Non-canonical proline analogs reveal mechanistic trends among GLIC and other ligand-gated ion channels

Abstract

A no. of characteristics set proline apart from the other natural amino acids, including its increased propensity to exist in a cis-amide conformation, its lack of a backbone hydrogen bond-donor, and its unusual cyclic side chain. Because of this uniqueness, proline sites in proteins are often highly conserved and crit. for maintaining proper structure and function. We have previously evaluated prolines from several membrane proteins, including those that induce helical kinks or bulges, and those that undergo cis-trans isomerization to allow global conformational transitions. Non-canonical amino acid mutagenesis has been an indispensible tool for identifying the features of each proline that contribute to overall protein function, thereby enabling a detailed chem.-scale description of biochem. processes. Here, we dissect the importance of essential prolines in the bacterial receptor Gloeobacter violaceus ligand-gated ion channel (GLIC), an important functional model for the eukaryotic Cys-loop receptor class of ligand-gated ion channels. The first high-resoln. structures of mammalian receptors have only recently become available, while there are now over 40 published structures of GLIC. These structures suggest specific conformational transitions that may occur during channel activation. If these gating motions are conserved throughout the family, a detailed understanding of the process holds promise for the development of strategies to tune receptor activity. Receptors were expressed in Xenopus oocytes, and examd. using whole-cell electrophysiol. 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 det. specific structural requirements. The data revealed unique requirements at each position, and show that Pro119, Pro203 and Pro299 obey previously obsd. phenotypes for cis-preferring, bulge-inducing, and kink-inducing prolines, resp. However, an unusual phenotype was displayed by Pro198, a non-conserved site, and possible interpretations will be discussed.