Ligand- and Subunit-specific Conformational Changes in the Ligand-binding Domain and the TM2-TM3 Linker of α1 β2 γ2 GABAA Receptors

Cys-loop receptor ligand binding sites are located at subunit interfaces where they are lined by loops A-C from one subunit and loops D-F from the adjacent subunit. Agonist binding induces large conformational changes in loops C and F. However, it is controversial as to whether these conformational changes are essential for gating. Here we used voltage clamp fluorometry to investigate the roles of loops C and F in gating the alpha 1 beta 2 gamma 2 GABA(A) receptor. Voltage clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. Previous attempts to define the roles of loops C and F using this technique have focused on homomeric Cys-loop receptors. However, the problem with studying homomeric receptors is that it is difficult to eliminate the possibility of bound ligands interacting directly with attached fluorophores at the same site. Here we show that ligands binding to the beta 2-alpha 1 interface GABA binding site produce conformational changes at the adjacent subunit interface. This is most likely due to agonist-induced loop C closure directly altering loop F conformation at the adjacent alpha 1-beta 2 subunit interface. However, as antagonists and agonists produce identical alpha 1 subunit loop F conformational changes, these conformational changes appear unimportant for gating. Finally, we demonstrate that TM2-TM3 loops from adjacent beta 2 subunits in alpha 1 beta 2 receptors can dimerize via K24'C disulfides in the closed state. This result implies unexpected conformational mobility in this crucial part of the gating machinery. Together, this information provides new insights into the activation mechanisms of Cys-loop receptors.