Abstract
Membrane proteins constitute a large fraction of all proteins, yet very little is known about their structure and conformational transitions. A fundamental question that remains obscure is how protein domains that are in direct contact with the membrane lipids move during the conformational change of the membrane protein. Important structural and functional information of several lipid-exposed transmembrane domains of the acetylcholine receptor (AChR) and other ion channel membrane proteins have been provided by the tryptophan-scanning mutagenesis. Here, we use the tryptophan-scanning mutagenesis to monitor the conformational change of the alphaM3 domain of the muscle-type AChR. The perturbation produced by the systematic tryptophan substitution along the alphaM3 domain were characterized through two-electrode voltage clamp and 125I-labeled alpha-bungarotoxin binding. The periodicity profiles of the changes in AChR expression (closed state) and ACh EC50 (open-channel state) disclose two different helical structures; a thinner-elongated helix for the closed state and a thicker-shrunken helix for the open-channel state. The existence of two different helical structures suggest that the conformational transition of the alphaM3 domain between both states resembles a spring motion and reveals that the lipid-AChR interface plays a key role in the propagation of the conformational wave evoked by agonist binding. In addition, the present study also provides evidence about functional and structural differences between the alphaM3 domains of the Torpedo and muscle-type receptors AChR.
Highlights
The acetylcholine receptor (AChR)2 belongs to the superfamily of the ligand-gated ion channel receptors that are of fundamental importance in the chemical synaptic transmission throughout the nervous system [1,2,3,4]
The other four mutations displayed an apparent reduction in AChR expression levels (i.e. F280W, V285W, S288W, and T292W), a detailed statistical analysis shown in Table 1 suggests that the expression of these mutants is not significantly different from wild type (WT)
The other three mutations (I286W, I289W, and I291W) showed 200 – 400% higher AChR expression levels compared with WT, suggesting a facilitation of oligomerization and/or assembly induced by these mutations
Summary
Important structural and functional information of several lipid-exposed transmembrane domains of the acetylcholine receptor (AChR) and other ion channel membrane proteins have been provided by the tryptophan-scanning mutagenesis. In the present study we used the tryptophan-scanning mutagenesis (TrpScanM) approach to monitor the conformational changes experienced by the muscle-type AChR ␣M3 domain (Fig. 1A) This approach has been used successfully for inward rectifier potassium channels [41,42,43], voltage-activated potassium channels (44 –50), nicotinic AChR channels [25, 32, 34, 35], glutamate receptor channels [51], ␥-aminobutyric acid type A receptor channels [52, 53], voltage-gated sodium channels [54], N-methyl-D-aspartate receptor channels [55], P2X4 receptor channels [56], mechanosensitive channels MscL [57], human. The comparison of the TrpScanM data between the ␣M3 domain of the muscle-type and Torpedo AChRs reveals localized conformational differences that could contribute to the functional differences between these receptors
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
