Spontaneous Mobility of GABAA Receptor M2 Extracellular Half Relative to Noncompetitive Antagonist Action

Abstract

The ␥-aminobutyric acid type A receptor ␤ 3 homopentamer is spontaneously open and highly sensitive to many noncompetitive antagonists(NCAs)andZn Ј to Glu 20 Ј) has a proposed NCA interaction site at S17ЈC in an ␣ 1 subunit (6) and a Zn 2ϩ site at His 17 Ј and Glu 20 Ј in the ␤ 3 subunit (5). Disulfide trapping experiments with heteromeric receptors (␣ 1 ␤ 1 or ␣ 1 ␤ 1 ␥ 2 ) have helped to define the structure and mobility of the extracellular half of M2. The following cases of spontaneous or induced disulfide bond formation have been observed (in M2 unless stated otherwise): ␣ 1 12ЈC with several Cys mutants in the intrasubunit M3 (7); ␥ 2 14ЈC with both ␣ 1 15ЈC and ␣ 1 16ЈC and also ␥ 2 13ЈC and ␣ 1 13ЈC (8); ␣-␤ or ␤-␤ spontaneous disulfide cross-linking at 17Ј and 20Ј (9); nonadjacent ␤-␤ disulfide bridging by ␤ 1 20ЈC (10). These relationships indicate that the M2 segments can undergo significant rotational and translational movement.The ␤ 3 homopentamer is ideal for defining the open state channel structure relative to NCA action because it is self-assembling, spontaneously open, and symmetrical with high sensitivities to NCAs and Zn 2ϩ (3,11,12). To further understand M2 structure and NCA action, here we have examined the role of the extracellular half of the M2 segments in NCA and Zn 2ϩ binding involving site-directed mutagenesis with Cys, Ser, and Ala replacements in M2 and M3 using two NCA radioligands. The structural aspects were studied with SCAM (substituted Cys accessibility method) and disulfide trapping (3, 13). This investigation establishes the conformational flexibility of the extracellular half of M2 segments in the ␤ 3 homopentamer and suggests that NCAs access their binding sites both directly, through the channel pore, and indirectly, through the interface of adjacent subunits. The relationships considered lead to a model of the ␤ 3 homopentameric GABA A receptor M2 segments relative to NCA action (shown in Fig. 1). 3 Positions in the M2 segment are identified by a system of index numbers in which the absolutely conserved basic residue at the N-terminal end of M2 is numbered 0Ј, i.e. GABA A R ␤ 3 Arg 250 . Residues toward the C terminus are numbered 1Ј, 2Ј, etc., and toward the N terminus are numbered Ϫ1Ј, Ϫ2Ј, etc. EXPERIMENTAL PROCEDURES Mutagenesis

Ginkgolide X Is a Potent Antagonist of Anionic Cys-loop Receptors with a Unique Selectivity Profile at Glycine Receptors

“…A likely explanation for this observation is that the mutations have rendered the receptors constitutively active or hypersensitive to trace concentrations of glycine in the assay buffer. This hypothesis is supported by numerous previous studies demonstrating the importance of the 10Ј and 13Ј M2 residues for proper gating of both cationic and anionic Cys-loop receptors (52)(53)(54)(55)(56)(57)(58)(59). The compositions of the 10Ј and/or 13Ј M2 rings in homomeric ␣1…”

Genetic Polymorphisms in Organic Cation Transporter 1 (OCT1) in Chinese and Japanese Populations Exhibit Altered Function

“…Flow cytometric analysis was used to determine surface expression measured as membrane immunofluorescence using GFP chimeras of OCT1 and its variants (Chen et al, 2006). Stably transfected HEK293 cells were harvested and washed with PBS.…”

GABAA receptor open-state conformation determines non-competitive antagonist binding

“…The β subunit is essential for NCA sensitivity in the recombinant multiple-subunit receptors (Ratra et al, 2001). The β 3 is the only single subunit highly sensitive to NCA binding at current knowledge (Chen et al, 2006a, b; Ratra et al, 2001). It is surprising that β1 and β2 are insensitive to NCAs (Ratra and Casida, 2001; Ratra et al, 2001).…”

“…The β3 subunit assembles to form homomeric surface receptors in somatic cells, but human β1 subunits do not (Taylor et al, 1999). NCA binding studies have focused on TM2 of the GABA A R (Buhr et al, 2001; Chen et al, 2006a, b; Dibas et al, 2002; Jursky et al, 2000; Perret et al, 1999). There are high homologies between β subunits, particularly in TM2.…”

“…The β3 and β2 subunits contain N15', while the β1 contains S15'. This residue faces away from the ion channel pore and into a water-filled cavity that appears capable of accommodating drugs (Chen et al, 2006b, Miller and Smart 2010). Mutation at 15’ of the β subunit is known to affect anesthetics and alcohol action to potentiate GABA A receptor-mediated electrical responses (Belelli et al, 1997; Hemmings et al, 2005; Jurd et al, 2002).…”