Effects of Zn2+ on wild and mutant neuronal alpha7 nicotinic receptors.
Zn2+ is a key structural/functional component of many proteins and is present at high concentrations in the brain and retina, where it modulates ligand-gated receptors. Therefore, a study was made of the effects of zinc on homomeric neuronal nicotinic receptors expressed in Xenopus oocytes after injection of cDNAs encoding the chicken wild or mutant alpha7 subunits. In oocytes expressing wild-type receptors, Zn2+ alone did not elicit appreciable membrane currents. Acetylcholine (AcCho) elicited large currents (IAcCho) that were reduced by Zn2+ in a reversible and dose-dependent manner, with an IC50 of 27 microM and a Hill coefficient of 0.4. The inhibition of IAcCho by Zn2+ was competitive and voltage-independent, a behavior incompatible with a channel blockade mechanism. In sharp contrast, in oocytes expressing a receptor mutant, with a threonine-for-leucine 247 substitution (L247Talpha7), subnanomolar concentrations of Zn2+ elicited membrane currents (IZn) that were reversibly inhibited by the nicotinic receptor blockers methyllycaconitine and alpha-bungarotoxin. Cell-attached single-channel recordings showed that Zn2+ opened channels that had a mean open time of 5 ms and a conductance of 48 pS. At millimolar concentrations Zn2+ reduced IAcCho and the block became stronger with cell hyperpolarization. Thus, Zn2+ is a reversible blocker of wild-type alpha7 receptors, but becomes an agonist, as well as an antagonist, following mutation of the highly conserved leucine residue 247 located in the M2 channel domain. We conclude that Zn2+ is a modulator as well as an activator of homomeric nicotinic alpha7 receptors.
Laboratorio di Biofisica, Centro Ricerca Sperimentale Istituto Regina Elena, via delle Messi d' Oro 156 I00158 Rome, Italy. Palma@ifo.it
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Miledi
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Eusebi
D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D002645:Chickens; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000378:metabolism*; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D017354:Point Mutation; D011978:Receptors, Nicotinic / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014981:Xenopus; D015032:Zinc / Q000494:pharmacology*; D064569:alpha7 Nicotinic Acetylcholine Receptor
D018076:DNA, Complementary; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; D064569:alpha7 Nicotinic Acetylcholine Receptor; D015032:Zinc; D000109:Acetylcholine