Charged amino acids of the N-terminal domain are involved in coupling binding and gating in alpha7 nicotinic receptors.
Binding of agonists to nicotinic acetylcholine receptors generates a sequence of conformational changes resulting in channel opening. Previously, we have shown that the aspartate residue Asp-266 at the M2-M3 linker of the alpha7 nicotinic receptor is involved in connecting binding and gating. High resolution structural data suggest that this region could interact with the so-called loops 2 and 7 of the extracellular N-terminal region. In this case, certain charged amino acids present in these loops could integrate together with Asp-266 and other amino acids, a mechanism involved in channel activation. To test this hypothesis, all charged residues in these loops, Asp-42, Asp-44, Glu-45, Lys-46, Asp-128, Arg-130, and Asp-135, were substituted with other amino acids, and expression levels and electrophysiological responses of mutant receptors were determined. Mutants at positions Glu-45, Lys-46, and Asp-135 exhibited poor or null functional responses to different nicotinic agonists regardless of significant membrane expression, whereas D128A showed a gain of function effect. Because the double reverse charge mutant K46D/D266K did not restore receptor function, a gating mechanism controlled by the pairwise electrostatic interaction between these residues is not likely. Rather, a network of interactions formed by residues Lys-46, Asp-128, Asp-135, Asp-266, and possibly others appears to link agonist binding to channel gating.
Sala
Francisco
F
Instituto de Neurociencias, Universidad Miguel Hernández-Consejo Superior de Investigaciones Cientificas, Apartado 18, 03550-Sant Joan d'Alacant, Alicante, Spain.
Mulet
José
J
Sala
Salvador
S
Gerber
Susana
S
Criado
Manuel
M
D019943:Amino Acid Substitution; D024342:Amino Acids, Acidic; D024361:Amino Acids, Basic; D000818:Animals; D002417:Cattle; D004594:Electrophysiology; D015640:Ion Channel Gating; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D016415:Sequence Alignment; D055672:Static Electricity; D064569:alpha7 Nicotinic Acetylcholine Receptor
D024342:Amino Acids, Acidic; D024361:Amino Acids, Basic; D021122:Protein Subunits; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor