Structure of the Pore-forming Transmembrane Domain of a Ligand-gated Ion Channel

Abstract

The structure of the pore-forming transmembrane domain of the nicotinic acetylcholine receptor from Torpedo has been investigated by infrared spectroscopy. Treatment of affinity-purified receptor with either Pronase or proteinase K digests the extramembranous domains (roughly 75% of the protein mass), leaving hydrophobic membrane-imbedded peptides 3-6 kDa in size that are resistant to peptide 1H/2H exchange. Infrared spectra of the transmembrane domain preparations exhibit relatively sharp and symmetric amide I and amide II band contours centered near 1655 and 1545 cm-1 respectively, in both 1H2O and 2H2O. The amide I band is very similar to the amide I bands observed in the spectra of α-helical proteins, such as myoglobin and bacteriorhodopsin, that lack β structure and exhibit much less β-sheet character than is observed in proteins with as little as 20% β sheet. Curve-fitting estimates 75-80% α-helical character, with the remaining peptides likely adopting extended and/or turn structures at the bilayer surface. Infrared dichroism spectra are consistent with transmembrane α-helices oriented perpendicular to the bilayer surface. The evidence strongly suggests that the transmembrane domain of the nicotinic receptor, the most intensively studied ligand-gated ion channel, is composed of five bundles of four transmembrane α-helices.