Ethanol sensitivity of recombinant homomeric and heteromeric AMPA receptor subunits expressed in Xenopus oocytes.
BACKGROUND
Ethanol is known to acutely inhibit AMPA receptor function, and sensitivity of AMPA receptors to ethanol is dependent on subunit composition in vivo and in vitro. A commonly used in vitro expression system for studying recombinant receptor subunits is the Xenopus laevis oocyte and two-electrode voltage-clamp electrophysiological recording. To date, ethanol sensitivity of injected receptor subunit complementary RNA (cRNA) has not been shown to be correlated with the actual expression of receptor subunits in oocytes. In this study, we compared ethanol sensitivity of homomeric and heteromeric AMPA receptor subunits microinjected into Xenopus oocytes and confirmed subunit expression in oocytes by immunoblot.


METHODS
cRNAs coding for the "flop" type AMPA GluR1 or GluR3 (homomeric), GluR2/GluR3 (heteromeric combination), and GluR1/2/3 (heteromeric combination) were microinjected in equimolar amounts of 16 to 20 ng into oocytes, which were studied for their sensitivity to ethanol. Oocytes injected with cRNA for homomeric or heteromeric subunit combinations were homogenized and the expressed subunits quantified with anti-GluR1, anti-GluR2, and anti-GluR2/3 antibodies.


RESULTS
Ethanol concentrations of 10 to 500 mM consistently inhibited currents activated in oocytes by 200 microM kainic acid. The expressed homomeric GluR1 receptor and heteromeric GluR1/2/3 receptor combination currents showed similar sensitivity to ethanol inhibition with half-maximal inhibition values of 170 +/- 12 mM and 176 +/- 8 mM, respectively. The expressed homomeric GluR3 receptor and heteromeric GluR2/3 receptor combination currents were differentially sensitive to ethanol inhibition with respective IC50 values of 238 +/- 9 mM and 338 +/- 16 mM.


CONCLUSIONS
The expressed homomeric and heteromeric "flop" type AMPA receptors were differentially sensitive to ethanol, which may in part explain differential ethanol sensitivity in native neurons.
Akinshola
B Emmanuel
BE
Department of Pharmacology, Howard University College of Medicine, Washington, DC 20059, USA. eakinshola@howard.edu
Yasuda
Robert P
RP
Peoples
Robert W
RW
Taylor
Robert E
RE
D000818:Animals; D004305:Dose-Response Relationship, Drug; D000431:Ethanol / Q000494:pharmacology*; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000502:physiology; D007608:Kainic Acid / Q000494:pharmacology; D009865:Oocytes; D021122:Protein Subunits / Q000819:agonists / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D018091:Receptors, AMPA / Q000819:agonists / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000819:agonists / Q000096:biosynthesis / Q000235:genetics; D014982:Xenopus laevis
D021122:Protein Subunits; D018091:Receptors, AMPA; D011994:Recombinant Proteins; D000431:Ethanol; D007608:Kainic Acid