TY - JOUR
T1 - Influence of subunit configuration on the interaction of picrotoxin-site ligands with recombinant GABA(A) receptors
AU - Bell-Horner, Cathy L.
AU - Dibas, Mohammed
AU - Huang, Ren Qi
AU - Drewe, John A.
AU - Dillon, Glenn H.
N1 - Funding Information:
This work was supported by NIH grant ES 07904 and by Texas Advanced Research Program grant 009768-027. We gratefully acknowledge Don Carter and Ron Gammill of Pharmacia-Upjohn for supplying stable cell lines and U-93631, respectively.
PY - 2000/3/10
Y1 - 2000/3/10
N2 - We have assessed the interaction of picrotoxin and a putative picrotoxin-site ligand [4-dimethyl-3-t-butylcarboxyl-4,5-dihydro (1,5-a) quinoxaline] (U-93631) with varying configurations of recombinant GABA(A) receptors, using the whole-cell patch clamp technique. In α2β2γ2 GABA(A) receptors, coapplication of picrotoxin with GABA had minimal effects on initial GABA-activated Cl- current amplitude, and subsequently enhanced decay of GABA-activated Cl- currents. The half-maximal inhibitory concentration (IC50) for picrotoxin in α2β2γ2 receptors was 10.3±1.6 μM. The α subunit isoform did not affect picrotoxin-induced inhibition, as IC50 values for α3β2γ2 (5.1±0.7 μM) and α6β2γ2 receptors (7.2±0.4 μM) were comparable to those obtained in α2β2γ2 receptors. Interestingly, in receptors lacking an α subunit (β2γ2 configuration), picrotoxin had a markedly lower IC50 (0.5±0.05 μM) compared to α-containing receptors. The inhibitory profile was generally similar for the presumed picrotoxin-site ligand U-93631, i.e., IC50 values were comparable in all αβγ-containing receptors, but the IC50 in β2γ2 receptors was greater than 10-fold lower. In addition, a modest but significant initial stimulation of GABA-activated current by U-93631 was observed in α2β2γ2 and β2γ2 receptors. A mutation in the second transmembrane domain, shown previously to abolish picrotoxin sensitivity, also greatly attenuated sensitivity to U-93631. Moreover, incubation of receptors with excess U-93631 hindered picrotoxin's ability to gain access to its binding site; both results indicate that U-93631 interacts at the picrotoxin site of the receptor. Our results indicate the presence of an α subunit hinders the ability of picrotoxin to block the GABA(A) receptor, and thus provides additional insight into the site of action of picrotoxin. In addition, we have shown that domains important for the actions of picrotoxin also affect U-93631. Thus, this compound should prove to be a useful ligand for analysis of the convulsant site of this receptor. Copyright (C) 2000 Elsevier Science B.V.
AB - We have assessed the interaction of picrotoxin and a putative picrotoxin-site ligand [4-dimethyl-3-t-butylcarboxyl-4,5-dihydro (1,5-a) quinoxaline] (U-93631) with varying configurations of recombinant GABA(A) receptors, using the whole-cell patch clamp technique. In α2β2γ2 GABA(A) receptors, coapplication of picrotoxin with GABA had minimal effects on initial GABA-activated Cl- current amplitude, and subsequently enhanced decay of GABA-activated Cl- currents. The half-maximal inhibitory concentration (IC50) for picrotoxin in α2β2γ2 receptors was 10.3±1.6 μM. The α subunit isoform did not affect picrotoxin-induced inhibition, as IC50 values for α3β2γ2 (5.1±0.7 μM) and α6β2γ2 receptors (7.2±0.4 μM) were comparable to those obtained in α2β2γ2 receptors. Interestingly, in receptors lacking an α subunit (β2γ2 configuration), picrotoxin had a markedly lower IC50 (0.5±0.05 μM) compared to α-containing receptors. The inhibitory profile was generally similar for the presumed picrotoxin-site ligand U-93631, i.e., IC50 values were comparable in all αβγ-containing receptors, but the IC50 in β2γ2 receptors was greater than 10-fold lower. In addition, a modest but significant initial stimulation of GABA-activated current by U-93631 was observed in α2β2γ2 and β2γ2 receptors. A mutation in the second transmembrane domain, shown previously to abolish picrotoxin sensitivity, also greatly attenuated sensitivity to U-93631. Moreover, incubation of receptors with excess U-93631 hindered picrotoxin's ability to gain access to its binding site; both results indicate that U-93631 interacts at the picrotoxin site of the receptor. Our results indicate the presence of an α subunit hinders the ability of picrotoxin to block the GABA(A) receptor, and thus provides additional insight into the site of action of picrotoxin. In addition, we have shown that domains important for the actions of picrotoxin also affect U-93631. Thus, this compound should prove to be a useful ligand for analysis of the convulsant site of this receptor. Copyright (C) 2000 Elsevier Science B.V.
KW - Cl channel
KW - Convulsant
KW - Picrotoxin
KW - Recombinant GABA(A) receptor
UR - http://www.scopus.com/inward/record.url?scp=0034629238&partnerID=8YFLogxK
U2 - 10.1016/S0169-328X(99)00330-7
DO - 10.1016/S0169-328X(99)00330-7
M3 - Article
C2 - 10719214
AN - SCOPUS:0034629238
SN - 0169-328X
VL - 76
SP - 47
EP - 55
JO - Molecular Brain Research
JF - Molecular Brain Research
IS - 1
ER -