Insights into Gating Motions of GLIC via Perturbation of Critical Prolines with Non-Canonical Amino Acid Probes

Abstract

Pentameric ligand-gated ion channels (pLGICs) are membrane proteins involved in fast synaptic transmission, and have been implicated in a number of neurodegenerative and psychological diseases in humans. These proteins have been the focus of extensive biochemical investigation, but high-resolution structures of mammalian receptors have only recently become available. The bacterial pLGIC Gloeobacter violaceus ligand-gated ion channel (GLIC), whose structure was first published in 2008, has served as a valuable model. There are now over 40 published structures obtained under various conditions, and these suggest specific conformational transitions that may occur during channel activation. If these gating motions are conserved throughout the pLGIC family, a detailed understanding of the process holds promise for the development of strategies to tune receptor activity. Here we discuss recent work in which we explore the role of prolines in the gating transitions of GLIC. Receptors were expressed in Xenopus oocytes, and examined using whole-cell electrophysiology. We first probed the importance of each proline residue in the receptor with an alanine scan. Pro119 in the Cys-loop, Pro198 and Pro203 in the M1 helix, and Pro299 in the M4 helix were sensitive to substitution. At these positions, we then used in vivo non-canonical amino acid mutagenesis with a range of proline analogs to determine specific structural requirements. The data revealed unique requirements at each position, and show that Pro119, Pro203 and Pro299 obey previously observed phenotypes for cis-preferring, bulge-inducing, and kink-inducing prolines respectively. However, an unusual phenotype was displayed by Pro198, a non-conserved site, and possible interpretations will be discussed.

Additional details