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Site-specific fluorescence reveals distinct structural changes induced in the human rho 1 GABA receptor by inhibitory neurosteroids.

ABSTRACT: The rho 1 GABA receptor is inhibited by a number of neuroactive steroids. A previous study (J Pharmacol Exp Ther 323:236-247, 2007) focusing on the electrophysiological effects of inhibitory steroids on the rho 1 receptor found that steroid inhibitors could be divided into three major groups based on how mutations to residues in the M2 transmembrane domain modified inhibition. It was proposed that the steroids act through distinct mechanisms. We selected representatives of the three groups (pregnanolone, tetrahydrodeoxycorticosterone, pregnanolone sulfate, allopregnanolone sulfate, and beta-estradiol) and probed how these steroids, as well as the nonsteroidal inhibitor picrotoxinin, modify GABA-elicited fluorescence changes from the Alexa 546 C5 maleimide fluorophore attached to residues in the extracellular region of the receptor. The fluorophore responds with changes in quantum yield to changes in the environment, allowing it to probe for structural changes taking place during channel activation or modulation. The results indicate that the modulators have specific effects on fluorescence changes suggesting that distinct conformational changes accompany inhibition. The findings are consistent with the steroids acting as allosteric inhibitors of the rho 1 GABA receptor and support the hypothesis that divergent mechanisms underlie the action of inhibitory steroids on the rho 1 GABA receptor.

SUBMITTER: Li P

PROVIDER: S-EPMC2845946 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Site-specific fluorescence reveals distinct structural changes induced in the human rho 1 GABA receptor by inhibitory neurosteroids.

Li Ping P Khatri Alpa A Bracamontes John J Weiss David S DS Steinbach Joe Henry JH Akk Gustav G

Molecular pharmacology 20100108 4

The rho 1 GABA receptor is inhibited by a number of neuroactive steroids. A previous study (J Pharmacol Exp Ther 323:236-247, 2007) focusing on the electrophysiological effects of inhibitory steroids on the rho 1 receptor found that steroid inhibitors could be divided into three major groups based on how mutations to residues in the M2 transmembrane domain modified inhibition. It was proposed that the steroids act through distinct mechanisms. We selected representatives of the three groups (preg ...[more]

PMID: 20061447

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Project description:Background and purposeThe binding of transmitter to specialized binding pockets leads to rearrangements in the structure of the receptor eventually resulting in channel opening. We used voltage-clamp fluorometry to investigate the pharmacological basis and biophysical processes that underlie structural changes at the transmitter binding site of the rat ?1?2?2L GABA(A) receptor.Experimental approachSimultaneous electrophysiological and site-specific fluorescence measurements were conducted on receptors expressed in Xenopus oocytes and labelled with an environmentally-sensitive fluorophore, Alexa 546 maleimide, at the ?1L127C site.Key resultsReceptors activated by GABA demonstrate a concentration-dependent increase in fluorescence intensity, indicating that the environment surrounding the fluorophore becomes less polar upon activation. Qualitatively similar responses were observed with other GABA site ligands such as piperidine-4-sulphonic acid, muscimol, ?-alanine and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol. Fluorescence changes were not affected by the direction of current flow. During long applications of GABA significant desensitization developed, which was not accompanied by additional changes in fluorescence. Pentobarbital was an efficacious agonist of the labelled mutant receptor but did not cause changes in fluorescence. Direct activation by etomidate or the steroid allopregnanolone also did not result in fluorescence changes. Functional potentiation of GABA-activated receptors by allopregnanolone or etomidate enhanced both the GABA-elicited functional response and the fluorescence change. In contrast, potentiation by pentobarbital was not accompanied by an enhanced fluorescence response.Conclusions and implicationsThe data indicate that there is no direct correlation between current flow or position of the activation gate and the structural changes as detected by Alexa 546-labelled ?1L127C?2?2L GABA(A) receptors. Channel potentiation by pentobarbital qualitatively differs from potentiation by etomidate or allopregnanolone.

Dataset Information

Site-specific fluorescence reveals distinct structural changes induced in the human rho 1 GABA receptor by inhibitory neurosteroids.

Publications

Site-specific fluorescence reveals distinct structural changes induced in the human rho 1 GABA receptor by inhibitory neurosteroids.

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