title: Two rare variations, D478N and D478E, that occur at the same amino acid residue in nicotinic acetylcholine receptor (nAChR) α2 subunit influence nAChR function.
abstract: There occur two rare variations, Asp(D)478Asn(N) and Asp(D)478Glu(E), in the putative cytoplasmic amphipathic α-helices of human nicotinic acetylcholine receptor (nAChR) α2 subunit as a result of mutation in the 1st (G → A: rs141072985) and 3rd (C → A: rs56344740) nucleotide of its 478th triplet codon (GAC). We assessed the effects of these two variations on the function of α2β2- and α2β4-nAChRs as they could alter the electronegativity and/or the structure of the cytoplasmic 'portals' (framed by subunit amphipathic α-helices) necessary for obligate ion permeation from extracellular space to cytoplasm. We injected decreasing ratio of subunit cRNAs (α:β; 10:1, 1:1 and 1:10) into Xenopus oocytes to express putative low-sensitivity (LS; 10:1), intermediate-sensitivity (IS; 1:1) and high sensitivity (HS; 1:10) isoforms of wild type and variant α2β2- and α2β4-nAChRs. Two-electrode voltage clamp analyses indicate that the agonist (ACh or nicotine) induced peak current responses (Imax) of α2β2-nAChR isoforms and those of α2β4-nAChR isoforms are increased (1.3-4.7-fold) as a result of D478E variation. The α2 subunit D478N variation only increases the Imax of IS (∼2-fold) or HS (1.4-2.1-fold) α2β2-nAChRs. Concentration-response curves constructed indicate no effect on agonist sensitivities of LS and HS isoforms of α2β2- or α2β4-nAChRs as a result of either variation in α2 subunit. Between the two variant nAChRs, α2(D478E)*-nAChR isoforms generally yield higher Imax than those of respective α2(D478N)*-nAChR isoforms. These effects could be attributed to alteration in cytoplasmic 'portals' and/or ion permeation through it owing to change in amino acid electronegativity (D → N) and side chain length (D → E) in nAChR α2 subunit.
authors.affiliation: Department of Psychiatry and Neurobehavioral Sciences, School of Medicine, University of Virginia, Charlottesville, VA 22903, USA.
authors.forename: Bhagirathi
authors.initials: B
authors.lastname: Dash
authors.affiliation: Department of Psychiatry and Neurobehavioral Sciences, School of Medicine, University of Virginia, Charlottesville, VA 22903, USA. Electronic address: ml2km@virginia.edu.
authors.forename: Ming D
authors.initials: MD
authors.lastname: Li
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D004305:Dose-Response Relationship, Drug; D014644:Genetic Variation; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008958:Models, Molecular; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009538:Nicotine / Q000494:pharmacology; D018722:Nicotinic Agonists / Q000494:pharmacology; D009865:Oocytes; D018408:Patch-Clamp Techniques; D011978:Receptors, Nicotinic / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014982:Xenopus laevis
chemical_list: D000109:Acetylcholine; D009419:Nerve Tissue Proteins; D009538:Nicotine; D018722:Nicotinic Agonists; D011978:Receptors, Nicotinic; C436489:CHRNA2 protein, human; C447159:CHRNB4 protein, human
keywords: Electrophysiology; Missense mutation; Nicotinic acetylcholine receptor; Receptor structure–function; Single nucleotide variation