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

Abstract

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 (ICCh) through non-selective cation channels (NSCCCh) and increments in global cytosolic Ca2+ concentration (Δ[Ca2+]c).In Na+-containing bath solution, ICCh fell from an initial phasic to a subsequent small (5 %) tonic component; Δ[Ca2+]c fell to zero. Tonic ICCh and [Ca2+]c became prominent after substitution of extracellular 140 mm Na+ by 140 mm Cs+. Tonic ICCh 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).Single channel currents of NSCCCh could be resolved in whole-cell recordings. Substitution of Na+ by Cs+ increased NSCCCh activity by one order of magnitude and slope conductance from 22 to 30 pS. Extracellular quinidine (3 μm) reversibly blocked the NSCCCh activity.Both tonic ICCh 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.A Ca2+ fractional current of tonic ICCh (fCa) of 0.009 was calculated by comparing the ratio Δ[Ca2+]c (corrected for simultaneous Ca2+ redistribution) over ICCh with depolarization-induced *Δ[Ca2+]c (Δ[Ca2+]c calculated from ICa induced by a 400 ms depolarization from −60 to 0 mV at 2 mm[Ca2+]o, 145 mm[Cs+]o) over ICa. fCa was 0.023 at [Ca2+]o = 4 mm.With 110 mm extracellular CaCl2 and 145 mm intracellular CsCl, ICCh reversed at +19.5 mV suggesting a permeability ratio PCa/PCs of 2.8.We conclude that Ca2+ influx through NSCCCh under physiological [Ca2+]o could induce Δ[Ca2+]c. The fCa was, however, much smaller than the one calculated from the reversal potential.