DEPENDENCE OF THE GABA(A) RECEPTOR GATING KINETICS ON THE ALPHA-SUBUNIT ISOFORM - IMPLICATIONS FOR STRUCTURE-FUNCTION RELATIONS AND SYNAPTIC TRANSMISSION

Citation
Kj. Gingrich et al., DEPENDENCE OF THE GABA(A) RECEPTOR GATING KINETICS ON THE ALPHA-SUBUNIT ISOFORM - IMPLICATIONS FOR STRUCTURE-FUNCTION RELATIONS AND SYNAPTIC TRANSMISSION, Journal of physiology, 489(2), 1995, pp. 529-543
Citations number
41
Categorie Soggetti
Physiology
Journal title
ISSN journal
00223751
Volume
489
Issue
2
Year of publication
1995
Pages
529 - 543
Database
ISI
SICI code
0022-3751(1995)489:2<529:DOTGRG>2.0.ZU;2-K
Abstract
1. To examine the dependence of gamma-aminobutyric acid (GABA(A)) rece ptor gating on the alpha-subunit isoform, we studied the kinetics of G ABA-gated currents (I-GABA) of receptors that differed in the alpha-su bunit subtype, alpha 1 beta 2 gamma 2S and alpha 3 beta 2 gamma 2S. cD NAs encoding rat brain subunits were co-expressed heterologously in HE K-293 cells and the resultant receptors studied with the whole-cell pa tch clamp technique and rapidly applied GABA pulses (5-10 s). 2. I-GAB A of both receptors showed a loosely similar dependence on GABA concen tration over a wide range (1-5000 mu M). Generally, I-GABA manifested activation reaching an early current peak, subsequent slower spontaneo us desensitization, and deactivation of open channels at pulse termina tion. Lowering GABA concentrations reduced peak currents and slowed ac tivation and desensitization kinetics. 3. The presence of alpha 3 alte red the peak I-GABA concentration-response relationship by shifting th e fitted Hill equation to tenfold greater GABA concentrations (GABA co ncentration at half amplitude: alpha 1, 7 mu M; and alpha 3, 75 mu M) without affecting Hill coefficients (alpha 1, 1.6; alpha 3, 1.5). Thes e findings indicate a reduction in the apparent activating site affini ty and are consistent with previous reports. 4. To investigate differe nces in gating, we normalized for apparent activating site affinities by analysing the time course of macroscopic gating at equi-activating GABA concentrations. The presence of alpha 3 slowed activation fourfol d (time to current peak (means +/- S.E.M.): alpha 1, 1.2 +/- 0.06 s (2 mu M); alpha 3, 4.7 +/- 0.5 s (20 mu M)), desensitization nearly twof old (reciprocal of time to 80% decay: alpha 1, 2.5 +/- 0.48 s(-1) (100 mu M); alpha 3, 1.5 +/- 0.15 s(-1) (1000 mu M)) and deactivation thre efold (monoexponential decay time constant: alpha 1, 0.22 +/- 0.026 s (2 mu M); alpha 3, 0.68 +/- 0.1 s (20 mu M)). 5. To gain an insight in to the gating mechanisms underlying macroscopic desensitization, we ex tended a previous gating model of GABA(A) receptor single-channel acti vity to include a desensitization pathway. Such a mechanism reproduced empirical alpha 1 beta 2 gamma 2S activation, desensitization and dea ctivation kinetics. 6. To identify molecular transitions underlying th e gating differences between alpha 1 beta 2 gamma 2S and alpha 3 beta 2 gamma 2S receptors, we explored parameter alterations of the alpha 1 beta 2 gamma 2S gating model that provided an accounting of alpha 3 b eta 2 gamma 2S empirical responses. Remarkably, alteration of rates an d rate constants involved in ligand binding alone allowed reproduction of alpha 3 beta 2 gamma 2S activation, desensitization and deactivati on. 7. These results indicate that substitution of the alpha 3 subunit , variant in an alpha 1 beta 2 gamma 2S receptor alters transition rat es involved in ligand binding that underlie changes in apparent activa ting site affinity and macroscopic current gating. Furthermore, they a rgue strongly that the structural determinants of these functional fea tures reside on the alpha-subunit.