Pursuing High-Resolution Structures of Nicotinic Acetylcholine Receptors: Lessons Learned from Five Decades.

Since their discovery, nicotinic acetylcholine receptors (nAChRs) have been extensively studied to understand their function, as well as the consequence of alterations leading to disease states. Importantly, these receptors represent pharmacological targets to treat a number of neurological and neurodegenerative disorders. Nevertheless, their therapeutic value has been limited by the absence of high-resolution structures that allow for the design of more specific and effective drugs. This article offers a comprehensive review of five decades of research pursuing high-resolution structures of nAChRs. We provide a historical perspective, from initial structural studies to the most recent X-ray and cryogenic electron microscopy (Cryo-EM) nAChR structures. We also discuss the most relevant structural features that emerged from these studies, as well as perspectives in the field.

Decoding pathogenesis of slow-channel congenital myasthenic syndromes using recombinant expression and mice models.

Despite the fact that they are orphan diseases, congenital myasthenic syndromes (CMS) challenge those who suffer from it by causing fatigable muscle weakness, in the most benign cases, to a progressive wasting of muscles that may sentence patients to a wheelchair or even death. Compared to other more common neurological diseases, CMS are rare. Nevertheless, extensive research in CMS is performed in laboratories such as ours. Among the diverse neuromuscular disorders of CMS, we are focusing in the slow-channel congenital myasthenic syndrome (SCS), which is caused by mutations in genes encoding acetylcholine receptor subunits. The study of SCS has evolved from clinical electrophysiological studies to in vitro expression systems and transgenic mice models. The present review evaluates the methodological approaches that are most commonly employed to assess synaptic impairment in SCS and also provides perspectives for new approaches. Electrophysiological methodologies typically employed by physicians to diagnose patients include electromyography, whereas patient muscle samples are used for intracellular recordings, single-channel recordings and toxin binding experiments. In vitro expression systems allow the study of a particular mutation without the need of patient intervention. Indeed, in vitro expression systems have usually been implicated in the development of therapeutic strategies such as quinidine- and fluoxetine-based treatments and, more recently, RNA interference. A breakthrough in the study of SCS has been the development of transgenic mice bearing the mutations that cause SCS. These transgenic mice models have actually been key in the elucidation of the pathogenesis of the SCS mutations by linking IP-3 receptors to calcium overloading, as well as caspases and calpains to the hallmark of SCS, namely endplate myopathy. Finally, we summarize our experiences with suspected SCS patients from a local perspective and comment on one aspect of the contribution of our group in the study of SCS.

Tryptophan scanning of the acetylcholine receptor's betaM4 transmembrane domain: decoding allosteric linkage at the lipid-protein interface with ion-channel gating.

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel protein that mediates fast excitatory synaptic transmission in the peripheral and central nervous systems. Changes in the structure and function of the AChR can lead to serious impairment of physiological processes. In this study, we combined site-directed mutagenesis, radioligand binding assays, electrophysiological recordings and Fourier analyses to characterize the functional role and structural aspects of the betaM4 transmembrane domain of the Torpedo AChR. We performed tryptophan replacements, from residues L438 through F455, along the betaM4 transmembrane domain. Expression levels of mutants F439W-G450W and F452W-I454W produced peak currents similar to or lower than those in wild-type (WT). Tryptophan substitutions at positions L438 and T451 led to a deficiency in either subunit expression or receptor assembly. Mutations L440W, V442W, C447W and S453W produced a gain-of-function response. Mutation F455W produced a loss of ion channel function. The periodicity profile of the normalized expression level (closed state) and EC(50) (open state) revealed a minor conformational change of 0.4 residues/turn of the betaM4 domain. These findings suggest that a minor movement of the betaM4 domain occurs during channel activation.

Fourier transform coupled to tryptophan-scanning mutagenesis: lessons from its application to the prediction of secondary structure in the acetylcholine receptor lipid-exposed transmembrane domains.

Although Fourier transform (FT) and tryptophan-scanning mutagenesis (TrpScanM) have been extremely useful for predicting secondary structures of membrane proteins, they are deemed to be low-resolution techniques. Herein, we describe the combined use of FT and TrpScanM (FT-TrpScanM) as a more reliable approach for the prediction of secondary structure. Five TrpScanM studies of the acetylcholine receptor lipid-exposed transmembrane domains (LETMDs) were revisited and analyzed by FT-TrpScanM. FT analysis of the raw data from the aforementioned TrpScanM studies supports and validates the conclusions derived from their tryptophan-periodicity profiles. Furthermore, by FT-TrpScanM, we were able to determine the minimum number of consecutive tryptophan substitutions necessary for more robust prediction of alpha-helical secondary structures and evaluate the quality of structure predictions by alpha-helical character curves. Finally, this study encourages future utilization of FT-TrpScanM to more reliably predict secondary structures of the membrane protein LETMDs.

Tryptophan-scanning mutagenesis in the alphaM3 transmembrane domain of the muscle-type acetylcholine receptor. A spring model revealed.

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.