Electrical fingerprintig reveals agonist binding sites required for activation of homo-pentameric cys-loop receptors

BOUZAT CECILIA; RAYES DIEGO; DE ROSA MARÍA JOSÉ; SINE STEVEN

Boston, Massachusetts

Congreso; 53th Biophysical Society Annual Meeting; 2009

Biophysical Society

Electrical Fingerprinting Reveals Agonist Binding Sites Required for Activation of Homo-pentameric Cys-loop Receptors   Cecilia Bouzat, Diego Rayes, María José De Rosa, and Steven M Sine 1Instituto de Investigaciones Bioquimicas, UNS-CONICET, Bahia Blanca, Argentina- 2Receptor Biology Laboratory, Departments of Physiology and Biomedical Engineering and Neurology, Mayo Clinic College of Medicine, Rochester MN, 55905     Ancestral neurotransmitter Cys-loop receptors were homo-pentamers harboring five identical agonist binding sites but most present day receptors are hetero-pentamers with only two binding sites. To understand why Cys-loop receptors evolved to utilize fewer than five binding sites, we disabled different numbers of sites and developed a method to monitor lifetimes of individual active receptors and the corresponding number of functional binding sites.  We find that maximal open-channel lifetime is achieved when the neurotransmitter occupies three non-consecutive binding sites. Occupancy of one site allows receptor activation, although the open state is unstable; occupancy of two non-consecutive sites produces a much longer-lived open state appropriate for efficient activation. However, occupancy of a third site further increases channel lifetime, thus providing optimal stabilization of the active state. Maximal activation of homomeric receptors by agonist occupancy of less than the five potential sites enhances the rate of channel opening and increases agonist sensitivity. The results reveal that allosteric requirements dictated the number and location of the agonist binding sites, and provide an indispensable framework for further progress in drug design.