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Characterization of Ca 2+ ‐activated 86 Rb + fluxes in rat C6 glioma cells: a system for identifying novel IK Ca ‐channel toxins
Author(s) -
deAllie Frank A.,
Bolsover Steven R.,
Nowicky Alex V.,
Strong Peter N.
Publication year - 1996
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1996.tb15215.x
Subject(s) - ionomycin , iberiotoxin , chemistry , charybdotoxin , tetraethylammonium , ouabain , ionophore , tetraethylammonium chloride , potassium channel , apamin , ion transporter , biophysics , membrane potential , conductance , intracellular , sodium , biochemistry , potassium , membrane , biology , mathematics , organic chemistry , combinatorics
1 The pharmacological characteristics of a putative Ca 2+ activated K + channel (IK Ca channel) in rat glioma C6 cells were studied in the presence of the Ca 2+ ionophore, ionomycin and various K + channel blockers, 86 Rb + being used as a radioisotopic tracer for K + . 2 The resting 86 Rb + influx into C6 cells was 318±20 pmol s −1 . The threshold for ionomycin activation of 86 Rb + influx was approx. 100 nM. At ionomycin concentrations above the activation threshold, the initial rate of 86 Rb + influx was proportional to ionophore concentration. Ionomycin‐activated 86 Rb + flux was saturable (EC 50 = 0.62±0.03 μ m ) and was not inhibited by ouabain. 3 Intracellular Ca 2+ increased within 30 s from a basal level of 42±2 nM to 233±17 nM, after addition of 2 μ m 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 (IC 50 = 0.12±0.34 mM) and tetraethylammonium chloride (IC 50 = 10±1.9 mM) inhibited 86 Rb + influx into C6 cells in a concentration‐dependent manner. Charybdotoxin (IC 50 = 0.5±0.02 nM) and iberiotoxin (IC 50 = 800±150 nM), as well as the crude venoms from the scorpions Leiurus quinquestriatus and Mesobuthus tamulus , also inhibited 86 Rb + influx. In contrast, apamin and toxin I had no inhibitory effects on 86 Rb + 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 86 Rb + 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 2+ activated K + channel activity.