Distinct conformational changes in activated agonist-bound and agonist-free glycine receptor subunits
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Distinct conformational changes in activated agonist-bound and agonist-free glycine receptor subunits. / Pless, Stephan Alexander; Lynch, Joseph W.
In: Journal of Neurochemistry, Vol. 108, No. 6, 03.2009, p. 1585-94.Research output: Contribution to journal › Journal article › Research › peer-review
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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
VL - 108
SP - 1585
EP - 1594
JO - Journal of Neurochemistry
JF - Journal of Neurochemistry
SN - 0022-3042
IS - 6
ER -
ID: 122597835