Cytoplasmic regions adjacent to the M3 and M4 transmembrane segments influence expression and function of alpha7 nicotinic acetylcholine receptors. A study with single amino acid mutants.
We studied the role of the cytoplasmic regions adjacent to the M3 and M4 transmembrane segments of alpha7 nicotinic receptors in the expression of functional channels. For this purpose, a total of 50 amino acids were mutated throughout the mentioned regions. Mutants close to M3, from Arg294 to Leu321, showed slight modifications in the levels of alpha-bungarotoxin binding sites and acetylcholine-evoked currents. Exceptions were mutants located at two clusters (His296 to Pro300 and Ile312 to Trp316), which exhibited low expression levels. In addition, some mutants showed altered functional responses. Many mutants close to M4 showed increased receptor expression, especially the ones located at the hydrophobic face of a putative amphipathic helix. This effect seems to be the consequence of a combination of increased receptor biosynthesis, higher transport efficiency and delayed degradation, such that we postulate that elements in the amphipathic domain strongly influence receptor stability. Finally, some mutants in this region showed altered functional responses: elimination of positively charged residues (Arg424 and Arg426) increased currents, whereas the opposite was observed upon suppression of negatively charged ones (Glu430 and Glu432). These results suggest that the cytoplasmic regions close to M3 and M4 play important structural and functional roles.
Castelán
Francisco
F
Instituto de Neurociencias de Alicante, Universidad Miguel Hernández-CSIC, Alicante, Spain.
Mulet
José
J
Aldea
Marcos
M
Sala
Salvador
S
Sala
Francisco
F
Criado
Manuel
M
D000109:Acetylcholine / Q000494:pharmacology; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D002038:Bungarotoxins / Q000493:pharmacokinetics; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression / Q000502:physiology*; D007456:Iodine Isotopes / Q000493:pharmacokinetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D008845:Microinjections / Q000379:methods; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism*; D009865:Oocytes; D018408:Patch-Clamp Techniques / Q000379:methods; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D017434:Protein Structure, Tertiary; D051381:Rats; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D013329:Structure-Activity Relationship; D014981:Xenopus; D064569:alpha7 Nicotinic Acetylcholine Receptor
D000596:Amino Acids; D002038:Bungarotoxins; C579165:Chrna7 protein, rat; D007456:Iodine Isotopes; D050505:Mutant Proteins; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; D000109:Acetylcholine