Magnitude of a conformational change in the glycine receptor beta1-beta2 loop is correlated with agonist efficacy
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Magnitude of a conformational change in the glycine receptor beta1-beta2 loop is correlated with agonist efficacy. / Pless, Stephan Alexander; Lynch, Joseph W.
In: The Journal of Biological Chemistry, Vol. 284, No. 40, 02.10.2009, p. 27370-6.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Magnitude of a conformational change in the glycine receptor beta1-beta2 loop is correlated with agonist efficacy
AU - Pless, Stephan Alexander
AU - Lynch, Joseph W
PY - 2009/10/2
Y1 - 2009/10/2
N2 - The efficacy of agonists at Cys-loop ion channel receptors is determined by the rate they isomerize receptors to a pre-open flip state. Once the flip state is reached, the shut-open reaction is similar for low and high efficacy agonists. The present study sought to identify a conformational change associated with the closed-flip transition in the alpha1-glycine receptor. We employed voltage-clamp fluorometry to compare ligand-binding domain conformational changes induced by the following agonists, listed from highest to lowest affinity and efficacy: glycine > beta-alanine > taurine. Voltage-clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. Agonist affinity and efficacy correlated inversely with maximum fluorescence magnitudes at labeled residues in ligand-binding domain loops D and E, suggesting that large conformational changes in this region preclude efficacious gating. However, agonist affinity and efficacy correlated directly with maximum fluorescence magnitudes from a label attached to A52C in loop 2, near the transmembrane domain interface. Because glycine experiences the largest affinity increase between closed and flip states, we propose that the magnitude of this fluorescence signal is directly proportional to the agonist affinity increase. In contrast, labeled residues in loops C, F, and the pre-M1 domain yielded agonist-independent fluorescence responses. Our results support the conclusion that a closed-flip conformation change, with a magnitude proportional to the agonist affinity increase from closed to flip states, occurs in the microenvironment of Ala-52.
AB - The efficacy of agonists at Cys-loop ion channel receptors is determined by the rate they isomerize receptors to a pre-open flip state. Once the flip state is reached, the shut-open reaction is similar for low and high efficacy agonists. The present study sought to identify a conformational change associated with the closed-flip transition in the alpha1-glycine receptor. We employed voltage-clamp fluorometry to compare ligand-binding domain conformational changes induced by the following agonists, listed from highest to lowest affinity and efficacy: glycine > beta-alanine > taurine. Voltage-clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. Agonist affinity and efficacy correlated inversely with maximum fluorescence magnitudes at labeled residues in ligand-binding domain loops D and E, suggesting that large conformational changes in this region preclude efficacious gating. However, agonist affinity and efficacy correlated directly with maximum fluorescence magnitudes from a label attached to A52C in loop 2, near the transmembrane domain interface. Because glycine experiences the largest affinity increase between closed and flip states, we propose that the magnitude of this fluorescence signal is directly proportional to the agonist affinity increase. In contrast, labeled residues in loops C, F, and the pre-M1 domain yielded agonist-independent fluorescence responses. Our results support the conclusion that a closed-flip conformation change, with a magnitude proportional to the agonist affinity increase from closed to flip states, occurs in the microenvironment of Ala-52.
KW - Alanine
KW - Animals
KW - Female
KW - Glycine
KW - Humans
KW - Isomerism
KW - Kinetics
KW - Models, Molecular
KW - Patch-Clamp Techniques
KW - Protein Structure, Secondary
KW - Protein Structure, Tertiary
KW - Receptors, Glycine
KW - Taurine
U2 - 10.1074/jbc.M109.048405
DO - 10.1074/jbc.M109.048405
M3 - Journal article
C2 - 19643731
VL - 284
SP - 27370
EP - 27376
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 40
ER -
ID: 122597801