Brain Neoplasms / Q000378:metabolism*; D002118:Calcium / Q000502:physiology*; D018999:Charybdotoxin / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005910:Glioma / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D010042:Ouabain / Q000494:pharmacology; D015221:Potassium Channels / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D012413:Rubidium / Q000378:metabolism*; D012414:Rubidium Radioisotopes; D012604:Scorpion Venoms / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D007476:Ionophores; D015221:Potassium Channels; D012414:Rubidium Radioisotopes; D012604:Scorpion Venoms; D018999:Charybdotoxin; D015759:Ionomycin; D010042:Ouabain; D012413:Rubidium; D002118:Calcium