The influence of an endogenous ß3 subunit on recombinant GABA(A) receptor assembly and pharmacology in WSS-1 cells and transiently transfected HEK293 cells

Paul A. Davies, Ewa B. Hoffmann, Holly J. Carlisle, Rachel F. Tyndale, Tim G. Hales

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Cell lines are commonly used for studying recombinant heterooligomeric ion channels with defined subunit composition. Such studies often ignore the contribution of endogenous proteins in the assembly of mature channels. We examined whether an endogenous subunit was required for the functional expression of γ-aminobutyric acid type A (GABA(A)) receptors in WSS-1 cells, HEK293 cells stably expressing recombinant α1 and γ2 subunits. Our pharmacological and RT-PCR analyses of GABA(A) receptors and their mRNAs in WSS-1 cells confirm the presence of α1 and γ2 subunits and suggest the existence of an endogenous ß3 subunit. Whole-cell GABA-evoked currents recorded from untransfected WSS-1 cells were blocked by bicuculline methiodide and enhanced by anesthetics and anticonvulsants including the subunit-selective compounds diazepam and loreclezole. These data suggest that, in addition to the γ2 subunit, WSS-1 cell receptors also contain ß2/3 subunits. RT-PCR revealed that WSS-1 cells and parental HEK293 cells contain ß3 mRNA. We examined the contribution of the ß3 subunit in the function of receptors formed by expression of α1 and γ2S subunits. Untransfected HEK293 cells were unresponsive to GABA. Cells transfected with α1 and γ2S cDNAs displayed small diazepam and loreclezole responsive GABA-activated currents. By contrast, the expression of α1 and γ2S cDNAs in the neuroblastoma NB41A3 cell line, that lacks ß subunit mRNAs, failed to produce functional receptors. These data reaffirm that α1 and γ2S subunits alone do not form functional GABA(A) receptors and that receptors of WSS-1 cells contain α1, ß3 and γ2S subunits.

Original languageEnglish
Pages (from-to)611-620
Number of pages10
JournalNeuropharmacology
Volume39
Issue number4
DOIs
Publication statusPublished - 15 Mar 2000

Fingerprint

HEK293 Cells
GABA-A Receptors
Pharmacology
gamma-Aminobutyric Acid
Diazepam
Messenger RNA
Complementary DNA
Aminobutyrates
Cell Line
Polymerase Chain Reaction
Neuroblastoma
Ion Channels
Anticonvulsants
Anesthetics

Keywords

  • Benzodiazepines
  • GABA(A) receptors
  • HEK293 cells
  • Loreclezole
  • NB41A3 cells
  • RT-PCR
  • WSS-1 cells

Cite this

@article{60e6b8bfe8994a0abcb19eeabfbca5a4,
title = "The influence of an endogenous {\ss}3 subunit on recombinant GABA(A) receptor assembly and pharmacology in WSS-1 cells and transiently transfected HEK293 cells",
abstract = "Cell lines are commonly used for studying recombinant heterooligomeric ion channels with defined subunit composition. Such studies often ignore the contribution of endogenous proteins in the assembly of mature channels. We examined whether an endogenous subunit was required for the functional expression of γ-aminobutyric acid type A (GABA(A)) receptors in WSS-1 cells, HEK293 cells stably expressing recombinant α1 and γ2 subunits. Our pharmacological and RT-PCR analyses of GABA(A) receptors and their mRNAs in WSS-1 cells confirm the presence of α1 and γ2 subunits and suggest the existence of an endogenous {\ss}3 subunit. Whole-cell GABA-evoked currents recorded from untransfected WSS-1 cells were blocked by bicuculline methiodide and enhanced by anesthetics and anticonvulsants including the subunit-selective compounds diazepam and loreclezole. These data suggest that, in addition to the γ2 subunit, WSS-1 cell receptors also contain {\ss}2/3 subunits. RT-PCR revealed that WSS-1 cells and parental HEK293 cells contain {\ss}3 mRNA. We examined the contribution of the {\ss}3 subunit in the function of receptors formed by expression of α1 and γ2S subunits. Untransfected HEK293 cells were unresponsive to GABA. Cells transfected with α1 and γ2S cDNAs displayed small diazepam and loreclezole responsive GABA-activated currents. By contrast, the expression of α1 and γ2S cDNAs in the neuroblastoma NB41A3 cell line, that lacks {\ss} subunit mRNAs, failed to produce functional receptors. These data reaffirm that α1 and γ2S subunits alone do not form functional GABA(A) receptors and that receptors of WSS-1 cells contain α1, {\ss}3 and γ2S subunits.",
keywords = "Benzodiazepines, GABA(A) receptors, HEK293 cells, Loreclezole, NB41A3 cells, RT-PCR, WSS-1 cells",
author = "Davies, {Paul A.} and Hoffmann, {Ewa B.} and Carlisle, {Holly J.} and Tyndale, {Rachel F.} and Hales, {Tim G.}",
year = "2000",
month = "3",
day = "15",
doi = "10.1016/S0028-3908(99)00163-X",
language = "English",
volume = "39",
pages = "611--620",
journal = "Neuropharmacology",
issn = "0028-3908",
publisher = "Elsevier",
number = "4",

}

The influence of an endogenous ß3 subunit on recombinant GABA(A) receptor assembly and pharmacology in WSS-1 cells and transiently transfected HEK293 cells. / Davies, Paul A.; Hoffmann, Ewa B.; Carlisle, Holly J.; Tyndale, Rachel F.; Hales, Tim G.

