Role of the outer beta-sheet in divalent cation modulation of alpha7 nicotinic receptors.
alpha-7 Nicotinic acetylcholine receptors (AChRs) exhibit a positive modulation by divalent cations similar to that observed in other AChRs. In the chick alpha7 AChR, this modulation involves a conserved glutamate in loop 9 (Glu172) that undergoes agonist-dependent movements during activation. From these observations, we hypothesized that movements of the nearby beta-sheet formed by the beta7, beta9, and beta10 strands may be involved in agonist activation and/or divalent modulation. To test this hypothesis, we examined functional properties of cysteine mutations of the beta7 and beta10 strands, alone or in pairs. We postulated that reduced flexibility or mobility of the beta7/beta9/beta10-sheet as a result of introduction of a disulfide bond between the beta strands would alter activation by agonists. Using a nondesensitizing alpha7 mutant background (L247T), we identified one mutant pair, K144C + T198C, that exhibited a unique characteristic: it was fully activated by divalent cations (Ca2+, Ba2+, or Sr2+) in the absence of acetylcholine (ACh). Divalent-evoked currents were blocked by the alpha7 antagonist methyllycaconitine and were abolished when Glu172 was mutated to glutamine. When the K144C + T198C pair was expressed in wild-type alpha7 receptors, activation required both ACh and divalent cations. We conclude that the introduction of a disulfide bond into beta7/beta9/beta10 lowers the energetic barrier between open and closed conformations, probably by reducing the torsional flexibility of the beta-sheet. In this setting, divalent cations, acting at the conserved glutamate in loop 9, act as full agonists or requisite coagonists.
Department of Pharmacology, CB# 7365, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365, USA. jmclaughlin@unc.edu
James T
JT
McLaughlin
Jie
J
Fu
Adrian D
AD
Sproul
Robert L
RL
Rosenberg
D000109:Acetylcholine / Q000494:pharmacology; D000157:Aconitine / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001464:Barium / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent / Q000737:chemistry / Q000494:pharmacology*; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D005260:Female; D008564:Membrane Potentials / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D018733:Nicotinic Antagonists / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D017433:Protein Structure, Secondary; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D013324:Strontium / Q000494:pharmacology; D014982:Xenopus laevis; D064569:alpha7 Nicotinic Acetylcholine Receptor
D002413:Cations, Divalent; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; C054634:methyllycaconitine; D001464:Barium; D003545:Cysteine; D000109:Acetylcholine; D002118:Calcium; D000157:Aconitine; D013324:Strontium