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Neuronal nicotinic acetylcholine receptor of the alpha 4/non-alpha (alpha 4/n alpha) type was reconstituted in Xenopus oocytes after nuclear injection of cDNA expression vectors. Functional neuronal receptor was only formed when the two subunits alpha 4 and n alpha were coinjected, neither alpha 4 nor n alpha 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 indistinguishable from those induced by AcCho. Five AcCho antagonists--neuronal bungarotoxin (kappa-bungarotoxin), tubocurarine (TC), hexamethonium bromide (Hex), decamethonium bromide (Dec), and mecamylamine (Mec)--have been tested. Neuronal bungarotoxin has no effect on the alpha 4/n alpha channel, whereas 2.5 microM TC reduces by half the current peak evoked by 1 microM 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 microM 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.

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