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TRPC4 proteins form receptor-operated cation channels that are activated in synergy by M2 and M3 ACh receptors coupled to Gq/11 and Gi/o proteins, respectively. These channels are widely expressed in the brain and smooth muscles where they perform a number of important functions, including control of GABA release from the dendrites and cholinergic excitation of smooth muscles. The biophysical properties of TRPC4 currents directly activated by GTPγS in mouse cells remain mostly unknown. We, thus, aimed to investigate these channels in mouse ileal myocytes where a prominent TRPC4-mediated cation current termed mICAT is observed, and to compare the behavior of this current to that of the better studied mICAT in guinea-pig myocytes. Although cation current responses to carbachol at –50 mV (i.e., at the value close to the normal resting potential in these cells) were highly similar, mICAT in the mouse lacked the permissive action of intracellular Ca2+ on channel opening. The slope factor of the muscarinic cation conductance, which is a defining property of voltage-dependent behavior, was identical in both species. There were differences in the potential at which the current peaked at negative potentials, but not in the maximal current densities. Major differences were found in the kinetics of mICAT voltagedependent relaxations, which were much faster in the mouse. The above rodent species employ two different strategies for the open probability increase by activated G-proteins; the mean open time was shorter in the mouse compared to that in the guinea-pig (15.1 ± 5.2 msec, n = 8, vs. 80.0 ± 19.7 msec, n = 9; P < 0.01). Correspondingly, the instantaneous frequency of channel opening was much higher in the mouse (154.1 ± 18.8 sec–1 vs. 70.2 ± 7.3 sec–1 in the guinea-pig; P < 0.001). These functional differences are discussed based on structural differences found in the corresponding TRPC4 amino acid sequences of the two rodent species, which are mainly clustered in the cytosolic C-terminus of TRPC4 protein.

Using a two-wave fluorescence probe, Fura-2, we studied changes in the intracellular concentration of calcium ions ([Ca2+]i) resulting from activation of muscarinic and purine receptors in single myocytes of the guinea-pig small intestine. Applications of the respective agonists added to the normal Krebs solution (1.0, 10.0, and 100.0 µM carbachol, CCh, as well as 10.0 and 100.0 µM ATP) induced a rise in the [Ca2+]i. Carbachol evoked an increase in the [Ca2+]i, including two components (a rapid and a plateaulike), while ATP under analogous conditions led only to a short-lasting rise in the [Ca2+]i. Transients induced by CCh or ATP applied in different concentrations, which exceeded a certain level, did not significantly differ from each other in their amplitudes, i.e., they were generated according to an all-or-none principle. In the nominally Ca-and Mg-free solution, CCh and ATP induced only rapid increases in the [Ca2+]i in myocytes. The absence of the slow component in the [Ca2+]i elevation upon the action of CCh under such conditions indicates that the effect of ATP, as compared with that of CCh, is not related to activation of the entry of Ca2+ ions into cells through voltage-operated calcium channels. After the addition of CCh, repeated application of CCh or ATP induced no effect, while application of CCh after the addition of ATP initiated a rise in the [Ca2+]i. These data show that intracellular calcium stores are depleted completely upon the action of CCh, while they are depleted only partially after the action of ATP. An inhibitor of phospholipase C (PLC), U-73122 (5.0 µM), completely blocked rises in the [Ca2+]i induced by both CCh and ATP; therefore, the release of Ca2+ ions from the intracellular calcium stores after application of these agonists is mediated by PLC. We hypothesize that the difference in the release of Ca2+ ions from the intracellular stores observed in our experiments upon activation of choline and purine receptors (partial and complete depletion of the stores upon the action of ATP and CCh, respectively) is responsible for the opposite functional effects of the above-mentioned neurotransmitters on smooth muscles.

A rise in the intracellular concentration of ionized calcium ([Ca2+]i) is a primary signal for contraction in all types of muscles. Recent progress in the development of imaging techniques, with special accent on fluorescence confocal microscopy, and new achievements in the synthesis of organelle- and ion-specific fluorochromes provide an experimental basis for studying the relationship between the structural organization of living smooth muscle cells (SMCs) and features of calcium signaling at the subcellular level. Applying fluorescent confocal imaging, patch-clamp recording, immunostaining, and flash photolysis techniques to freshly isolated SMCs, we have demonstrated that: (i) Ca2+ sparks are mediated by spontaneous clustered opening of ryanodine receptors (RyRs) and occur at the highest rate at preferred sites (frequent discharge sites, FDSs), the number of which depends on SMC type; (ii) FDSs are associated with sub-plasmalemmal sarcoplasmic reticulum (SR) elements, but not with polarized mitochondria; (iii) Ca2+ spark frequency increases with membrane depolarization in voltage-clamped SMCs or following neurotransmitter application to SMCs, in which the membrane potential was not controlled, leading to spark summation and resulting in a cell-wide increase in [Ca2+]i and myocyte contraction; (iv) cross-talk between RyRs and inositol trisphosphate receptors (IP3Rs) is an important determinant of the [Ca2+]i dynamics and recruits neighboring Ca2+-release sites to generate [Ca2+]i waves; (v) [Ca2+]i waves induced by depolarization of the plasma membrane or by noradrenaline or caffeine, but not by carbachol (CCh), originate at FDSs; (vi) Ca2+-dependent K+ and Cl- channels sense the local changes in [Ca2+]i during a Ca2+ spark and thereby may couple changes in [Ca2+]i within a microdomain to changes in the membrane potential, thus affecting the cell excitability; (vii) the muscarinic cation current (mI cat) does not “mirror” changes in [Ca2+]i, thus reflecting the complexity of [Ca2+]i — muscarinic cationic channel coupling; (viii) RyR-mediated Ca2+ release, either spontaneous or caffeine-induced, does not augment mI cat; (ix) intracellular flash release of Ca2+ is less effective in augmentation of mI cat than flash release of IP3, suggesting that IP3 may sensitize muscarinic cationic channels to Ca2+; (x) intracellular flash release of IP3 fails to augment mI cat in SMCs, in which [Ca2+]i was strongly buffered, suggesting that IP3 exerts no direct effect on muscarinic cationic channel gating, and that these channels sense an increase in [Ca2+]i rather than depletion of the IP3-dependent Ca2+ store; and (xi) predominant expression of IP3R type 1 in the peripheral SR provides a structural basis for a tight functional coupling between IP3R-mediated Ca2+ release and muscarinic cationic channel opening.

