Zn2+ inhibition of recombinant GABAA receptors: an allosteric, state-dependent mechanism determined by the gamma-subunit.

K. J. Gingrich, P. M. Burkat

Research output: Contribution to journalArticle

Abstract

1. The gamma-subunit in recombinant gamma-aminobutyric acid (GABAA) receptors reduces the sensitivity of GABA-triggered Cl- currents to inhibition by Zn2+ and transforms the apparent mechanism of antagonism from non-competitive to competitive. To investigate underlying receptor function we studied Zn2- effects on macroscopic and single-channel currents of recombinant alpha 1 beta 2 and alpha 1 beta 2 gamma 2 receptors expressed heterologously in HEK-293 cells using the patch-clamp technique and rapid solution changes. 2. Zn2+ present for > 60 s (constant) inhibited peak, GABA (5 microM)-triggered currents of alpha 1 beta 2 receptors in a concentration-dependent manner (inhibition equation parameters: concentration at half-amplitude (IC50) = 0.94 microM; slope related to Hill coefficient, S = 0.7) that was unaffected by GABA concentration. The gamma 2 subunit (alpha 1 beta 2 gamma 2 receptor) reduced Zn2+ sensitivity more than fiftyfold (IC50 = 51 microM, S = 0.86); increased GABA concentration (100 microM) antagonized inhibition by reducing apparent affinity (IC50 = 322 microM, S = 0.79). Zn2+ slowed macroscopic gating of alpha 1 beta 2 receptors by inducing a novel slow exponential component in the activation time course and suppressing a fast component of control desensitization. For alpha 1 beta 2 gamma 2 receptors, Zn2+ accelerated a fast component of apparent desensitization. 3. Zn2+ preincubations lasting up to 10 s markedly increased current depression and activation slowing of alpha 1 beta 2 receptors, but had little effect on currents from alpha 1 beta 2 gamma 2 receptors. 4. Steady-state fluctuation analysis of macroscopic alpha 1 beta 2 gamma 2 currents (n = 5) resulted in control (2 microM GABA) power density spectra that were fitted by a sum of two Lorentzian functions (relaxation times: 37 +/- 5.6 and 1.41 +/- 0.15 ms, means +/- S.E.M.). Zn2+ (200 microM) reduced the total power almost sixfold and accelerated the slow (23 +/- 2.8 ms, P < 0.05) without altering the fast (1.40 +/- 0.16 ms) relaxation time. The ratio (fast/slow) of Lorentzian areas was increased by Zn2+ (control, 3.39 +/- 0.55; Zn2+, 4.9 +/- 0.37, P < 0.05). 5. Zn2+ (500 microM) depression of previously activated current amplitudes (% control) for alpha 1 beta 2 gamma 2 receptors was independent of GABA concentration (5 microM, 13.2 +/- 0.72%; 100 microM, 12.2 +/- 2.9%, P < 0.8, n = 5). Both onset and offset inhibition time courses were biexponential. Onset rates were enhanced by Zn2+ concentration. Inhibition onset was also biexponential for preactivated alpha 1 beta 2 receptors with current depression more than fourfold less sensitive (5 microM GABA, IC50 = 3.8 microM, S = 0.84) relative to that in constant Zn2+. 6. The results lead us to propose a general model of Zn2+ inhibition of GABAA receptors in which Zn2+ binds to a single extracellular site, induces allosteric receptor inhibition involving two non-conducting states, site affinity is state-dependent, and the features of state dependence are determined by the gamma-subunit.

Original languageEnglish (US)
JournalThe Journal of physiology
Volume506
StatePublished - Feb 1 1998

Fingerprint

GABA-A Receptors
gamma-Aminobutyric Acid
Inhibitory Concentration 50
Allosteric Site
GABA Receptors
HEK293 Cells
Patch-Clamp Techniques

