Human neuronal threonine-for-leucine-248 alpha 7 mutant nicotinic acetylcholine receptors are highly Ca2+ permeable.
A cDNA coding for the human neuronal nicotinic alpha7 receptor subunit with Leu-248 mutated to threonine was expressed in Xenopus oocytes. When activated by acetylcholine (AcCho), the receptors expressed generated currents that had low desensitization, linear current-voltage relation, and high apparent affinity for both AcCho and nicotine. These characteristics are similar to those already described for the chick threonine-for-leucine-247 alpha7 nicotinic AcCho receptor (nAcChoR) mutant (L247Talpha7). These properties were all substantially maintained when the human L248Talpha7 mutant was transiently expressed in human Bosc 23 cells. Simultaneous whole-cell clamp and fluorescence measurements with the Ca(2+) indicator dye Fura-2 showed that nicotine induced a Ca(2+) influx in standard 2 mM Ca(2+) solution. The average fractional Ca(2+) current flowing through L248Talpha7 nAcChoRs was 6.7%, which is larger than that flowing through muscle alpha(beta)epsilon(delta) nAcChoRs (4.1%). The relative Ca(2+) permeability, determined in oocytes in the absence of Cl(-), was measured from the shift in reversal potential caused by increasing the external Ca(2+) concentration from 1 to 10 mM. The human wild-type alpha7 nAcChoR was found to be more permeable than the L248Talpha7 mutant to Ca(2+). Our findings indicate that the Ca(2+) permeability of the homomeric alpha7 nAcChoR is larger than that of the heteromeric neuronal nicotinic receptors studied to date and is possibly similar to that of the N-methyl-D-aspartate subtype of brain glutamate receptors.
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Istituto Pasteur-Fondazione Cenci Bolognetti and Dipartimento di Medicina Sperimentale e Patologia, Universita' di Roma "La Sapienza, " Viale Regina Elena 324, I00161 Roma, Italy.
Palma
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Mileo
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Miledi
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Eusebi
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D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D017931:DNA Primers; D006801:Humans; D017136:Ion Transport; D007930:Leucine / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000378:metabolism*; D018722:Nicotinic Agonists / Q000494:pharmacology; D018733:Nicotinic Antagonists / Q000494:pharmacology; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013912:Threonine / Q000235:genetics*
D017931:DNA Primers; D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; D013912:Threonine; D007930:Leucine; D002118:Calcium