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