Acetylcholine, the principal neurotransmitter of the parasympathetic nervous system, evokes smooth muscle excitation and contraction by acting at the muscarinic receptors which, in many tissues, including the gastrointestinal tract, are comprised of the M2 and M3 subtypes. The opening of ion channels selective for monovalent cations (e.g., Na+ and K+) is the major mechanism of cholinergic excitation. We have studied signal transduction pathways and single cationic channel properties using patch-clamp recording and Ca2+ imaging techniques in guinea-pig single ileal myocytes. Cationic channels were found to couple to both M2 and M3 receptors via the GTP-bound Goα and phospholipase C activation, respectively. When these primarily signaling links are established, cationic channel opening can be further potentiated by membrane depolarization and an increase in the intracellular Ca2+ concentration. A strong synergism exists between the receptor occupancy by the agonist and intrinsic voltage dependence of the current as the former can effectively modulate the voltage range of cationic channel activation, while membrane depolarization produces a strong sensitizing effect. However, at potentials close to 0 mV ion flux is terminated by channel flickery block, while further depolarization induces long-lasting channel inactivation. Channel flicker is not caused by intracellular Mg2+, polyamines, or any other freely diffusible molecule and is confined to potentials around 0 mV irrespective of the driving force. Thus, it appears to be an intrinsic channel property of physiological importance as it improves conditions for the action potential discharge and propagation. Similarly, intracellular Ca2+-dependent facilitation of channel opening is counteracted by a slower desensitization. Further, the most intriguing negative control was discovered in the experiments whereby all cellular G proteins were non-selectively and persistently activated by GTPγS infusion, in which case, over time, carbachol instead of activation caused strong and almost irreversible inhibition of the cationic current. In cell-attached and outside-out membrane patches exposed to 50 μM carbachol or 200 μM internal GTPγS, the activity of three types of cationic channels was observed. They had dissimilar conductances (10, 50, and 130 pS), voltage dependence, and kinetics. The properties of the 50 pS channel are consistent with the whole-cell current behavior, at least when [Ca2+] i is “clamped” at 100 nM. The voltage-independent component of the cationic conductance, which appears at higher levels of [Ca2+] i , is likely mediated by the 130 pS channel, while the role of the 10 pS channel at present is unclear. Thus, smooth muscle cationic channels can uniquely detect and integrate many of the most important physiological signals such as the active conformation of two different muscarinic receptors, their associated G proteins and enzymes, as well as membrane potential and [Ca2+] i levels. Moreover, some signals act in synergy, while most of them, depending on the intensity, can be either stimulatory or inhibitory.

1 Bogomolets Institute of Physiology, National Academy of Sciences of Ukraine, Kyiv, Ukraine. 2 St. George’s Hospital Medical School, London, Great Britain. Correspondence should be addressed to A. V. Zholos (e-mail: zholos@serv.biph.kiev.ua). A cation current (I cat ), which is selective with respect to monovalent cations, has been observed in several types of smooth muscles [1, 2]. In visceral smooth muscles, carbachol (CCh) produces depolarization by activation of the monovalent cation-selective channels and subsequent opening of the voltagegated Ca channels. So far, there is no information on the action of CCh on single smooth muscle cells of the murine ileum. Using symmetrical Cs solutions, both the external application of 50 μM CCh and the internal action of 200 μM GTPγS evoked an inward current with a peak amplitude exceeding 0.5 nA (n = 50). The current-voltage (I-V) relationship for this current was Ushaped at negative potentials, and the reversal potential, E rev , was close to 0 mV. At least two types of monovalent cation-selective channels with unitary conductances of 17 and 70 pS (n = 12) were identified in outside-out patches. The open probability (P o ) for the 70 pS channel increased with depolarization, and the U shape of the I-V relationship of the whole-cell current at negative potentials was mostly determined by the voltage-dependent P o typical of this channel. External application of 20 μM quinine completely blocked single channel currents evoked by CCh or GTPγS in the outside-out patch configuration. Single channel openings in outside-out patches were rather stable and could be observed for more than 10 min after patch excision. The number of active channels in a patch was usually from one to four, and outside-out patches generally had either no channels or more than one active channel. The effects of external Ca and Mg ions on the behavior of single channels were also tested. Addition of Mg and Ca at 2.0, 2.5, or 10 mM concentrations to the bath solution reduced the unitary cationic channel conductance from 70 pS down to 42 pS and 32 pS, respectively, and somewhat increased the open channel noise. Our data suggest that the CCh-evoked I cat in smooth muscle cells of the murine ileum is mediated by at least two types of cationic channels with conductances of 17 or 70 pS. The 70 pS channel plays a major role in the generation of the whole-cell CCh-evoked I cat , and the U-shaped I-V relationship at negative potentials is mostly determined by the voltage dependence of P o typical of this channel.