Chronic benzodiazepine treatment of cells expressing recombinant GABAA receptors uncouples allosteric binding:: studies on possible mechanisms

被引:57
作者
Ali, NJ [1 ]
Olsen, RW [1 ]
机构
[1] Univ Calif Los Angeles, Sch Med, Dept Mol & Med Pharmacol, Los Angeles, CA 90095 USA
关键词
benzodiazepine; GABA(A) receptor; internalization; phosphorylation; uncoupling;
D O I
10.1046/j.1471-4159.2001.00664.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
`Functional and behavioral tolerance to chronic benzodiazepine (BZ) exposure has been associated with an uncoupling of the BZ and GABA binding sites. As in rats exposed to BZ for periods of a week or longer, recombinant GABA(A) receptors (GABARs) expressed in Sf9 cells lose the normally observed allosteric enhancement of [H-3]flunitrazepam binding by GAGA agonists, which is measured in homogenized membranes after a few hours exposure to pharmacological doses of agonist BZ. Treatment of Sf9 cells expressing recombinant GABAR with various drugs that inhibit protein kinase A (PKA), but not protein kinase C (PKC), resulted in an uncoupling of the BZ and GABA binding sites; whereas promotion of phosphorylation by PKA, but not PKC, favored coupling and recoupling. However, mutation of the only PKA phosphorylation site expressed from among the subunits proved that direct phosphorylation of the GABAR was not involved in either coupling after chronic BZ exposure or reversal of uncoupling after exposure to the competitive BZ antagonist, flumazenil. Osmotic-shock of cell membrane homogenates to lyse intracellular compartments reversed uncoupling, and uncoupling can be replicated in untreated cells by performing membrane binding assays in an acidic environment, suggesting that GABARs become internalized into an acidic intracellular environment where normal BZ binding occurs, but that potentiation by GABA is hindered. The internalization of receptors was shown by immunofluorescence after chronic exposure to either BZ or the PKA inhibitor H-89.
引用
收藏
页码:1100 / 1108
页数:9
相关论文
共 43 条
[1]  
BARAK LS, 1994, J BIOL CHEM, V269, P2790
[2]   Intracellular trafficking of GABAA receptors [J].
Barnes, EM .
LIFE SCIENCES, 2000, 66 (12) :1063-1070
[3]   Molecular mechanisms of benzodiazepine-induced downregulation of GABA(A) receptor alpha(1) subunit protein in rat cerebellar granule cells [J].
Brown, MJ ;
Bristow, DR .
BRITISH JOURNAL OF PHARMACOLOGY, 1996, 118 (05) :1103-1110
[4]   ACTIVATORS OF PROTEIN-KINASE-C INCREASE THE PHOSPHORYLATION OF THE SYNAPSINS AT SITES PHOSPHORYLATED BY CAMP-DEPENDENT AND CA2+ CALMODULIN-DEPENDENT PROTEIN-KINASES IN THE RAT HIPPOCAMPAL SLICE [J].
BROWNING, MD ;
DUDEK, EM .
SYNAPSE, 1992, 10 (01) :62-70
[5]   Activation of protein kinase C induces γ-aminobutyric acid type a receptor internalization in Xenopus oocytes [J].
Chapell, R ;
Bueno, OF ;
Alvarez-Hernandez, X ;
Robinson, LC ;
Leidenheimer, NJ .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (49) :32595-32601
[6]   The γ-aminobutyric acid type A (GABAA) receptor-associated protein (GABARAP) promotes GABAA receptor clustering and modulates the channel kinetics [J].
Chen, L ;
Wang, HB ;
Vicini, S ;
Olsen, RW .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2000, 97 (21) :11557-11562
[7]   Cell surface stability of γ-aminobutyric acid type A receptors -: Dependence on protein kinase C activity and subunit composition [J].
Connolly, CN ;
Kittler, JT ;
Thomas, P ;
Uren, JM ;
Brandon, NJ ;
Smart, TG ;
Moss, SJ .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (51) :36565-36572
[8]   MOLECULAR MECHANISMS IN THE RECEPTOR ACTION OF BENZODIAZEPINES [J].
COSTA, E ;
GUIDOTTI, A .
ANNUAL REVIEW OF PHARMACOLOGY AND TOXICOLOGY, 1979, 19 :531-545
[9]  
Elster L, 2000, J NEUROSCI RES, V61, P193
[10]   Evidence for phosphorylation-dependent internalization of recombinant human p1 GABAC receptors [J].
Filippova, N ;
Dudley, R ;
Weiss, DS .
JOURNAL OF PHYSIOLOGY-LONDON, 1999, 518 (02) :385-399