Single amino acid residue in the extracellular portion of transmembrane segment 2 in the nicotinic alpha7 acetylcholine receptor modulates sensitivity to ketamine.
BACKGROUND
Ketamine inhibits the activation of both heteromeric and homomeric nicotinic acetylcholine receptors. The site of molecular interaction is unknown.


METHODS
The inhibition of alpha7 nicotinic acetylcholine receptors by ketamine was compared to that of 5-hydroxytryptamine-3A (5HT3A) receptors that are resistant to ketamine inhibition in Xenopus laevis oocytes. To determine whether the region of transmembrane segments 2 and 3 is relevant for ketamine inhibition of nicotinic receptors, the authors identified single amino acid residues that differ in the sequence alignment of the two proteins. They created 22 mutant alpha7 nicotinic receptors that contain the single homologous amino acid residue in the 5HT3A sequence.


RESULTS
Of the 22 mutant alpha7 nicotinic receptors tested, only one (alpha7 A258S) was significantly resistant to 20 microM ketamine. The ketamine concentration response relationship for the alpha7 A258S mutant was shifted to the right with the IC50 for ketamine increased from 17 +/- 2 for wild type to 30 +/- 3 microM in the mutant (P < 0.001). Agonist activation was unchanged by the mutation. The homologous amino acid residue in the 5HT3A receptor was mutated to the alanine that occurs in the wild-type nicotinic receptor. This mutation made the previously insensitive 5HT3A receptor sensitive to ketamine (P < 0.001).


CONCLUSIONS
Conservative mutation of a single amino acid in the extracellular transmembrane segment 2 domain induces resistance to ketamine inhibition in the alpha7 nicotinic receptor and sensitivity to inhibition in the 5HT3A receptor. This region may represent a ketamine binding site in the alpha7 nicotinic receptor, or it may be an important transduction site for ketamine action.
Ho
Kenny K
KK
Department of Anesthesiology, Columbia University, New York, New York 10032, USA.
Flood
Pamela
P
D019943:Amino Acid Substitution; D000596:Amino Acids / Q000235:genetics / Q000502:physiology*; D000818:Animals; D004351:Drug Resistance; D004594:Electrophysiology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology*; D005110:Extracellular Space / Q000502:physiology; D006801:Humans; D007649:Ketamine / Q000494:pharmacology*; D007700:Kinetics; D009154:Mutation / Q000502:physiology; D018733:Nicotinic Antagonists / Q000494:pharmacology*; D009865:Oocytes / Q000187:drug effects; D018408:Patch-Clamp Techniques; D011978:Receptors, Nicotinic / Q000187:drug effects* / Q000235:genetics; D044406:Receptors, Serotonin, 5-HT3 / Q000187:drug effects; D014982:Xenopus laevis; D064569:alpha7 Nicotinic Acetylcholine Receptor
D000596:Amino Acids; C579167:Chrna7 protein, human; D018691:Excitatory Amino Acid Antagonists; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; D044406:Receptors, Serotonin, 5-HT3; D064569:alpha7 Nicotinic Acetylcholine Receptor; D007649:Ketamine