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

Abstract

1. Ca2+ microfluorometry (100 microM K5 fura-2) and the voltage-clamp technique were combined to study the effect of carbachol (CCh, 50 microM) in inducing currents (ICCh) through non-selective cation channels (NSCCCh) and increments in global cytosolic Ca2+ concentration (Delta[Ca2+]c). 2. In Na+-containing bath solution, ICCh fell from an initial phasic to a subsequent small (5 %) tonic component; Delta[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 Delta[Ca2+]c were insensitive to intracellular heparin (3 mg ml-1) and ryanodine (4 microM), i.e. they did not depend on Ca2+ release from sarcoplasmic reticulum (SR). 3. 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 microM) reversibly blocked the NSCCCh activity. 4. Both tonic ICCh and tonic Delta[Ca2+]c (a) followed a similar time course of activation, desensitization and facilitation, (b) were reversibly blocked by 3 microM quinidine, and (c) persisted upon block of SR Ca2+ release. 5. A Ca2+ fractional current of tonic ICCh (fCa) of 0.009 was calculated by comparing the ratio Delta[Ca2+]c (corrected for simultaneous Ca2+ redistribution) over ICCh with depolarization-induced *Delta[Ca2+]c (Delta[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. 6. 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. 7. We conclude that Ca2+ influx through NSCCCh under physiological [Ca2+]o could induce Delta[Ca2+]c. The fCa was, however, much smaller than the one calculated from the reversal potential.

Figure 5. Ca²⁺ dependence of cytoplasmic Ca²⁺ removal processes

A, I_Ca induced by a 400 ms depolarization from −60 to 0 mV. Note that the time scale is faster than those of following figures. B, *Δ[Ca²⁺]_c caused by I_Ca. Decay of *Δ[Ca²⁺]_c was fitted by a single-exponential function (smooth dashed line). C, the exponential function in B was differentiated to obtain the time course of the Ca²⁺ removal process (smooth dashed line). D, rate of Ca²⁺ removal plotted against the corresponding global *[Ca²⁺]_c (▪). Differentiated results of raw data were plotted together in C (noisy line) and D (scattered open circles). Note that the removal rate of [Ca²⁺]_c depends linearly on [Ca²⁺]_c (straight line). Heparin (4 mg ml⁻¹) and ryanodine (4 μm) were dialysed from the pipette.

Figure 1. Whole-cell currents (I_CCh) and Δ[Ca²⁺]_c induced by 50 μm CCh modified by substitution of extracellular Na⁺ by Cs⁺

A, in Na⁺ Tyrode solution, 50 μm CCh induced phasic responses, i.e. both I_CCh and Δ[Ca²⁺]_c rapidly decayed. Substitution of Na⁺ by Cs⁺ recovered a tonic I_CCh and a tonic Δ[Ca²⁺]_c. KCl solution was in the electrode and 1 μm verapamil was in the bath solution to block L-type Ca²⁺ channels. Top trace: holding potential −40 mV, 2 s pulses to 0 mV at 0.1 Hz. B, dialysis of Cs⁺ electrode solution containing 3 mg ml⁻¹ heparin blocks the phasic response. CCh induces a tonic I_CCh and a tonic Δ[Ca²⁺]_c that rise along a slow time course (holding potential −60 mV). C, Cs⁺ electrode solution containing 4 μm ryanodine and 3 mg ml⁻¹ heparin. Lack of response to 10 mm caffeine suggests that SR has been functionally removed; nevertheless CCh induced tonic I_CCh and tonic Δ[Ca²⁺]_c of the usual amplitude (holding potential −60 mV).

