Prokaryotic Cys-Loop Receptor Homologs as Mechanistic Models for Channel Function

Abstract

Pentameric ligand-gated ion channels are a large class of proteins involved in electrochemical signal transduction. Signal transduction is a result of two processes: (1) ligand binding to the receptor and (2) a conformational wave that opens a pore far from the binding site (>20Å). Recently, two prokaryotic members of this family were identified, one from Gloeobacter violaceus (GLIC), and one from Erwinia chrysanthemi (ELIC). These receptors provide a valuable platform for the study of ligand binding and channel gating in the Cys-loop receptor family. Receptors were expressed heterologously in Xenopus laevis oocytes, and whole-cell voltage-clamp electrophysiology was used as a reporter for ligand binding and channel gating. To examine the proton-binding event in GLIC, unnatural amino acids were incorporated via nonsense-suppression with chemically acylated tRNA. This approach provides a subtle probe of intrasubunit interactions with the highly sensitive H11' site, which has been previously identified as necessary for the proton sensitivity of GLIC. A unique pair of proline residues at the extracellular terminus of the M1 helix were also investigated to examine potential intersubunit interactions that may play a role in channel gating. Despite the availability of putative "open" and "closed" X-ray structures of GLIC and ELIC, the mechanism of gating is not yet clear. Gating is a dynamic process initiated with ligand binding and resulting in conformational changes and pore opening tens of angstroms away. To fully understand this process will require a dynamic picture of the events that occur during the gating of ion channels, which we ultimately aim to uncover using time-resolved fluorescence energy transfer. Progress towards the development of temporal control for triggering channel opening/closing will be discussed.

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