Pulsed Electron Paramagnetic Resonance of Spin Labeled Human GABA(A)Rs Functionally Reconstituted in Lipid Bilayers Suggests there is Space for Lipids between the Subunits

Abstract

GABA(A)Rs are members of the Cys-loop superfamily of ion channel receptors. They are pentamers containing up to three different homologous subunits arranged pseudo symmetrically around a central anion–conducting pore. The binding sites of GABA and many drugs are located between specific subunits. Human α1β3 GABA(A)Rs, expressed in HEK cells, were spin labeled during purification in the etomidate binding site on the third transmembrane helix of the β3-subunit (β3 M3 M286C) and functionally reconstituted into detergent–free asolectin bilayers. The separation between the tethered nitroxides, measured by double electron electron resonance (DEER), is related to the intersubunit separation and the local conformation of the spin label. In the desensitized state, two pairs of nitroxide separations were observed. Each pair contains a nearest and a next nearest neighbor subunit separation that obeys pentameric symmetry constraints. Rotamer analysis was performed on homology models based on published crystal structures of several homologous anion–conducting, Cys-loop homo-pentamers. These predicted well the longer of the two pentameric pairs, but not the shorter. However, both pairs of separations can be accommodated by models, in which the subunits are systematically moved radially away from the ion pore, so increasing the subunit spacing (side of the pentamer). An increase of 0.75 to 1 A is optimal. This analysis suggested that one pair of pentamerically–related nitroxide separations corresponds to a population of spin labels in the lipid-protein interface and the other to that between the subunits. The optimal increase in subunit spacing is the minimum needed to dock lipids into the subunit interface, suggesting that this might occur in the bilayer state but not in the crystal structures. Supported by GM58448 and GM35215.