Tryptophan-scanning Mutagenesis in the αM3 Transmembrane Domain of the Muscle-type Acetylcholine Receptor

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

“…Thus, the AChR serves as an excellent model to test the validity of the FT-TrpScanM approach and provides reassurance on its capacity to generate trustworthy predictions of the secondary structures of membrane protein LETMDs in general. Furthermore, FT-TrpScanM supports and validates the conclusion from previous TrpScanM [1][2][3][4][5] and biochemical [28][29][30] studies, which predicted that the LETMDs are arranged as α-helical structures. In addition, using this approach, we were able to provide additional structural information, estimate mean periodicities, determine the minimum number of consecutive tryptophan substitutions required to reliably predict α-helical structures, and assess the quality of structure predictions.…”

“…It is noteworthy that we took advantage of the recently reported structure of the Torpedo AChR, which was determined at 4 Å resolution using cryo-electron microscopy in order to corroborate our predictions [26]. Interestingly, the TrpScanM predicted α-helical structures for the LETMDs [1][2][3][4][5] even when a lower-resolution cryoelectron microscopy structure mistakenly predicted the LETMDs to be arranged as β-sheets [27]. Thus, the AChR serves as an excellent model to test the validity of the FT-TrpScanM approach and provides reassurance on its capacity to generate trustworthy predictions of the secondary structures of membrane protein LETMDs in general.…”

“…In this scenario, the tryptophan-scanning mutagenesis (TrpScanM) has emerged as another option for predicting the secondary structure and packing arrangement of the lipid-exposed transmembrane domains (LETMDs), which can then be used to model their overall spatial orientation [1]. Although TrpScanM has been successfully applied to several ion-channel proteins such as nicotinic AChR channels [1][2][3][4][5], inward rectifier potassium channels [6][7][8], voltage-activated potassium channels [9][10][11][12][13][14][15], glutamate receptor channels [16], γ-aminobutyric acid type A receptor channels [17,18], voltage-gated sodium channels [19], N-methyl-D-aspartate receptor channels [20], P2X 4 receptor channels [21] mechanosensitive channels MscL [22,23], human ether-a-go-go-related gene (HERG) K + channels [24] and epithelial Na + channels [25], the TrpScanM is, nevertheless, deemed a low-resolution method for predicting secondary structure and packing arrangement because it does not provide direct structural information. The rationale for choosing TrpScanM is that tryptophan systematic substitutions in the transmembrane domain of the ion-channel protein should result in a loss of function and/or functional expression when facing the interior of the protein.…”

Molecular Determinants of Ivermectin Sensitivity at the Glycine Receptor Chloride Channel

“…Tryptophan substitution has been employed at GABA A Rs to elucidate transmembrane domain binding sites for alcohols and anesthetics (30,31), at nAChRs to define transmembrane helical structure and dynamics (32,33) and at P 2 X receptors to investigate movements induced by ivermectin (34). Glycine dose-response relationships were quantitated for all these mutant GlyRs to establish whether the substitutions were well tolerated by the receptor.…”

Potential role of caveolin-1-positive domains in the regulation of the acetylcholine receptor's activable pool: Implications in the pathogenesis of a novel congenital myasthenic syndrome

“…39,[46][47][48] The normalized maximum response corresponds to the maximum current (I max ) divided by the total number of receptors as determined by 125 I-labaled α-BTX binding assays. Therefore, if only a fraction of the total number of receptors contributes to the activatable pool, then I max will be lower than expected, resulting in a decreased normalized maximum response.…”