The net orientation of nicotinic receptor transmembrane alpha-helices in the resting and desensitized states

Abstract

The net orientation of nicotinic acetylcholine receptor transmembrane alpha-helices has been probed in both the activatable resting and nonactivatable desensitized states using linear dichroism Fourier-transform infrared spectroscopy. Infrared spectra recorded from reconstituted nicotinic acetylcholine receptor membranes after 72 h exposure to (2)H2O exhibit an intense amide I component band near 1655 cm(-1) that is due predominantly to hydrogen-exchange-resistant transmembrane peptides in an alpha-helical conformation. The measured dichroism of this band is 2.37, suggesting a net tilt of the transmembrane alpha-helices of roughly 40 degrees from the bilayer normal, although this value overestimates the tilt angle because the measured dichroism at 1655 cm(-1) also reflects the dichroism of overlapping amide I component bands. Significantly, no change in the net orientation of the transmembrane alpha-helices is observed upon agonist binding. In fact, the main changes in structure and orientation detected upon desensitization involve highly solvent accessible regions of the polypeptide backbone. Our data are consistent with a capping of the ligand binding site by the solvent accessible C-loop with little change in the structure of the transmembrane domain in the desensitized state. Changes in structure at the interface between the ligand-binding and transmembrane domains may uncouple binding from gating.

FIGURE 1

A comparison of polarized FTIR spectra of PC/PA/Chol 3:1:1 membranes collected at −5°C (top traces) and at 22.5°C (middle traces), as well as spectra of the reconstituted nAChR in PC/PA/Chol 3:1:1 membranes at 22.5°C (bottom traces). FTIR spectra of the membranes were recorded with either perpendicular polarized light (dashed lines) or parallel polarized light (solid lines). The spectra collected with perpendicular polarized light were multiplied by a factor of two to account for differences in the evanescent field strength (R_ISO). (A) Acyl chain C-H stretching bands. (B) Lipid ester carbonyl stretching band. (C) Deconvolved lipid ester carbonyl stretching band. Spectra were deconvolved between 1800 and 1600 cm⁻¹ using gamma factor 10 and smoothing 80%.

FIGURE 2

The dependence of the PC/PA/Chol 3:1:1 membrane acyl chain C-H symmetric stretching vibration dichroic ratio (A) and order parameter (B) on temperature.

FIGURE 3

Polarized FTIR spectra of reconstituted nAChR PC/PA/Chol 3:1:1 membrane films. FTIR spectra collected at 22.5°C with perpendicular polarized light (dashed lines) and parallel polarized light (solid lines) are illustrated. The FTIR spectra recorded with perpendicular polarized light were multiplied by a factor of 2 to account for differences in the evanescent field strength (R_ISO).

FIGURE 4

Deconvolved polarized FTIR spectra of reconstituted nAChR PC/PA/Chol 3:1:1 membrane films recorded in the absence (A) and presence (B) of 1 mM Carb. Deconvolved FTIR spectra collected with perpendicular polarized light (dashed lines) were multiplied by a factor of two (R_ISO) to account for differences in the evanescent field strength. FTIR spectra collected with parallel polarized light are illustrated by the solid lines. All four spectra were recorded sequentially from the same nAChR film.

FIGURE 5

Carb-difference spectra recorded from the nAChR in PC/PA/Chol 3:1:1 membranes. (A) Carb-difference spectra recorded with unpolarized infrared light. The top trace was recorded in ¹H₂O buffer, whereas the middle trace was recorded in ²H₂O buffer. The bottom trace is a control showing the difference between spectra both recorded in the absence of Carb. (B) Carb-difference spectra recorded with parallel (top trace and solid line in bottom traces) and perpendicular (middle trace and dashed line in bottom traces) polarized infrared light. The perpendicular polarized difference spectrum has been scaled to account for the difference in evanescent field strength (R_ISO).