Point mutations in the M2 region of the alpha, beta, or gamma subunit of the GABAA channel that abolish block by picrotoxin.
Site-directed mutagenesis and the two-electrode voltage-clamp techniques were used to evaluate the site of action of picrotoxin on rat alpha 1 beta 2 gamma 2 containing GABAA receptors expressed in Xenopus oocytes. Following a sequence comparison between GABAA subunits and the picrotoxin-insensitive glycine beta subunit, the following mutations were made near the center of the M2 region of the alpha 1, beta 2, and gamma 2 GABAA subunits: alpha 1(T261F/T267A), beta 2(T246F/T252A), and gamma 2(T271F/T277A). Wild type (alpha 1 beta 2 gamma 2) GABA channels had an IC50 for picrotoxin of 1.3 +/- O.3 microM. In contrast, alpha 1 beta 2 gamma 2 channels that contained any one of the mutated alpha 1, beta 2, or gamma 2 subunits produced currents that were insensitive to picrotoxin (0.1-100 microM). The single mutant beta 2(T246F), in combination with wild type alpha and gamma subunits, also conferred picrotoxin-insensitivity. In contrast, combinations containing beta 2(T252A) were blocked by picrotoxin with an IC50 of 1.4 +/- 0.4 microM. In some instances, the EC50 to GABA was slightly altered in the mutant receptors; but no change was observed in EC50 or potentiation by the allosteric modulator, alprazolam. The data in this study suggest that picrotoxin's site of action is within the channel pore; however the mechanism by which picrotoxin blocks current remains unknown.
Gurley
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Neurogen Corporation, Branford, CT 06405, USA.
Amin
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Ross
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PC
Weiss
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White
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004351:Drug Resistance / Q000235:genetics; D005260:Female; D058787:GABA-A Receptor Antagonists; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D010852:Picrotoxin / Q000494:pharmacology; D017354:Point Mutation; D011487:Protein Conformation; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry / Q000235:genetics*; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
D058787:GABA-A Receptor Antagonists; D010852:Picrotoxin; D011963:Receptors, GABA-A; D011978:Receptors, Nicotinic