Characterization of two mutations, M287L and Q266I, in the α1 glycine receptor subunit that modify sensitivity to alcohols.
Glycine receptors (GlyRs) are inhibitory ligand-gated ion channels. Ethanol potentiates glycine activation of the GlyR, and putative binding sites for alcohol are located in the transmembrane (TM) domains between and within subunits. To alter alcohol sensitivity of GlyR, we introduced two mutations in the GlyR α1 subunit, M287L (TM3) and Q266I (TM2). After expression in Xenopus laevis oocytes, both mutants showed a reduction in glycine sensitivity and glycine-induced maximal currents. Activation by taurine, another endogenous agonist, was almost abolished in the M287L GlyR. The ethanol potentiation of glycine currents was reduced in the M287L GlyR and eliminated in Q266I. Physiological levels of zinc (100 nM) potentiate glycine responses in wild-type GlyR and also enhance the ethanol potentiation of glycine responses. Although zinc potentiation of glycine responses was unchanged in both mutants, zinc enhancement of ethanol potentiation of glycine responses was absent in M287L GlyRs. The Q266I mutation decreased conductance but increased mean open time (effects not seen in M287L). Two lines of knockin mice bearing these mutations were developed. Survival of homozygous knockin mice was impaired, probably as a consequence of impaired glycinergic transmission. Glycine showed a decreased capacity for displacing strychnine binding in heterozygous knockin mice. Electrophysiology in isolated neurons of brain stem showed decreased glycine-mediated currents and decreased ethanol potentiation in homozygous knockin mice. Molecular models of the wild-type and mutant GlyRs show a smaller water-filled cavity within the TM domains of the Q266I α1 subunit. The behavioral characterization of these knockin mice is presented in a companion article (J Pharmacol Exp Ther 340:317-329, 2012).
Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, TX 78712-0159, USA.
Cecilia M
CM
Borghese
Yuri A
YA
Blednov
Yu
Y
Quan
Sangeetha V
SV
Iyer
Wei
W
Xiong
S John
SJ
Mihic
Li
L
Zhang
David M
DM
Lovinger
James R
JR
Trudell
Gregg E
GE
Homanics
R Adron
RA
Harris
D000438:Alcohols / Q000494:pharmacology*; D019943:Amino Acid Substitution / Q000502:physiology*; D000818:Animals; D001667:Binding, Competitive / Q000502:physiology; D001933:Brain Stem / Q000166:cytology / Q000378:metabolism; D002478:Cells, Cultured; D004357:Drug Synergism; D055724:Electrophysiological Phenomena / Q000187:drug effects / Q000502:physiology; D000431:Ethanol / Q000494:pharmacology; D005260:Female; D005445:Flunitrazepam / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D055879:Gene Knock-In Techniques / Q000379:methods; D005838:Genotype; D005998:Glycine / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects* / Q000502:physiology; D051379:Mice; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008958:Models, Molecular; D016472:Motor Neuron Disease / Q000235:genetics / Q000401:mortality / Q000503:physiopathology; D009154:Mutation / Q000502:physiology*; D009474:Neurons / Q000187:drug effects / Q000502:physiology; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D010641:Phenotype; D018009:Receptors, Glycine / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013116:Spinal Cord / Q000378:metabolism; D013331:Strychnine / Q000378:metabolism; D013654:Taurine / Q000494:pharmacology; D014162:Transfection; D014982:Xenopus laevis; D015032:Zinc / Q000494:pharmacology
D000438:Alcohols; C114561:GLRB protein, human; D018009:Receptors, Glycine; D013654:Taurine; D000431:Ethanol; D005445:Flunitrazepam; D013331:Strychnine; D015032:Zinc; D005998:Glycine