Distinct conformational changes in activated agonist-bound and agonist-free glycine receptor subunits

TY - JOUR

T1 - Distinct conformational changes in activated agonist-bound and agonist-free glycine receptor subunits

AU - Pless, Stephan Alexander

AU - Lynch, Joseph W

PY - 2009/3

Y1 - 2009/3

N2 - Ligand binding to Cys-loop receptors produces either global conformational changes that lead to activation or local conformational changes that do not. We found that the fluorescence of a fluorophore tethered to R271C in the extracellular M2 region of the alpha1 glycine receptor increases during glycine activation but not during ivermectin activation. This prompted the hypothesis that this signal reports a glycine-mediated conformational change not essential for activation. We tested this by investigating whether the fluorescence signal depended on whether the fluorophore was attached to a glycine-free or a glycine-bound subunit. Agonist-free subunits were created by incorporating T204A and R65K mutations, which disrupted glycine binding to both (+) and (-) subunit interfaces. In heteromeric receptors comprising wild-type and R65K,T204A,R271C triple-mutant subunits, the fluorescence response exhibited a drastically reduced glycine sensitivity relative to the current response. Two conclusions can be drawn from this. First, because the labeled glycine-free subunits were activated by glycine binding to neighboring wild-type subunits, our results provide evidence for a cooperative activation mechanism. However, because the fluorescent label on glycine-free subunits does not reflect movements at the channel gate, we conclude that glycine binding also produces a local non-concerted conformational change that is not essential for receptor activation.

AB - Ligand binding to Cys-loop receptors produces either global conformational changes that lead to activation or local conformational changes that do not. We found that the fluorescence of a fluorophore tethered to R271C in the extracellular M2 region of the alpha1 glycine receptor increases during glycine activation but not during ivermectin activation. This prompted the hypothesis that this signal reports a glycine-mediated conformational change not essential for activation. We tested this by investigating whether the fluorescence signal depended on whether the fluorophore was attached to a glycine-free or a glycine-bound subunit. Agonist-free subunits were created by incorporating T204A and R65K mutations, which disrupted glycine binding to both (+) and (-) subunit interfaces. In heteromeric receptors comprising wild-type and R65K,T204A,R271C triple-mutant subunits, the fluorescence response exhibited a drastically reduced glycine sensitivity relative to the current response. Two conclusions can be drawn from this. First, because the labeled glycine-free subunits were activated by glycine binding to neighboring wild-type subunits, our results provide evidence for a cooperative activation mechanism. However, because the fluorescent label on glycine-free subunits does not reflect movements at the channel gate, we conclude that glycine binding also produces a local non-concerted conformational change that is not essential for receptor activation.

KW - Allosteric Regulation

KW - Amino Acid Sequence

KW - Amino Acids

KW - Animals

KW - Binding Sites

KW - Biophysical Processes

KW - Electric Stimulation

KW - Female

KW - Gene Expression

KW - Humans

KW - Membrane Potentials

KW - Microinjections

KW - Models, Biological

KW - Mutation

KW - Oocytes

KW - Patch-Clamp Techniques

KW - Protein Binding

KW - Protein Subunits

KW - RNA, Messenger

KW - Receptors, Glycine

KW - Xenopus laevis

U2 - 10.1111/j.1471-4159.2009.05930.x

DO - 10.1111/j.1471-4159.2009.05930.x

M3 - Journal article

C2 - 19166513

SN - 1471-4159

VL - 108

SP - 1585

EP - 1594

JO - Journal of Neurochemistry

JF - Journal of Neurochemistry

IS - 6

ER -