Characterization of Ca2+‐activated 86Rb+ fluxes in rat C6 glioma cells: a system for identifying novel IKCa‐channel toxins
Open Access
- 1 February 1996
- journal article
- Published by Wiley in British Journal of Pharmacology
- Vol. 117 (3) , 479-487
- https://doi.org/10.1111/j.1476-5381.1996.tb15215.x
Abstract
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 μm) 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 μm 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 Ca2+ -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.Keywords
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