Single-Chennel kinetic analysis for activation and desensitization of homomeric 5-HT3A receptors.

JEREMÍAS CORRADI; FERNANDA GUMILAR; CECILIA BOUZAT

Bahía Blanca

Workshop; Workshop: Neuronal Communication: From Structure to physiology; 2008

Sociedad Argentina de Investigación en Neurociéncias

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 the mutations in the 5-HT3A subunit to obtain a high-conductance form so that sngle-channel currents can be detected. At all 5-HT concentrations (> 0.1 microM) channel activity appear as opening events in quick succession forming burst, which, in turn, coalesce into clusters. By combining single-channel and macroscopic data we fenerated a detailed kinetic model that perfectly describes activation, deactivation and desensitization. The model shows that full activarion arises from receptors with three molecules of agonist bound. It also reveals an earlier conformational change of th fully-liganded receptor (flipping) thar occurs while the channel is still closed. From this pre-open states, which conforms the cluster whose duration parallels the time constant of desensitizarion. This suggests that at a synapse the lifetime of the elementary response of 5-HT3A receptors is determined mainly by by desensitization. Since the desensitized state is a stable state, the ineter-response latency is expected to be prolonged. The present kinetic model provides a foundation for studyung molecular mechanisms of drug action. We show that mutations at caline 10´ of M4 affect opening and closing rates whithin 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.