Activation and blocking of neuronal nicotinic acetylcholine receptor reconstituted in Xenopus oocytes.

Abstract

Neuronal nicotinic acetylcholine receptor of the a4/non-a (a4/na) type was reconstituted in Xenopus oocytes after nuclear injection of cDNA expression vectors. Functional neuronal receptor was only formed when the two subunits a4 and na were coiniected, neither a4 nor na alone being effective. Responses to bath application of acetylcholine (AcCho) have been measured in voltage clamp. AcCho doses as low as 10 nM induce currents of up to 50 nA. Dose-response studies indicate a Kd of about 0.77 x 10-6 M and a Hill coefficient of 1.5, thus predicting more than one AcCho binding site per receptor molecule. The current-voltage relationship of AcCho-induced currents presents a strong inward rectification. Responses to AcCho were compared to those of three other agonists: L-nicotine, carbachol, and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP). Sensitivities to AcCho, nicotine, and DMPP are quite similar. Sensitivity to carbachol is much lower, but the currents are otherwise indisinguishable from those induced by AcCho. Five AcCho antagonistsneuronal bungarotoxin (K-bungarotoxin), tubocurarine (TC), hexamethonium bromide (Hex), decamethonium bromide (Dec), and mecamylamine (Mec)-have been tested. Neuronal bungarotoxin has no effect on the a4/na channel, whereas 2.5 ,M TC reduces by half the current peak evoked by 1 ,uM AcCho. The block by TC is independent of membrane voltage. By contrast, the block of AcCho-induced currents by Hex or Dec is strongly voltage dependent, suggesting that these substances enter the channel. The block by Mec is detectable at concentrations as low as 100 nM when applied together with 1 ,uM AcCho and is voltage independent. Hex, Dec, and Mec are effective only when AcCho is present. While the effects of all other agents are fully reversible, the Mec block is persistent.The nicotinic acetylcholine (AcCho) receptor (nAcChoR) mediating synaptic transmission at the neuromuscular junction of vertebrates has long been a favored model system in the study of ligand-gated ion channels. Because of the efforts of many, much is now known about its biochemistry, electrophysiology, and molecular biology (1). In contrast, little is known about the different types of neuronal nAcChoRs that recently have been identified in the central and peripheral nervous system by using antibody and cDNA probes derived from muscle receptor (2-8). At least six neuronal genes have been cloned, sequenced, and found to encode proteins homologous to the four related subunits assembled in the mature muscle receptor (2, 4-8). As functional nAcChoRs can be reconstituted in Xenopus oocytes by cytoplasmic injection of complementary RNA (2, 7, 8) or by nuclear injection of cDNA expression vectors (9), it is now possible to characterize the physiology and pharmacology of the numerous receptor types resulting from the assembly of cloned subunits. Such studies have revealed that functional neuronal nAcChoRs assemble from two subunits only (an a subunit and a non-a-subunit in our nomenclature), whereas four different subunits...

Functional interactions between nicotinic and P2X channels in short‐term cultures of guinea‐pig submucosal neurons

“…Thus, hexamethonium blocked about 50% of the outward currents but inhibited about 90% of the inward currents (see above). This voltage-dependent effect of hexamethonium has been employed to support the idea that this drug blocks nicotinic channels by entering into the open pores (Ascher et al 1979;Bertrand et al 1990). …”

“…Fourth, in cells with no initial response to ACh or in which IACh had been blocked with hexamethonium, IACh+ATP has exactly the same amplitude and kinetics as IATP. It is also likely that this inhibitory interaction not only requires activation of receptors but might actually require channels to be open, since hexamethonium, a substance that is believed to be a nicotinic channel blocker (Ascher et al 1979;Bertrand et al 1990), prevents ACh effects on IATP. At least four different observations indicate that IACh+ATP are carried through both nicotinic and P2X channels and not only through one population of these channels.…”

Inward rectification of acetylcholine‐elicited currents in rat phaeochromocytoma cells.

“…Inward rectification of ACh-elicited currents has been reported in mammalian peripheral ganglion cells (Derkach, Selyanko & Skok, 1983;Mathie, Cull-Candy & Colquhoun, 1987;Yawo, 1989;Fieber & Adams, 1991), chromaffin cells (Hirano, Kidokoro & Ohmori, 1987;Maconochie, 1990), and some central nervous system cells (Mulle & Changeux, 1990;Zhang & Feltz, 1990). In addition, macroscopic currents of neuronal nicotinic receptors composed of ac7 or a4 and na subunits reconstituted in Xenopus ooctyes have been reported to be inwardly rectifying (Bertrand, Ballivet & Rungger, 1990;Couturier, Bertrand, Matter, Hernandez, Bertrand, Millar, Valera, Barkas & Ballivet, 1990). In contrast to muscle-type receptors where the weak inward rectification can be explained by the voltage dependence of the apparent channel closing rate (Dionne & Stevens, 1975), the mechanisms underlying the inward rectification of ACh-elicited currents in ganglion cells have not been fully elucidated.…”

Neuronal specificity of the alpha 7 nicotinic acetylcholine receptor promoter develops during morphogenesis of the central nervous system.

“…Ten different genes encoding neuronal nAChR subunits have been cloned and characterized in the chicken (a2 -(x8, nc I-naz3; Nef et al, 1988;Couturier et al, 1990a,b;Schoepfer et al, 1988Schoepfer et al, , 1990Hernandez,M.-C., in preparation) and in the rat (a2-a5, ,32-34; Boulter et al, 1986Boulter et al, , 1990Goldman et al, 1987;Wada et al, 1988;Deneris et al, 1988Deneris et al, , 1989Duvoisin et al, 1989;Isenberg and Meyer, 1989). Reconstitution studies in the Xenopus oocyte system have shown that the a2, a3 and a4 subunits can each lead to assembly of a functional nAChR in association with either na 1 or na3 and that each of these six receptors has specific physiological and pharmacological properties Wada et al, 1988;Bertrand et al, 1990;Couturier et al, 1990a). In contrast, the a7 and a8 subunits form homomeric receptors.…”