Ca2+ influx through carbachol-activated non-selective cation channels in guinea-pig gastric myocytes

Abstract

1. Ca2+ microfluorometry (100 μM K5 fura-2) and the voltage-clamp technique were combined to study the effect of carbachol (CCh, 50 μM) in inducing currents (I(CCh)) through nonselective cation channels (NSC(CCh)) and increments in global cytosolic Ca2+ concentration (Δ[Ca2+](c)). 2. In Na+-containing bath solution, I(CCh) fell from an initial phasic to a subsequent small (5%) tonic component; Δ[Ca2+](c) fell to zero. Tonic I(CCh) and [Ca2+](c) became prominent after substitution of extracellular 140 mM Na+ by 140 mM Cs+. Tonic I(CCh) and Δ[Ca2+](c) were insensitive to intracellular heparin (3 mg ml-1) and ryanodine (4 μM), i.e. they did not depend on Ca2+ release from sarcoplasmic reticulum (SR). 3. Single channel currents of NSC(CCh) could be resolved in whole-cell recordings. Substitution of Na+ by Cs+ increased NSC(CCh) activity by one order of magnitude and slope conductance from 22 to 30 pS. Extracellular quinidine (3 μM) reversibly blocked the NSC(CCh) activity. 4. Both tonic I(CCh) and tonic Δ[Ca2+](c) (a) followed a similar time course of activation, desensitization and facilitation, (b) were reversibly blocked by 3 μM quinidine, and (c) persisted upon block of SR Ca2+ release. 5. A Ca2+ fractional current of tonic I(CCh) (f(Ca)) of 0.009 was calculated by comparing the ratio Δ[Ca2+](c) (corrected for simultaneous Ca2+ redistribution) over I(CCh) with depolarization-induced *Δ[Ca2+](c) (Δ[Ca2+](c) calculated from I(Ca) induced by a 400 ms depolarization from -60 to 0 mV at 2 mM [Ca2+](o), 145 mM [Cs+](o)) over I(Ca).f(Ca) was 0.023 at [Ca2+](o) = 4 mM. 6. With 110 mM extracellular CaCl2 and 145 mM intracellular CsCl, I(CCh) reversed at +19.5 mV suggesting a permeability ratio P(Ca)/P(Cs) of 2.8. 7. We conclude that Ca2+ influx through NSC(CCh) under physiological [Ca2+](o) could induce Δ[Ca2+](c). The f(Ca) was, however, much smaller than the one calculated from the reversal potential.