In: Neuropharmacology, Vol. 39, No. 4, 15.03.2000, p. 611-620.

Research output: Contribution to journalArticle

TY - JOUR

T1 - The influence of an endogenous ß3 subunit on recombinant GABA(A) receptor assembly and pharmacology in WSS-1 cells and transiently transfected HEK293 cells

AU - Davies, Paul A.

AU - Hoffmann, Ewa B.

AU - Carlisle, Holly J.

AU - Tyndale, Rachel F.

AU - Hales, Tim G.

PY - 2000/3/15

Y1 - 2000/3/15

N2 - Cell lines are commonly used for studying recombinant heterooligomeric ion channels with defined subunit composition. Such studies often ignore the contribution of endogenous proteins in the assembly of mature channels. We examined whether an endogenous subunit was required for the functional expression of γ-aminobutyric acid type A (GABA(A)) receptors in WSS-1 cells, HEK293 cells stably expressing recombinant α1 and γ2 subunits. Our pharmacological and RT-PCR analyses of GABA(A) receptors and their mRNAs in WSS-1 cells confirm the presence of α1 and γ2 subunits and suggest the existence of an endogenous ß3 subunit. Whole-cell GABA-evoked currents recorded from untransfected WSS-1 cells were blocked by bicuculline methiodide and enhanced by anesthetics and anticonvulsants including the subunit-selective compounds diazepam and loreclezole. These data suggest that, in addition to the γ2 subunit, WSS-1 cell receptors also contain ß2/3 subunits. RT-PCR revealed that WSS-1 cells and parental HEK293 cells contain ß3 mRNA. We examined the contribution of the ß3 subunit in the function of receptors formed by expression of α1 and γ2S subunits. Untransfected HEK293 cells were unresponsive to GABA. Cells transfected with α1 and γ2S cDNAs displayed small diazepam and loreclezole responsive GABA-activated currents. By contrast, the expression of α1 and γ2S cDNAs in the neuroblastoma NB41A3 cell line, that lacks ß subunit mRNAs, failed to produce functional receptors. These data reaffirm that α1 and γ2S subunits alone do not form functional GABA(A) receptors and that receptors of WSS-1 cells contain α1, ß3 and γ2S subunits.

AB - Cell lines are commonly used for studying recombinant heterooligomeric ion channels with defined subunit composition. Such studies often ignore the contribution of endogenous proteins in the assembly of mature channels. We examined whether an endogenous subunit was required for the functional expression of γ-aminobutyric acid type A (GABA(A)) receptors in WSS-1 cells, HEK293 cells stably expressing recombinant α1 and γ2 subunits. Our pharmacological and RT-PCR analyses of GABA(A) receptors and their mRNAs in WSS-1 cells confirm the presence of α1 and γ2 subunits and suggest the existence of an endogenous ß3 subunit. Whole-cell GABA-evoked currents recorded from untransfected WSS-1 cells were blocked by bicuculline methiodide and enhanced by anesthetics and anticonvulsants including the subunit-selective compounds diazepam and loreclezole. These data suggest that, in addition to the γ2 subunit, WSS-1 cell receptors also contain ß2/3 subunits. RT-PCR revealed that WSS-1 cells and parental HEK293 cells contain ß3 mRNA. We examined the contribution of the ß3 subunit in the function of receptors formed by expression of α1 and γ2S subunits. Untransfected HEK293 cells were unresponsive to GABA. Cells transfected with α1 and γ2S cDNAs displayed small diazepam and loreclezole responsive GABA-activated currents. By contrast, the expression of α1 and γ2S cDNAs in the neuroblastoma NB41A3 cell line, that lacks ß subunit mRNAs, failed to produce functional receptors. These data reaffirm that α1 and γ2S subunits alone do not form functional GABA(A) receptors and that receptors of WSS-1 cells contain α1, ß3 and γ2S subunits.

KW - Benzodiazepines

KW - GABA(A) receptors

KW - HEK293 cells

KW - Loreclezole

KW - NB41A3 cells

KW - RT-PCR

KW - WSS-1 cells

UR - http://www.scopus.com/inward/record.url?scp=0033625084&partnerID=8YFLogxK

U2 - 10.1016/S0028-3908(99)00163-X

DO - 10.1016/S0028-3908(99)00163-X

M3 - Article

C2 - 10728882

AN - SCOPUS:0033625084

VL - 39

SP - 611

EP - 620

JO - Neuropharmacology

JF - Neuropharmacology

SN - 0028-3908

IS - 4

ER -