Lateral diffusion, function, and expression of the slow channel congenital myasthenia syndrome αC418W nicotinic receptor mutation with changes in lipid raft components.

Lipid rafts, specialized membrane microdomains in the plasma membrane rich in cholesterol and sphingolipids, are hot spots for a number of important cellular processes. The novel nicotinic acetylcholine receptor (nAChR) mutation αC418W, the first lipid-exposed mutation identified in a patient that causes slow channel congenital myasthenia syndrome was shown to be cholesterol-sensitive and to accumulate in microdomains rich in the membrane raft marker protein caveolin-1. The objective of this study is to gain insight into the mechanism by which lateral segregation into specialized raft membrane microdomains regulates the activable pool of nAChRs. We performed fluorescent recovery after photobleaching (FRAP), quantitative RT-PCR, and whole cell patch clamp recordings of GFP-encoding Mus musculus nAChRs transfected into HEK 293 cells to assess the role of cholesterol and caveolin-1 (CAV-1) in the diffusion, expression, and functionality of the nAChR (WT and αC418W). Our findings support the hypothesis that a cholesterol-sensitive nAChR might reside in specialized membrane microdomains that upon cholesterol depletion become disrupted and release the cholesterol-sensitive nAChRs to the pool of activable receptors. In addition, our results in HEK 293 cells show an interdependence between CAV-1 and αC418W that could confer end plates rich in αC418W nAChRs to a susceptibility to changes in cholesterol levels that could cause adverse drug reactions to cholesterol-lowering drugs such as statins. The current work suggests that the interplay between cholesterol and CAV-1 provides the molecular basis for modulating the function and dynamics of the cholesterol-sensitive αC418W nAChR.

Tryptophan scanning mutagenesis reveals distortions in the helical structure of the δM4 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor.

The lipid-protein interface is an important domain of the nicotinic acetylcholine receptor (nAChR) that has recently garnered increased relevance. Several studies have made significant advances toward determining the structure and dynamics of the lipid-exposed domains of the nAChR. However, there is still a need to gain insight into the mechanism by which lipid-protein interactions regulate the function and conformational transitions of the nAChR. In this study, we extended the tryptophan scanning mutagenesis (TrpScanM) approach to dissect secondary structure and monitor the conformational changes experienced by the δM4 transmembrane domain (TMD) of the Torpedo californica nAChR, and to identify which positions on this domain are potentially linked to the regulation of ion channel kinetics. The difference in oscillation patterns between the closed- and open-channel states suggests a substantial conformational change along this domain as a consequence of channel activation. Furthermore, TrpScanM revealed distortions along the helical structure of this TMD that are not present on current models of the nAChR. Our results show that a Thr-Pro motif at positions 462-463 markedly bends the helical structure of the TMD, consistent with the recent crystallographic structure of the GluCl Cys-loop receptor which reveals a highly bent TMD4 in each subunit. This Thr-Pro motif acts as a molecular hinge that delineates two gating blocks in the δM4 TMD. These results suggest a model in which a hinge-bending motion that tilts the helical structure is combined with a spring-like motion during transition between the closed- and open-channel states of the δM4 TMD.

Fourier transform coupled tryptophan scanning mutagenesis identifies a bending point on the lipid-exposed δM3 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor (nAChR) is a member of a family of ligand-gated ion channels that mediate diverse physiological functions, including fast synaptic transmission along the peripheral and central nervous systems. Several studies have made significant advances toward determining the structure and dynamics of the lipid-exposed domains of the nAChR. However, a high-resolution atomic structure of the nAChR still remains elusive. In this study, we extended the Fourier transform coupled tryptophan scanning mutagenesis (FT-TrpScanM) approach to gain insight into the secondary structure of the δM3 transmembrane domain of the Torpedo californica nAChR, to monitor conformational changes experienced by this domain during channel gating, and to identify which lipid-exposed positions are linked to the regulation of ion channel kinetics. The perturbations produced by periodic tryptophan substitutions along the δM3 transmembrane domain were characterized by two-electrode voltage clamp and (125)I-labeled α-bungarotoxin binding assays. The periodicity profiles and Fourier transform spectra of this domain revealed similar helical structures for the closed- and open-channel states. However, changes in the oscillation patterns observed between positions Val-299 and Val-304 during transition between the closed- and open-channel states can be explained by the structural effects caused by the presence of a bending point introduced by a Thr-Gly motif at positions 300-301. The changes in periodicity and localization of residues between the closed-and open-channel states could indicate a structural transition between helix types in this segment of the domain. Overall, the data further demonstrate a functional link between the lipid-exposed transmembrane domain and the nAChR gating machinery.