Single-Channel Kinetic Analysis for Activation and Desensitization of Homomeric 5-HT3A Receptors

CORRADI JEREMÍAS; GUMILAR FERNANDA; BOUZAT CECILIA

Boston, Massachusetts

Congreso; 53th Biophysical Society Annual Meeting; 2009

Biophysical Society

Single-Channel Kinetic Analysis for Activation and Desensitization of Homomeric 5-HT3A Receptors Jeremías Corradi, Fernanda Gumilar, and Cecilia Bouzat. Instituto de Investigaciones Bioquímicas de Bahía Blanca, Argentina.   The 5-HT3A receptor is a member of the Cys-loop family of ligand-gated ion channels. Due to its low conductance, kinetic analysis of this receptor has been restricted to the macroscopic level. We introduced mutations in the 5-HT3A subunit to obtain a high-conductance form so that single-channel currents can be detected. At all 5-HT concentrations (>0.1 µM) channel activity appears as opening events in quick succession forming bursts, which, in turn, coalesce into clusters. By combining single-channel and macroscopic data we generated a detailed kinetic model that perfectly describes activation, deactivation and desensitization. The model shows that full activation arises from receptors with three molecules of agonist bound. It also reveals an earlier conformational change of the fully-liganded receptor (flipping) that occurs while the channel is still closed. From this pre-open state the receptor enters into an open-closed cycle involving three open states, which conforms the cluster whose duration parallels the time constant of desensitization. This suggests that at a synapse the lifetime of the elementary response of 5-HT3A receptors is determined mainly by desensitization. Since the desensitized state is a stable state, the inter-response latency is expected to be prolonged. The present kinetic model provides a foundation for studying molecular mechanisms of drug action. We show that mutations at valine 10′ of M4 affect opening and closing rates within the open-closed cycle. This reveals that the outermost transmembrane domain is important for appropriate gating and shows a high conservation of M4 function among members of this superfamily.