Figure 2. Currents through single CCh-activated non-selective cation channels (NSC_CCh) recorded under whole-cell configuration

A and B, after severe desensitization of I_CCh (several long-lasting CCh applications) single channel currents are discernible on a magnified current scale (membrane potential −60 mV). In the same cell, 2 μm CCh was applied in Cs⁺ Tyrode solution (A) and 15 μm CCh was applied in Na⁺ Tyrode solution (B). Ab and Bb show NSC_CCh recordings on an expanded time scale. C, amplitude histograms of NSC_CCh for 15 s sampling periods and least square fit by a sum of Gaussian distributions in Cs⁺ Tyrode solution (Ca) and Na⁺ Tyrode solution (Cb). Cc, I–V curves of open channel current recorded in Cs⁺ Tyrode solution (▪) and Na⁺ Tyrode solution (•). Extrapolated lines crossed the horizontal axis at −1.6 and −0.5 mV in Cs⁺ and Na⁺ Tyrode solution, respectively. D, reversible block of NSC_CCh by bath-applied quinidine (3 μm) (different cell).

Figure 3. Suppression of I_CCh and tonic Δ[Ca²⁺]_c by quinidine (top) or by changing membrane potential (bottom)

A, 3 μm quinidine reversibly blocked CCh-induced I_CCh and Δ[Ca²⁺]_c along a parallel time course (symmetrical CsCl solutions, heparin present). B, the first control CCh response at −40 mV was followed by a 2 min washout, then CCh was applied a second time. In the continued presence of CCh the clamp potential was changed as indicated in the upper trace. Note: amplitude of tonic Δ[Ca²⁺]_c (lower trace) followed the amplitude of tonic I_CCh and not the Ca²⁺ driving force.

Figure 4. Desensitization and Ca²⁺ facilitation of the CCh response. I_CCh and Δ[Ca²⁺]_c are compared with depolarization-induced I_Ca and *Δ[Ca²⁺]_c

The cell was exposed to 50 μm CCh four times: I_CCh and Δ[Ca²⁺]_c during the first and fourth exposure are shown. Because of desensitization, the fourth response started at a very slow rate (start marked by arrows labelled 4). Before, during and after the fourth CCh application, 800 ms clamp steps from −60 to 0 mV induced I_Ca and *Δ[Ca²⁺]_c (marked by asterisks). Uppermost traces show I_Ca on an expanded time scale. Note: the small and slow fourth CCh response was facilitated by superimposed I_Ca and spiky *Δ[Ca²⁺]_c. Note: *Δ[Ca²⁺]_c due to I_Ca decayed rapidly in contrast to superimposed tonic I_CCh and tonic Δ[Ca²⁺]_c that lasted as long as CCh was present. Symmetrical CsCl conditions, 0.3 μm Bay K 8644 present in the bath.

Figure 6. Evaluation of fractional Ca²⁺ current, f_Ca, of I_CCh

A, CCh-induced I_CCh and Δ[Ca²⁺]_c (same myocyte as in Fig. 5). B, to evaluate f_Ca, data were transformed and plotted according to eqn (5): Δ[Ca²⁺]_c(t) +ΣR_CaΔt =f_Ca (*Δ[Ca²⁺]_c/∫I_Cadt) ΣI_CChΔt. Ba, ordinate Δ[Ca²⁺]_c(t) +ΣR_CaΔt: removal-corrected accumulation of fura-2-measured [Ca²⁺]_c plus Δ[Ca²⁺]_c(t) is plotted against experimental time. Bb, CCh-induced accumulated Σ[Ca²⁺]_c(t) was calculated from ΣI_CChΔt by multiplication with the ratio *Δ[Ca²⁺]_c/∫I_Cadt (from Fig. 5) and plotted against experimental time. Note: in Ba and Bb CCh application started 3 s after zero time. Bc, plot according to eqn (5). Removal-corrected measured [Ca²⁺]_c(t) (Ba) was plotted versus calculated Σ[Ca²⁺]_c(t) (Bb) for 1 s time intervals. Linear regression yielded a slope of 0.0098 which identified f_Ca for this cell.

Figure 7. Voltage dependence and reversal potential of I_CCh in 110 mm CaCl₂ bathing solution

A, ramp-like repolarizations (0.11 V s⁻¹, see inset) were applied before and during stimulation with CCh (50 μm). B, difference current during hyperpolarization was plotted as a function of membrane potential.