Contribution of agonist binding sites and coupling regions to activation and desensitization of homomeric cys-loop receptors

CECILIA BOUZAT; NATALIA ANDERSEN; JEREMÍAS CORRADI; STEVEN M SINE

San Diego, California

Congreso; 56th Annual Meeting of the Biophysical Society; 2012

Biophysical Society

Homomeric receptors are the simplest structural class of receptors of the Cys-loop superfamily and are therefore invaluable models for probing fundamental relationships between structure and function. To determine how many of the five agonist binding sites are required to be occupied by the agonist for maximal open channel stability we applied an electrical fingerprinting strategy in the homomeric receptor model, a7-5HT3A. To vary the number of functional agonist binding sites we installed mutations that prevent agonist binding, and to report the presence of the mutant subunit, we installed mutations that alter single-channel conductance. We find that receptors can be activated by occupancy of only one agonist binding site but open channel lifetime is brief, and occupancy of three non-consecutive sites is required for maximal open channel lifetime. The conformational changes initiated at the binding site are propagated to the gate through the extracellular-transmembrane interface, known as coupling region. We show that structural differences in the coupling region of homomeric a7 and 5-HT3A receptors account for the large differences in open channel lifetime and rate of desensitization between these homomeric members of the superfamily. By applying the electrical fingerprinting strategy, we determine that each coupling region in the pentamer contributes an equal increment to the stability of the open channel. We also determine minimal requirements for channel opening regardless of stability of the open state, and find that receptors can open with one functional binding site and two contiguous and functional coupling regions, or with five functional binding sites and only one functional coupling region. The overall findings show that whereas the agonist binding sites contribute inter-dependently and asymmetrically to open channel stability, the coupling regions contribute independently and symmetrically.