Two rare variations, D478N and D478E, that occur at the same amino acid residue in nicotinic acetylcholine receptor (nAChR) α2 subunit influence nAChR function

Neuropharmacology. 2014 Oct;85:471-81. doi: 10.1016/j.neuropharm.2014.05.014. Epub 2014 Jun 17.

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.

Keywords: Electrophysiology; Missense mutation; Nicotinic acetylcholine receptor; Receptor structure–function; Single nucleotide variation.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Acetylcholine / pharmacology
  • Animals
  • Dose-Response Relationship, Drug
  • Genetic Variation*
  • Humans
  • Membrane Potentials / drug effects
  • Membrane Potentials / physiology
  • Models, Molecular
  • Nerve Tissue Proteins / metabolism
  • Nicotine / pharmacology
  • Nicotinic Agonists / pharmacology
  • Oocytes
  • Patch-Clamp Techniques
  • Receptors, Nicotinic / genetics*
  • Receptors, Nicotinic / metabolism*
  • Sequence Homology, Amino Acid
  • Transfection
  • Xenopus laevis

Substances

  • CHRNA2 protein, human
  • CHRNB4 protein, human
  • Nerve Tissue Proteins
  • Nicotinic Agonists
  • Receptors, Nicotinic
  • nicotinic receptor beta2
  • Nicotine
  • Acetylcholine