Propofol modulation of α1 glycine receptors does not require a structural transition at adjacent subunits that is crucial to agonist-induced activation

TY - JOUR

T1 - Propofol modulation of α1 glycine receptors does not require a structural transition at adjacent subunits that is crucial to agonist-induced activation

AU - Lynagh, Timothy

AU - Kunz, Alexander

AU - Laube, Bodo

PY - 2013/11/20

Y1 - 2013/11/20

N2 - Pentameric glycine receptors (GlyRs) couple agonist binding to activation of an intrinsic ion channel. Substitution of the R271 residue impairs agonist-induced activation and is associated with the human disease hyperekplexia. On the basis of a homology model of the α1 GlyR, we substituted residues in the vicinity of R271 with cysteines, generating R271C, Q226C, and D284C single-mutant GlyRs and R271C/Q226C and R271C/D284C double-mutant GlyRs. We then examined the impact of interactions between these positions on receptor activation by glycine and modulation by the anesthetic propofol, as measured by electrophysiological experiments. Upon expression in Xenopus laevis oocytes, D284C-containing receptors were nonfunctional, despite biochemical evidence of successful cell surface expression. At R271C/Q226C GlyRs, glycine-activated whole-cell currents were increased 3-fold in the presence of the thiol reductant dithiothreitol, whereas the ability of propofol to enhance glycine-activated currents was not affected by dithiothreitol. Biochemical experiments showed that mutant R271C/Q226C subunits form covalently linked pentamers, showing that intersubunit disulfide cross-links are formed. These data indicate that intersubunit disulfide links in the transmembrane domain prevent a structural transition that is crucial to agonist-induced activation of GlyRs but not to modulation by the anesthetic propofol and implicate D284 in the functional integrity of GlyRs.

AB - Pentameric glycine receptors (GlyRs) couple agonist binding to activation of an intrinsic ion channel. Substitution of the R271 residue impairs agonist-induced activation and is associated with the human disease hyperekplexia. On the basis of a homology model of the α1 GlyR, we substituted residues in the vicinity of R271 with cysteines, generating R271C, Q226C, and D284C single-mutant GlyRs and R271C/Q226C and R271C/D284C double-mutant GlyRs. We then examined the impact of interactions between these positions on receptor activation by glycine and modulation by the anesthetic propofol, as measured by electrophysiological experiments. Upon expression in Xenopus laevis oocytes, D284C-containing receptors were nonfunctional, despite biochemical evidence of successful cell surface expression. At R271C/Q226C GlyRs, glycine-activated whole-cell currents were increased 3-fold in the presence of the thiol reductant dithiothreitol, whereas the ability of propofol to enhance glycine-activated currents was not affected by dithiothreitol. Biochemical experiments showed that mutant R271C/Q226C subunits form covalently linked pentamers, showing that intersubunit disulfide cross-links are formed. These data indicate that intersubunit disulfide links in the transmembrane domain prevent a structural transition that is crucial to agonist-induced activation of GlyRs but not to modulation by the anesthetic propofol and implicate D284 in the functional integrity of GlyRs.

KW - activation mechanism

KW - cross-linking

KW - Cys-loop receptor

KW - Ligand-gated ion channel

KW - propofol

KW - transmembrane domain

UR - http://www.scopus.com/inward/record.url?scp=84888369754&partnerID=8YFLogxK

U2 - 10.1021/cn400134p

DO - 10.1021/cn400134p

M3 - Journal article

C2 - 23992940

AN - SCOPUS:84888369754

SN - 1948-7193

VL - 4

SP - 1469

EP - 1478

JO - ACS Chemical Neuroscience

JF - ACS Chemical Neuroscience

IS - 11

ER -