1 The effects on the whole-cell carbachol-induced muscarinic cationic current (mIcat) of antibodies against the a-subunits of various G proteins, as well as the effect of a Gbg subunit, were studied in single guinea-pig ileal smooth muscle cells voltage-clamped at À50 mV. Ionized intracellular calcium concentration, [Ca 2+ ] i , was clamped at 100 nM using a 1,2-bis(2-aminophenoxyl-ethane-N,N,N 0 ,N 0 -tetraacetic acid)/Ca 2+ mixture. 2 Application of ascending concentrations of carbachol (1 -300 mM) activated mIcat (mean amplitude 0.83 nA at 300 mM carbachol; EC 50 8 mM; Hill slope 1.0). A 20 min or longer intracellular application via the pipette solution of G i3 /G o or G o antibodies resulted in about a 70% depression of the maximum response without change in the EC 50 value. In contrast, antibodies against a-subunits of G i1 , G i1 /G i2 , G i3 , G q /G 11 or G s protein over a similar or longer period did not significantly reduce mIcat. Antibodies to common Gb or infusion of the Gbg subunit itself had no effect on mIcat.3 If cells were exposed briefly to carbachol (50 or 100 mM) at early times (o3 min) after infusion of antibodies to Ga i3 /Ga o or to Ga o had begun, carbachol responses remained unchanged even after 20 -60 min; that is, the depression of mIcat by these antibodies was prevented. 4 These data show that Ga o protein couples the muscarinic receptor to the cationic channel in guinea-pig ileal longitudinal smooth muscle and that Gbg is not involved. They also show that prior activation of the muscarinic receptor presumably causes a long-lasting postactivation change of the G protein, which is not reflected in mIcat, but acts to hinder antibody binding.

1 The eects of SK&F 96365 on cationic current evoked either by activating muscarinic receptors with carbachol or by intracellularly applied GTPgS (in the absence of carbachol) were studied using patch-clamp recording techniques in single guinea-pig ileal smooth muscle cells. 2 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 anity of the blocker. The inhibition was similar for both carbachol-and GTPgSevoked currents suggesting that the cationic channel rather than the muscarinic receptor was the primary site of the SK&F 96365 action. 3 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 ®tted 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. 4 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 713 mV. 5 Increasing the duration of voltage steps at 780 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. 6 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.

Transmembrane ion currents in isolated single smooth muscle cells (SMC) from the guinea pig taenia coli were investigated using a whole-cell mode of the patch-clamp technique. Currents induced by depolarizing shifts in the membrane potential from its holding level of --60 mV contained an initial inward phase (Ca 2+ current), which in 30-40 msec was followed by an outward phase. It was shown that outward current was carried by K ions and consisted at least of three components: one CaZ+-lndependent K + current of delayed rectifier (KV) and two Ca2+-dependent K + currents. The latter can be further divided into the apamin-sensitive (SK) and charybdotoxinsensitive (BK) currents. It was found that relative contributions of these three components in total outward current at 0 mV were 35-45%, 5-15%, and 45-55% for KV, SK, and BK currents, respectively. A potential-dependent current carried by CI ions was also found. This CI-current had inward direction within the range of potentials below the chloride equilibrium potential (EO) and outward direction above the Eel. The magnitude of Ci" current was significantly lower than the magnitude of total K + current.

The effects of a blocker, SK&F 96365, on cationic currents evoked either by activation of the muscarinic receptors with carbachol or by intracellular application of GTPyS were studied using patch-clamp recording techniques in single guinea pig ileal smooth muscle cells.SK&F 96365 reversibly inhibited the muscarinic receptor-initiated cationic current in a concentration-, time-, and voltage-dependent manner, producing concomitant alteration of the steady-state I-V relationship shape, which could be interpreted assuming that membrane depolarization increased the affinity of the blocker. The inhibition pattern was similar for both carbachol-and GTP'~S-evoked currents, suggesting that the cationic channel, rather than the muscarinic receptor, was the primary site for the SK&F 96365 action. With long-lasting voltage steps in the presence of SK&F 96365, membrane depolarization induced additional rapid inhibition, which was more slowly relieved during Bogomolets Institute of Physiology, National Academy of Sciences of Ukraine, Kyiv, Ukraine. 2 St. George's Hospital Medical School, London, Great Britain. membrane depolarization. Both inhibition and disinhibition could be well fitted by a single exponential function with the time constants decreasing with depolarization (e-fold per about 12 mV) for inhibition and approximately linearly decreasing with hyperpolarization for disinhibition.At a constant SK&F 96365 concentration, the proportion of cationic current inhibition was a sigmoid function of the membrane potential, with the potential of half-maximum increase positive to -30 mV and the slope factor of about-13 mV. With voltage steps from-80 to 80 mV of increasing durations, normalized inhibition increased and was almost identical at both potentials, providing pharmacological evidence for the same cationic channel populations being involved in the cationic current generation at negative and positive potentials.We conclude that the mode of SK&F 96365 action, which is distinctive and unique for the muscarinic receptor cationic channel, shows that this blocker can be a valuable pharmacological tool in future molecular biology studies of this channel.