Intramembrane proton binding site linked to activation of bacterial pentameric ion channel.

摘要: Prokaryotic orthologs of eukaryotic Cys-loop receptor channels recently emerged as structural and mechanistic surrogates to investigate this superfamily intercellular signaling proteins. Here, we examine proton activation the prokaryotic ortholog GLIC using patch clamp electrophysiology, mutagenesis, molecular dynamics (MD) simulations. Whole-cell current recordings from human embryonic kidney (HEK) 293 cells expressing show half-maximal at pH 6, close pKa histidine, implicating three native His residues in sensing linked activation. The mutation H235F abolishes activation, H277Y is without effect, all nine mutations His-127 prevent expression on cell surface. In crystal structure, His-235 transmembrane (TM) α-helix 2, hydrogen bonds main chain carbonyl oxygen Ile-259 TM 3. MD simulations that when protonated, bond persists, channel remains open conformation, whereas deprotonated, dissociates, closes. Mutations proximal Tyr-263, which also links α-helices 2 3 via a bond, alter sensitivity over 1.5 unit range. Tyr-263 bonding capacity His-235. overall findings region novel binding site