Dependence of the GABA(A) receptor gating kinetics on the α-subunit isoform: Implications for structure-function relations and synaptic transmission

K. J. Gingrich, W. A. Roberts, R. S. Kass

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Abstract

1. To examine the dependence of γ-aminobutyric acid (GABA(A)) receptor gating on the a-subunit isoform, we studied the kinetics of GABA-gated currents (I(GABA)) of receptors that differed in the α-subunit subtype, α1β2γ2S and α3β2γ2S. cDNAs encoding rat brain subunits were co-expressed heterologously in HEK-293 cells and the resultant receptors studied with the whole-cell patch clamp technique and rapidly applied GABA pulses (5-10 s). 2. I(GABA) of both receptors showed a loosely similar dependence on GABA concentration over a wide range (1-5000 μM). Generally, I(GABA) manifested activation reaching an early current peak, subsequent slower spontaneous desensitization, and deactivation of open channels at pulse termination. Lowering GABA concentrations reduced peak currents and slowed activation and desensitization kinetics. 3. The presence of α3 altered the peak I(GABA) concentration-response relationship by shifting the fitted Hill equation to tenfold greater GABA concentrations (GABA concentration at half amplitude: α1, 7 μM; and α3, 75 μM) without affecting Hill coefficients (α1, 1.6; α3, 1.5). These findings indicate a reduction in the apparent activating site affinity and are consistent with previous reports. 4. To investigate differences 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 α3 slowed activation fourfold (time to current peak (means ± S.E.M.: α1, 1.2 ± 0.06 s (2 μM); α3, 4.7 ± 0.5 s (20 μM)), desensitization nearly twofold (reciprocal of time to 80% decay: α1, 2.5 ± 0.48 s-1 (100 μM); α3, 1.5 ± 0.15 s-1 (1000 μM)) and deactivation threefold (monoexponential decay time constant: α1, 0.22 ± 0.026 s (2 μM); α3, 0.68 ± 0.1 s (20 μM)). 5. To gain an insight into the gating mechanisms underlying macroscopic desensitization, we extended a previous gating model of GABA(A) receptor single-channel activity to include a desensitization pathway. Such a mechanism reproduced empirical α1β2γ2S activation, desensitization and deactivation kinetics. 6. To identify molecular transitions underlying the gating differences between α1β2γ2S and α3β2γ2S receptors, we explored parameter alterations of the α1β2γ2S gating model that provided an accounting of α3β2γ2S empirical responses. Remarkably, alteration of rates and rate constants involved in ligand binding alone allowed reproduction of α3β2γ2S activation, desensitization and deactivation. 7. These results indicate that substitution of the α3 subunit, variant in an α1β2γ2S receptor alters transition rates involved in ligand binding that underlie changes in apparent activating site affinity and macroscopic current gating. Furthermore, they argue strongly that the structural determinants of these functional features reside on the α-subunit.

Original languageEnglish (US)
Pages (from-to)529-543
Number of pages15
JournalJournal of Physiology
Volume489
Issue number2
Publication statusPublished - 1995

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ASJC Scopus subject areas

  • Physiology

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