Alanine-Scanning Mutagenesis in the Signature Bisulfide Loop of the Glycine Receptor apha1 Subunit: Critical Residues for Activation and Modulation

摘要

The glycine receptor enables the generation of inhibitory postsynaptic currents at synapses via neurotransmitter-dependent activation.These receptors belong to the ligand-gated ion channel gene superfamily,in which all members are comprised of five subunits,each of which possesses a signature 13-residue disulfide loop (Cys loop) in the extracellular domain.In this study,we used alanine-scanning mutagenesis of the residues between C138 and C152 of the Cys loop of the glycine receptor al subunit to identify residues critical for receptor activation and allosteric modulation.Mutation of LI42,F145,or PI46 to alanine produced decreases in the potency,maximal amplitude,and Hill coefficient for currents elicited by glycine and impaired receptor activation by the agonist taurine.These residues,along with D148,are positionally conserved in the family of LGIC subunits.Mutation at several other positions had little or no effect.The inhaled anesthetics halothane and isoflurane potentiate submaximal agonist responses at wild-type receptors,via an allosteric site.The mutations LI42A,F145A,PI46A,and D148A abolished positive modulation by these anesthetics,in some cases revealing a small inhibitory effect.A molecular model of the glycine receptor alphal subunit suggests that the Cys loop is positioned in a region of the receptor at the interface between the extracellular and transmembrane domains and that the critical functional residues identified here lie along the face of a predominantly hydrophobic surface.The present data implicate the Cys loop as an important functional moiety in the process of glycine receptor activation and allosteric regulation by anesthetics.