Activation of a muscarinic receptor selectively inhibits a rapidly inactivated Ca2+ current in rat sympathetic neurons.

Abstract

Sympathetic neurons dissociated from the superior cervical ganglion of 2-day-old rats were studied by whole-cell patch clamp and by fura-2 measurements of the cytosolic free Ca2+ concentration, [Ca21],.Step depolarizations in the presence of tetrodotoxin and hexamethonium triggered two Ca2+ currents that differed in the voltage dependence of activation and kinetics of inactivation. These currents resemble the L and N currents previously described in chicken sensory neurons [Nowycky, M. C., Fox, A. P. & Tsien, R. W. (1985) Nature (London) 316, 440442]. Treatment with acetylcholine resulted in the rapid (within seconds), selective, and reversible inhibition of the rapidly inactivated, N-type current, whereas the long-lasting L-type current remained unaffected. The high sensitivity to blocker drugs (atropine, pirenzepine) indicated that this effect of acetylcholine was due to a muscarinic M1 receptor. Intracellular perfusion with nonhydrolyzable guanine nucleotide analogs or pretreatment of the neurons with pertussis toxin had profound effects on the Ca2+ current modulation. Guanosine 5'-[y-thio]triphosphate caused the disappearance of the N-type current (an effect akin to that of acetylcholine, but irreversible), whereas guanosine 5'-[fithio]diphosphate and pertussis toxin pretreatment prevented the acetylcholine-induced inhibition. In contrast, cAMP, applied intracellularly together with 3-isobutyl-1-methylxanthine, as well as activators and inhibitors of protein kinase C, were without effect. Acetylcholine caused shortening of action potentials in neurons treated with tetraethylammonium to partially block K+ channels. Moreover, when applied to neurons loaded with the fluorescent indicator fura-2, acetylcholine failed to appreciably modify [Ca2+]1 at rest but caused a partial blunting of the initial [Ca2+], peak induced by depolarization with high K+. This effect was blocked by muscarinic antagonists and pertussis toxin and was unaffected by protein kinase activators. Thus, muscarinic modulation of the N-type Ca2+ channels appears to be mediated by a pertussis toxin-sensitive guanine nucleotide-binding protein and independent of both cAMP-dependent protein kinase and protein kinase C.In various cellular systems the function of voltage-gated Ca2l channels is known to be modulated by intracellular events [phosphorylations by cAMP-and cGMP-dependent protein kinases (1-3), and protein kinase C (4, 5)] triggered by the activation of the receptors for various neurotransmitters [e.g., norepinephrine, y-aminobutyric acid, serotonin, adenosine, and acetylcholine (AcCho) (6-13)]. Studies carried out during the last few years have shown that Ca2+ channels are heterogeneous. In sensory neurons of the chicken dorsal root ganglion (DRG), three types of channels (L, N, and T) have been identified that differ in their unitary conductance, voltage dependence ofactivation, and kinetics of inactivation (14). Because of their different properties, these channels may play different physiological roles, and it is therefore imp...

Reconstitution of Muscarinic Modulation of the KCNQ2/KCNQ3 K+Channels That Underlie the Neuronal M Current

“…Although stimulation of M 1 receptors in many other cells causes release of Ca 2ϩ from IP 3 -sensitive stores via the actions of PLC (Felder, 1995), in rat SCG neurons muscarinic agonists do not induce an observable increase in [Ca 2ϩ ] i (Wanke et al, 1987;Beech et al, 1991). To see if stimulation of expressed M 1 receptors in tsA cells induces [Ca 2ϩ ] i rises, we exposed undialyzed cells to oxo-M and monitored [Ca 2ϩ ] i with indo-1 loaded as the cell-permeant indo-1 AM ester.…”

“…The M current is thought to play an important role in neuronal excitability, and its suppression increases responses to excitatory synaptic inputs (Jones et al, 1995;Wang and McKinnon, 1995). Modulation of the M current by muscarinic receptor agonists and by angiotensin (Constanti and Brown, 1981; Marrion, 1997;Shapiro et al, 1994a) is mediated by a G-protein of the G q /11 class (Delmas et al, 1998; Haley et al, 1998) via a diffusible cytoplasmic second messenger (Selyanko et al, 1992) that is yet to be identified.Although the buffering of intracellular free Ca 2ϩ ([Ca 2ϩ ] i ) to very low levels prevents muscarinic suppression of the M current in rat sympathetic neurons , no transients or rises of [Ca 2ϩ ] i have been detected on mAChR (Wanke et al, 1987;Beech et al, 1991) or angiotensin (Shapiro et al, 1994a) receptor stimulation. Such results have argued against a [Ca 2ϩ ] i signal as the unidentified cytoplasmic messenger.…”

“…Although the buffering of intracellular free Ca 2ϩ ([Ca 2ϩ ] i ) to very low levels prevents muscarinic suppression of the M current in rat sympathetic neurons , no transients or rises of [Ca 2ϩ ] i have been detected on mAChR (Wanke et al, 1987;Beech et al, 1991) or angiotensin (Shapiro et al, 1994a) receptor stimulation. Such results have argued against a [Ca 2ϩ ] i signal as the unidentified cytoplasmic messenger.…”

Muscarinic responses of rat basolateral amygdaloid neurons recorded in vitro.

“…Nevertheless, it should be noted that muscarinic reduction of calcium currents has been reported in both the CNS (striatal neurons: Misgeld, Calabresi & Dodt, 1986; cultured hippocampal cells; Gahwiler & Brown, 1987) and in the periphery (myenteric neurons;North & Tokimasa, 1983; sympathetic ganglion cells: Belluzzi, Sacchi & Wanke, 1985;Wanke, Ferroni, Malgaroli, Ambrosini, Pozzan & Meldolesi, 1987).…”

Structural and functional aspects of calcium homeostasis in eukaryotic cells

“…We have mentioned above the stimulatory effects of CAMP and the GTP-binding proteins on L-type Ca' + VOCs. The complex kinetics and heterogeneity of N-type channels, on the other hand, makes it difficult to establish which types of Ca" VOCs are the targets for the widespread inhibitory modulation of neuronal-Ca2 + currents by neurotransmitters such as noradrenaline (via a2 adrenoreceptors) [146 -1511; 4-aminobutyric acid (via B receptors) [146,1471; dopamine (via D2 receptors) [149]; acetylcholine (via M I receptors) [37,74,1521; adenosine (via A1 receptors) [153]; opioids (via k and 6 receptors) [154-1561; somatostatin (via unknown receptors) [150,1541. In a few cases one can safely conclude that the targets for inhibitory modulation are N-type Ca'+ VOCs [37,150,151,1531.…”