Evidence that the TM1-TM2 Loop Contributes to the rho 1 GABA Receptor Pore

Considerable evidence indicates the second transmembrane domain (TM2) of the gamma -aminobutyric acid (GABA) receptor lines the integral ion pore. To further delineate the structures that constitute the ion pore and selectivity filter of the rho 1 GABA receptor, we used the substituted cysteine accessibility method with charged reagents to identify anion- and cation-accessible surfaces. Twenty-one consecutive residues were mutated to cysteine, one at a time, in the presumed intracellular end of the first transmembrane domain (TM1; Ala super(271)-Met super(276)), the entire linker connecting TM1 to TM2 (Leu super(277)-Arg super(287)), and the presumed intracellular end of TM2 (Ala super(288)-Ala super(291)). Positively (MTSEA super(+)) and negatively (pCMBS super(-)) charged sulfhydryl reagents, as well as Cd super(2+), were added extracellularly to test accessibility of the engineered cysteines. Four of the mutants, all at the intracellular end of TM2 (R287C, V289C, P290C, A291C), were accessible to positively charged reagents, whereas seven mutants (A271C, T272C, L277C, W279C, V280C, P290C, A291C) were functionally modified by negatively charged pCMBS super(-). These seven modified residues were at the intracellular end of TM2, in the TM1-TM2 linker, and at the intracellular end of TM1. In nearly all cases (excluding P290C), the rate and the degree of modification were state-dependent, with greater accessibility in the presence of agonist. Select cysteine mutants were combined with a point mutation (A291E) that converted the pore from chloride-to non-selective. In this case, positively charged reagents could modify residues in the TM1-TM2 linker (Leu super(277) and Val super(280)), supporting the notion that the modifying reagents were reaching their target through the pore. Taken together, our results suggest that, up to its intracellular end, the TM2 domain is not charge selective. In addition, we propose that the TM1-TM2 linker and the intracellular end of TM1 are along the pathway of the permeating ion. These findings may lend new insights into the structure of the GABA receptor pore.