Voltage-dependent inhibition of the muscarinic cationic current in guinea-pig ileal cells by SK&F 96365

Abstract

The effects of SK&F 96365 on cationic current evoked either by activating muscarinic receptors with carbachol or by intracellularly applied GTPgammaS (in the absence of carbachol) were studied using patch-clamp recording techniques in single guinea-pig ileal smooth muscle cells. SK&F 96365 reversibly inhibited the muscarinic receptor cationic current in a concentration-, time- and voltage-dependent manner producing concomitant alteration of the steady-state I-V relationship shape which could be explained by assuming that increasing membrane positivity increased the affinity of the blocker. The inhibition was similar for both carbachol- and GTPgammaS-evoked currents suggesting that the cationic channel rather than the muscarinic receptor was the primary site of the SK&F 96365 action. Increased membrane positivity induced additional rapid inhibition of the cationic current by SK&F 96365 which was more slowly relieved during membrane repolarization. Both the inhibition and disinhibition time course could be well fitted by a single exponential function with the time constants decreasing with increasing positivity for the inhibition (e-fold per about 12 mV) and approximately linearly decreasing with increasing negativity for the disinhibition. At a constant SK&F 96365 concentration, the degree of cationic current inhibition was a sigmoidal function of the membrane potential with a potential of half-maximal increase positive to about +30 mV and a slope factor of about -13 mV. Increasing the duration of voltage steps at -80 or at 80 mV, increased the percentage inhibition; the degree of inhibition was almost identical at both potentials providing evidence that the same cationic channel was responsible for the cationic current both at negative and at positive potentials. It is concluded that the distinctive and unique mode of SK&F 96365 action on the muscarinic receptor cationic channel is a valuable tool in future molecular biology studies of this channel.

Figure 1

Effects of SK&F 96365 on carbachol-activated cationic current in single guinea-pig ileal smooth muscle cells. (A) Steady-state current-voltage relationships measured by 6 s duration voltage ramps in the presence of 50 μM carbachol and SK&F 96365 at the indicated concentrations. (B) Relative amplitude of the muscarinic cationic current in a single experiment at 80 and −80 mV plotted against SK&F 96365 concentration on a semilogarithmic scale and fitted according to equation 1. Dotted lines indicate the IC₅₀ values. (C) Voltage dependence of the IC₅₀ values.

Figure 2

Voltage dependence of the inhibitory effect of SK&F 96365 on carbachol-activated cationic current. (A,B) time- and concentration dependence of the inhibition at two test potentials. SK&F 96365 was applied at ascending concentrations in the presence of 50 μM carbachol while rectangular voltage steps to 80 mV from the holding potential of −40 mV (0.6 duration) were applied every 5 s. Dotted lines in B indicate background current amplitude at corresponding potentials measured before carbachol application. (C) concentration-effect curves at 80 mV (IC₅₀=4.1 μM, P=1.8) and −40 mV (IC₅₀=10.9 μM, P=1.0).

Figure 3

Membrane depolarization induced rapid additional inhibition of the cationic current by SK&F 96365. Carbachol was applied at the moment indicated and was maintained at 50 μM throughout the experiment. SK&F 96365 applied at −40 mV partially inhibited cationic current with a time constant of 9.4 s. Upon voltage step to 80 mV additional strong inhibition with a time constant of 0.6 s occurred which was relieved after repolarization to −40 mV with a time constant of 5.6 s (superimposed dotted lines show single exponential fittings). Note the 7 min gap in the record to wash out SK&F 96365.

Figure 4

Voltage dependence and kinetics of the inhibition at fixed SK&F 96365 concentration. (A) cationic current evoked by 50 μM carbachol at −40 mV in control (left) and following 20 s duration voltage steps to different test potentials (note that only the last 5 s segment is shown for each trace during the test pulse). All traces were recorded in the presence of 10 μM SK&F 96365. Dotted line indicates background current amplitude at −40 mV before agonist application. (B) per cent of depolarization-induced inhibition of the cationic current plotted against test potential for the data shown in A. The values were calculated as a/b×100% with a and b measured as shown in A. Data points were fitted by a Boltzmann distribution with the potential of half-maximal inhibition of 18 mV and slope factor of −18 mV. (C) per cent inhibition of the cationic current evoked by intracellular 200 μM GTPγS application (no carbachol in the bath) depending on test potential in the presence of 30 μM SK&F 96365. Voltage protocol was similar to that illustrated in A. Best-fit values for the potential of half-maximal inhibition and slope factor were +38 mV and −13 mV, respectively. In the same cell the time constant of the inhibition during test depolarization decreased e-fold per 11.9 mV.

Figure 5

Voltage dependence of the disinhibition kinetics. (A) GTPγS-evoked cationic current relaxations at various test potentials following voltage step to 80 mV in control and in the presence of 30 μM SK&F 96365. (B) mean values for the disinhibition time constant plotted against test potential (n=5). Superimposed current traces shown in the inset were obtained using GTPγS to activate the cationic conductance before and after SK&F 96365 application. Inhibition by 30 μM SK&F 96365 at 80 mV and disinhibition at −70 mV developed with the time constants indicated. The horizontal dotted lines indicate zero current level.

Figure 6

Comparison of fractional current inhibition of GTPγS-evoked cationic current by SK&F 96365 at 80 and at −80 mV. (A) superimposed current traces and voltage protocol used in this experiment to study GTPγS-induced cationic current in the presence of 30 μM SK&F 96365. Dotted lines show background current amplitude at 80 mV (upper line) and −80 mV (lower line) measured immediately after break-through before the GTPγS effect had developed. (B) per cent inhibition of the current at 80 mV and −80 mV calculated from the traces shown in A (circles) and for another cell studied with the same protocol (squares). Note the same time scale in both panels.