ASJC Scopus subject areas

  • Physiology

Cite this

@article{3a0fd599f3b042209ae77e6f6a601c0c,
title = "Zn2+ inhibition of recombinant GABAA receptors: an allosteric, state-dependent mechanism determined by the gamma-subunit.",
abstract = "1. The gamma-subunit in recombinant gamma-aminobutyric acid (GABAA) receptors reduces the sensitivity of GABA-triggered Cl- currents to inhibition by Zn2+ and transforms the apparent mechanism of antagonism from non-competitive to competitive. To investigate underlying receptor function we studied Zn2- effects on macroscopic and single-channel currents of recombinant alpha 1 beta 2 and alpha 1 beta 2 gamma 2 receptors expressed heterologously in HEK-293 cells using the patch-clamp technique and rapid solution changes. 2. Zn2+ present for > 60 s (constant) inhibited peak, GABA (5 microM)-triggered currents of alpha 1 beta 2 receptors in a concentration-dependent manner (inhibition equation parameters: concentration at half-amplitude (IC50) = 0.94 microM; slope related to Hill coefficient, S = 0.7) that was unaffected by GABA concentration. The gamma 2 subunit (alpha 1 beta 2 gamma 2 receptor) reduced Zn2+ sensitivity more than fiftyfold (IC50 = 51 microM, S = 0.86); increased GABA concentration (100 microM) antagonized inhibition by reducing apparent affinity (IC50 = 322 microM, S = 0.79). Zn2+ slowed macroscopic gating of alpha 1 beta 2 receptors by inducing a novel slow exponential component in the activation time course and suppressing a fast component of control desensitization. For alpha 1 beta 2 gamma 2 receptors, Zn2+ accelerated a fast component of apparent desensitization. 3. Zn2+ preincubations lasting up to 10 s markedly increased current depression and activation slowing of alpha 1 beta 2 receptors, but had little effect on currents from alpha 1 beta 2 gamma 2 receptors. 4. Steady-state fluctuation analysis of macroscopic alpha 1 beta 2 gamma 2 currents (n = 5) resulted in control (2 microM GABA) power density spectra that were fitted by a sum of two Lorentzian functions (relaxation times: 37 +/- 5.6 and 1.41 +/- 0.15 ms, means +/- S.E.M.). Zn2+ (200 microM) reduced the total power almost sixfold and accelerated the slow (23 +/- 2.8 ms, P < 0.05) without altering the fast (1.40 +/- 0.16 ms) relaxation time. The ratio (fast/slow) of Lorentzian areas was increased by Zn2+ (control, 3.39 +/- 0.55; Zn2+, 4.9 +/- 0.37, P < 0.05). 5. Zn2+ (500 microM) depression of previously activated current amplitudes ({\%} control) for alpha 1 beta 2 gamma 2 receptors was independent of GABA concentration (5 microM, 13.2 +/- 0.72{\%}; 100 microM, 12.2 +/- 2.9{\%}, P < 0.8, n = 5). Both onset and offset inhibition time courses were biexponential. Onset rates were enhanced by Zn2+ concentration. Inhibition onset was also biexponential for preactivated alpha 1 beta 2 receptors with current depression more than fourfold less sensitive (5 microM GABA, IC50 = 3.8 microM, S = 0.84) relative to that in constant Zn2+. 6. The results lead us to propose a general model of Zn2+ inhibition of GABAA receptors in which Zn2+ binds to a single extracellular site, induces allosteric receptor inhibition involving two non-conducting states, site affinity is state-dependent, and the features of state dependence are determined by the gamma-subunit.",
author = "Gingrich, {K. J.} and Burkat, {P. M.}",
year = "1998",
month = "2",
day = "1",
language = "English (US)",
volume = "506",
journal = "Journal of Physiology",
issn = "0022-3751",
publisher = "Wiley-Blackwell",

}

TY - JOUR

T1 - Zn2+ inhibition of recombinant GABAA receptors

T2 - an allosteric, state-dependent mechanism determined by the gamma-subunit.

AU - Gingrich, K. J.

AU - Burkat, P. M.

PY - 1998/2/1

Y1 - 1998/2/1

N2 - 1. The gamma-subunit in recombinant gamma-aminobutyric acid (GABAA) receptors reduces the sensitivity of GABA-triggered Cl- currents to inhibition by Zn2+ and transforms the apparent mechanism of antagonism from non-competitive to competitive. To investigate underlying receptor function we studied Zn2- effects on macroscopic and single-channel currents of recombinant alpha 1 beta 2 and alpha 1 beta 2 gamma 2 receptors expressed heterologously in HEK-293 cells using the patch-clamp technique and rapid solution changes. 2. Zn2+ present for > 60 s (constant) inhibited peak, GABA (5 microM)-triggered currents of alpha 1 beta 2 receptors in a concentration-dependent manner (inhibition equation parameters: concentration at half-amplitude (IC50) = 0.94 microM; slope related to Hill coefficient, S = 0.7) that was unaffected by GABA concentration. The gamma 2 subunit (alpha 1 beta 2 gamma 2 receptor) reduced Zn2+ sensitivity more than fiftyfold (IC50 = 51 microM, S = 0.86); increased GABA concentration (100 microM) antagonized inhibition by reducing apparent affinity (IC50 = 322 microM, S = 0.79). Zn2+ slowed macroscopic gating of alpha 1 beta 2 receptors by inducing a novel slow exponential component in the activation time course and suppressing a fast component of control desensitization. For alpha 1 beta 2 gamma 2 receptors, Zn2+ accelerated a fast component of apparent desensitization. 3. Zn2+ preincubations lasting up to 10 s markedly increased current depression and activation slowing of alpha 1 beta 2 receptors, but had little effect on currents from alpha 1 beta 2 gamma 2 receptors. 4. Steady-state fluctuation analysis of macroscopic alpha 1 beta 2 gamma 2 currents (n = 5) resulted in control (2 microM GABA) power density spectra that were fitted by a sum of two Lorentzian functions (relaxation times: 37 +/- 5.6 and 1.41 +/- 0.15 ms, means +/- S.E.M.). Zn2+ (200 microM) reduced the total power almost sixfold and accelerated the slow (23 +/- 2.8 ms, P < 0.05) without altering the fast (1.40 +/- 0.16 ms) relaxation time. The ratio (fast/slow) of Lorentzian areas was increased by Zn2+ (control, 3.39 +/- 0.55; Zn2+, 4.9 +/- 0.37, P < 0.05). 5. Zn2+ (500 microM) depression of previously activated current amplitudes (% control) for alpha 1 beta 2 gamma 2 receptors was independent of GABA concentration (5 microM, 13.2 +/- 0.72%; 100 microM, 12.2 +/- 2.9%, P < 0.8, n = 5). Both onset and offset inhibition time courses were biexponential. Onset rates were enhanced by Zn2+ concentration. Inhibition onset was also biexponential for preactivated alpha 1 beta 2 receptors with current depression more than fourfold less sensitive (5 microM GABA, IC50 = 3.8 microM, S = 0.84) relative to that in constant Zn2+. 6. The results lead us to propose a general model of Zn2+ inhibition of GABAA receptors in which Zn2+ binds to a single extracellular site, induces allosteric receptor inhibition involving two non-conducting states, site affinity is state-dependent, and the features of state dependence are determined by the gamma-subunit.

