Abstract
The role of the outermost transmembrane α-helix in both the maturation and function of the prokaryotic pentameric ligand-gated ion channels, GLIC and ELIC, was examined by Ala scanning mutagenesis, deletion mutations, and mutant cycle analyses. Ala mutations at the M4-M1/M3 interface lead to loss-of-function phenotypes in GLIC, with the largest negative effects occurring near the M4 C terminus. In particular, two aromatic residues at the M4 C terminus form a network of π-π and/or cation-π interactions with residues on M3 and the β6-β7 loop that is essential for both maturation and function. M4-M1/M3 interactions appear to be optimized in GLIC with even subtle structural changes at this interface leading to detrimental effects. In contrast, mutations along the M4-M1/M3 interface of ELIC typically lead to gain-of-function phenotypes, suggesting that these interactions in ELIC are not optimized for channel function. In addition, no cluster of interacting residues involving the M4 C terminus, M3, and the β6-β7 loop was found, suggesting that the M4 C terminus plays little role in ELIC maturation or function. This study shows that M4 makes distinct contributions to the maturation and gating of these two closely related homologs, suggesting that GLIC and ELIC exhibit divergent features of channel function.
Highlights
The role of the outermost M4 transmembrane ␣-helix in prokaryotic pentameric ligand-gated ion channel function is unknown
Through Ala scanning mutagenesis, M4 deletion mutations, and mutant cycle analyses, we show that the M4-M1/M3 interactions are optimized in GLIC for channel function, whereas in ELIC they are not
We identify a cluster of interacting residues on the M4 C terminus, M3, and the 6-7 loop that is essential for GLIC maturation and function, with no analogous cluster located in ELIC
Summary
The role of the outermost M4 transmembrane ␣-helix in prokaryotic pentameric ligand-gated ion channel (pLGIC) function is unknown. One model proposes that lipids potentiate gating by enhancing M4 interactions with the adjacent ␣-helices, M1 and M3, to promote effective interactions between the M4 C terminus and the Cys-loop [14], a structure at the interface between the extracellular domain (ECD) and TMD that plays a key role in coupling agonist binding to channel gating (26 –29). ELIC has not yet been crystallized in open and closed structures, even though structures of ELIC have been solved in the presence and absence of bound agonist [36, 40] This raises the possibility that crystallized ELIC, with weakened or no interactions between the M4 C terminus and adjacent structures at the ECD/TMD interface, adopts a conformation that does not undergo agonist-induced conformational transitions [37]. Our study shows that M4 contributes differently to the maturation and gating of these two closely related homologs, and suggests that GLIC and ELIC exhibit divergent features of channel function/modulation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
