TY - JOUR
T1 - Benzodiazepines induce a conformational change in the region of the γ-aminobutyric acid type a receptor α1-subunit M3 membrane-spanning segment
AU - Williams, Daniel B.
AU - Akabas, Myles H.
PY - 2000
Y1 - 2000
N2 - Benzodiazepine binding to γ-aminobutyric acid type A (GABA(A)) receptors allosterically modulates GABA binding and increases the currents induced by submaximal GABA concentrations. Benzodiazepines induce conformational changes in the GABA-binding site in the extracellular domain, but it is uncertain whether these conformational changes extend into the membrane-spanning domain where the channel gate is located. Alone, benzodiazepines do not open the channel. We used the substituted-cysteine-accessibility method to investigate diazepam-induced conformational changes in the region of the α1-subunit M3 membrane-spanning segment. In the absence of diazepam or GABA, pCMBS- did not react at a measurable rate with cysteine-substitution mutants between α1 Phe296 and α1Glu303. In the presence of 100 nM diazepam, pCMBS- reacted with α1F296C, α1F298C, and α1L301C but not with the other cysteine mutants between α1Phe296 and α1Glu303. These three mutants are a subset of the five residues that we previously showed reacted with pCMBS- applied in the presence of GABA. The pCMBS- reaction rates with these three cysteine mutants were similar in the presence of diazepam and GABA. Thus, diazepam, which binds to the extracellular domain, induces a conformational change in the membrane-spanning domain that is similar to a portion of the change induced by GABA. Because diazepam does not open the channel, these results provide structural evidence that the diazepam-bound state represents an intermediate conformation distinct from the open and resting/closed states of the receptor. The diazepam-induced conformational change in the M3 segment vicinity may be related to the mechanism of allosteric potentiation.
AB - Benzodiazepine binding to γ-aminobutyric acid type A (GABA(A)) receptors allosterically modulates GABA binding and increases the currents induced by submaximal GABA concentrations. Benzodiazepines induce conformational changes in the GABA-binding site in the extracellular domain, but it is uncertain whether these conformational changes extend into the membrane-spanning domain where the channel gate is located. Alone, benzodiazepines do not open the channel. We used the substituted-cysteine-accessibility method to investigate diazepam-induced conformational changes in the region of the α1-subunit M3 membrane-spanning segment. In the absence of diazepam or GABA, pCMBS- did not react at a measurable rate with cysteine-substitution mutants between α1 Phe296 and α1Glu303. In the presence of 100 nM diazepam, pCMBS- reacted with α1F296C, α1F298C, and α1L301C but not with the other cysteine mutants between α1Phe296 and α1Glu303. These three mutants are a subset of the five residues that we previously showed reacted with pCMBS- applied in the presence of GABA. The pCMBS- reaction rates with these three cysteine mutants were similar in the presence of diazepam and GABA. Thus, diazepam, which binds to the extracellular domain, induces a conformational change in the membrane-spanning domain that is similar to a portion of the change induced by GABA. Because diazepam does not open the channel, these results provide structural evidence that the diazepam-bound state represents an intermediate conformation distinct from the open and resting/closed states of the receptor. The diazepam-induced conformational change in the M3 segment vicinity may be related to the mechanism of allosteric potentiation.
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U2 - 10.1124/mol.58.5.1129
DO - 10.1124/mol.58.5.1129
M3 - Article
C2 - 11040062
AN - SCOPUS:0033789013
SN - 0026-895X
VL - 58
SP - 1129
EP - 1136
JO - Molecular Pharmacology
JF - Molecular Pharmacology
IS - 5
ER -