Relative impact of residues at the intracellular and extracellular ends of the human GABAC ρ1 receptor M2 domain on picrotoxinin activity

The relative impact on picrotoxinin activity of residues at the intracellular (2′ and 6′ residues) and extracellular (15′ and 17′ residues) ends of the second transmembrane (M2) domain of the human γ-aminobutyric acid-C (GABAC) ρ1 receptor was investigated. A series of GABAC ρ1 subunits were produced containing either single or multiple mutations at the positions of interest. Wild-type and mutant subunits (containing one or more of the following mutations: P2′S, T6′M, I15′N, G17′H) were expressed in Xenopus oocytes and characterized using agonists, partial agonists and antagonists. Changes in agonist activity were observed for mutant receptors. Most notably, mutation at the 2′ position resulted in decreased agonist potency, while mutation at the 15′ and 17′ residues increased agonist potency. The affinity of the competitive antagonist (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid (TPMPA) was unchanged compared to wild-type at all mutant receptors. Of the four residues studied, mutation of residues at the 2′ and 6′ positions had the greatest impact on picrotoxinin activity. Inclusion of the P2′S mutation typically produced receptors with increased picrotoxinin potency, while the T6′M mutation reduced picrotoxinin potency. Picrotoxinin is a mixed antagonist at wild-type and all mutant receptors, with the exception of the double mutant ρ1P2′S/T6′M receptors at which the non-competitive component was isolated. It is proposed that the contribution of M2 domain residues to picrotoxinin activity is potentially two-fold: (1) their role as a potential picrotoxinin binding site within the pore; and (2) they are critical for receptor activation properties of the receptor, thus may alter the allosteric mechanism of picrotoxinin.