AB - 1. The gamma-subunit in recombinant gamma-aminobutyric acid (GABAA) receptors reduces the sensitivity of GABA-triggered Cl- currents to inhibition by Zn2+ and transforms the apparent mechanism of antagonism from non-competitive to competitive. To investigate underlying receptor function we studied Zn2- effects on macroscopic and single-channel currents of recombinant alpha 1 beta 2 and alpha 1 beta 2 gamma 2 receptors expressed heterologously in HEK-293 cells using the patch-clamp technique and rapid solution changes. 2. Zn2+ present for > 60 s (constant) inhibited peak, GABA (5 microM)-triggered currents of alpha 1 beta 2 receptors in a concentration-dependent manner (inhibition equation parameters: concentration at half-amplitude (IC50) = 0.94 microM; slope related to Hill coefficient, S = 0.7) that was unaffected by GABA concentration. The gamma 2 subunit (alpha 1 beta 2 gamma 2 receptor) reduced Zn2+ sensitivity more than fiftyfold (IC50 = 51 microM, S = 0.86); increased GABA concentration (100 microM) antagonized inhibition by reducing apparent affinity (IC50 = 322 microM, S = 0.79). Zn2+ slowed macroscopic gating of alpha 1 beta 2 receptors by inducing a novel slow exponential component in the activation time course and suppressing a fast component of control desensitization. For alpha 1 beta 2 gamma 2 receptors, Zn2+ accelerated a fast component of apparent desensitization. 3. Zn2+ preincubations lasting up to 10 s markedly increased current depression and activation slowing of alpha 1 beta 2 receptors, but had little effect on currents from alpha 1 beta 2 gamma 2 receptors. 4. Steady-state fluctuation analysis of macroscopic alpha 1 beta 2 gamma 2 currents (n = 5) resulted in control (2 microM GABA) power density spectra that were fitted by a sum of two Lorentzian functions (relaxation times: 37 +/- 5.6 and 1.41 +/- 0.15 ms, means +/- S.E.M.). Zn2+ (200 microM) reduced the total power almost sixfold and accelerated the slow (23 +/- 2.8 ms, P < 0.05) without altering the fast (1.40 +/- 0.16 ms) relaxation time. The ratio (fast/slow) of Lorentzian areas was increased by Zn2+ (control, 3.39 +/- 0.55; Zn2+, 4.9 +/- 0.37, P < 0.05). 5. Zn2+ (500 microM) depression of previously activated current amplitudes (% control) for alpha 1 beta 2 gamma 2 receptors was independent of GABA concentration (5 microM, 13.2 +/- 0.72%; 100 microM, 12.2 +/- 2.9%, P < 0.8, n = 5). Both onset and offset inhibition time courses were biexponential. Onset rates were enhanced by Zn2+ concentration. Inhibition onset was also biexponential for preactivated alpha 1 beta 2 receptors with current depression more than fourfold less sensitive (5 microM GABA, IC50 = 3.8 microM, S = 0.84) relative to that in constant Zn2+. 6. The results lead us to propose a general model of Zn2+ inhibition of GABAA receptors in which Zn2+ binds to a single extracellular site, induces allosteric receptor inhibition involving two non-conducting states, site affinity is state-dependent, and the features of state dependence are determined by the gamma-subunit.

UR - http://www.scopus.com/inward/record.url?scp=0031991571&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0031991571&partnerID=8YFLogxK

M3 - Article

C2 - 9503325

VL - 506

JO - Journal of Physiology

JF - Journal of Physiology

SN - 0022-3751

ER -