Characterization of Ca super(2+)-activated super(86)Rb super(+) fluxes in rat C6 glioma cells: A system for identifying novel IK sub(Ca)-channel toxins

The pharmacological characteristics of a putative Ca super(2+) activated K super(+) channel (IK sub(Ca) channel) in rat glioma C6 cells were studied in the presence of the Ca super(2+) ionophore, ionomycin and various K super(+) channel blockers, super(86)Rb super(+) being used as a radioisotopic tracer for K super(+). The resting super(86)Rb super(+) influx into C6 cells was 318 plus or minus 20 pmol s super(-1). The threshold for ionomycin activation of super(86)Rb super(+) influx was approx. 100 nM. At ionomycin concentrations above the activation threshold, the initial rate of super(86)Rb super(+) influx was proportional to ionophore concentration. Ionomycin-activated super(86)Rb super(+) flux was saturable (EC sub(50) = 0.62 plus or minus 0.03 mu M) and was not inhibited by ouabain. Intracellular Ca super(2+) increased within 30 s from a basal level of 42 plus or minus 2 nM to 233 plus or minus 17 nM, after addition of 2 mu M ionomycin. During this period, intracellular pH fell from 7.03 plus or minus 0.04 to 6.87 plus or minus 0.03 and the cell hyperpolarized from -34 plus or minus 10 mV to -76 plus or minus 2 mV. Single channel conductance measurements on inside-out patches in physiological K super(+) solutions identified a 14 plus or minus 3 pS Ca super(2+)-activated K super(+) current between -25 mV and +50 mV. In symmetrical (100 mM) K super(+), the single channel conductance was 26 pS. Externally applied quinine (IC sub(50) = 0.12 plus or minus 0.34 mM) and tetraethylammonium chloride (IC sub(50) = 10 plus or minus 1.9 mM) inhibited super(86)Rb super(+) influx into C6 cells in a concentration-dependent manner. Charybdotoxin (IC sub(50) = 0.5 plus or minus 0.02 nM) and iberiotoxin (IC sub(50) = 800 plus or minus 150 nM), as well as the crude venoms from the scorpions Leiurus quinquestriatus and Mesobuthus tamulus, also inhibited super(86)Rb super(+) influx. In contrast, apamin and toxin I had no inhibitory effects on super(86)Rb super(+) 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. The ionomycin-activated super(86)Rb super(+) 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, Ca super(2+) activated K super(+) channel activity.