Determinants of zinc potentiation on the alpha4 subunit of neuronal nicotinic receptors.
We have shown previously that the function of neuronal nicotinic acetylcholine receptors can be modulated by zinc. This modulation varies from potentiation to inhibition, depending on receptor subunit composition and zinc concentration, with the alpha4beta2 and alpha4beta4 receptors displaying the most dramatic potentiation. In this study, we used site-directed mutagenesis to identify glutamate 59 and histidine 162 on the rat alpha4 subunit as potential mediators of zinc potentiation. By modeling the extracellular domain of the receptor pentamer, we locate these residues to two subunit-subunit interfaces that alternate with the two acetylcholine-binding interfaces. Substitution of a cysteine at either position allows additional reduction of zinc potentiation upon treatment with the methanethiosulfonate reagents N-biotinoylaminoethyl methanethiosulfonate (MTSEA-biotin) and [2-(trimethylammonium)ethyl] methanethiosulfonate. Mutagenesis and methanethiosulfonate treatment are most effective at position 162, and the presence of zinc hinders the reaction of MTSEA-biotin with the substituted cysteine at this position, suggesting that alpha4His162 participates in forming a coordination site for zinc. Mutagenesis and methanethiosulfonate treatment are less effective at position 59, suggesting that whereas alpha4Glu59 may be near the zinc coordination site, it may not be participating in coordination of the zinc ion. It is noteworthy that the position of alpha4Glu59 within the neuronal nAChR is identical to that of a residue that lines the benzodiazepine-binding site on GABA(A) receptors. We suggest that the zinc potentiation sites on neuronal nAChRs are structurally and functionally similar to the benzodiazepine-binding sites on GABA(A) receptors.
Hsiao
Bernard
B
Department of Molecular and Cellular Pharmacology, Miller School of Medicine, University of Miami, Miami, FL 33101, USA.
Mihalak
Karla B
KB
Repicky
Sarah E
SE
Everhart
Drew
D
Mederos
Ana H
AH
Malhotra
Arun
A
Luetje
Charles W
CW
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D015640:Ion Channel Gating; D008969:Molecular Sequence Data; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis; D015032:Zinc / Q000494:pharmacology*
D011978:Receptors, Nicotinic; D015032:Zinc; D002118:Calcium