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Research has been conducted on the tryptophan substitutions at the M4 domain of Torpedo californica. The functional consequences on the channel gating have been examined.

Fifteen mutants were generated with tryptophan substitutions within the betaTM3 domain, between residues R282W and 1296W. None of these tryptophan mutants produced peak currents larger than that of wild type. Five of the mutants, L284W, F286W, 1287W, V295W, and 1296W were expressed at levels greater than 15 percent of the wild type.

Alpha-TM3 domain of the muscle-type AChR is studied in detail by examining functional consequences of single tryptophan substitutions at five conserved positions. The results indicated that tryptophan substitution directly affects ion-channel gating, primarily the channel closing rate.

The functional and structural implications of tryptophan scanning mutagenesis in the alpha M3 transmembrane domain of the Torpedo nicotinic acetylcholine receptor (AChR) are discussed. The results suggests that alpha M3 contributes to the gating machinery of the nocotinic AChR receptor and that alpha M3 is comprised of a mixture of two types of helical structures.

Thyroid hormones - particularly triiodothyronine, T3 - play a critical role in the morphological transformations comprising metamorphosis in larval bullfrogs (Rana catesbeiana). Traditional staging criteria for anuran larvae incompletely distinguish physiological and behavioral changes during growth. We therefore first developed a new parameter to describe larval growth, the developmental index (DI), which is simply the ratio between the tail length of the larva and its head diameter. Using the DI we were able to identify two distinct populations classifying the larvae during growth along a continuous linear scale with a cutoff value of DI at 2.8. Classification based on the DI, used in this study, proved an effective complement to existing classifications based on developmental staging into pre- or pro-metamorphic stages. Exposure to T3 in the water induced a rapid (beginning within 5 min) and significant decrease (~20-40%) in locomotor activity, measured as total distance traversed and velocity. The largest decrease occurred in more developed larvae (DI< 2.8). To determine correlated changes in the neuromuscular junctions during metamorphosis and apoptotic tail loss, miniature endplate currents from tail muscle were recorded during acute exposure to a hypertonic solution, which simulates an apoptotic volume decrease. Our results support a role for T3 in regulating larval locomotor activity during development, and suggest an enhanced response to volume depletion at the neuromuscular junction of older larvae (DI<2.8) compared to younger animals (DI≥2.8). We discuss the significance of the possible role of an apoptotic volume decrease at the level of the neuromuscular junction. [ABSTRACT FROM AUTHOR]

The periodicity of structural and functional effects induced by tryptophan scanning mutagenesis has been successfully used to define function and secondary structure of various transmembrane domains of the acetylcholine receptor of Torpedo californica. We expand the tryptophan scanning of the AchR of T. californica to the γM4 transmembrane domain (γTM4) by introducing tryptophan, at residues 451–462, along the γTM4. Wild type (WT) and mutant AChR were expressed in Xenopus laevis oocytes. Using [125I]α-bungarotoxin binding assays and voltage clamp, we determined that the nAChR expression, EC50, and Hill coefficient values for WT are 1.8 ± 0.4 fmol, 30.3 ± 1.1 μM, and 1.8 ± 0.3, respectively. Mutations L456W, F459W, and G462W induce a significant increase in nAChR expression (2.8 ± 0.5, 3.6 ± 0.6, and 3.0 ± 0.5 fmol, respectively) when compared with WT. These data suggest that these residues are important for AChR oligomerization. Mutations A455W, L456W, F459W, and G462W result in a significant decrease in EC50 (19.5 ± 1.7, 11.4 ± 0.7, 16.4 ± 3.8, and 19.1 ± 2.6 μM, respectively), thus suggesting a gain in function when compared with WT. In contrast, mutation L458W induced an increase in EC50 (42.8 ± 6.8 pM) or loss in function when compared with WT. The Hill coefficient values were the same for WT and all of the mutations studied. The periodicity in function (EC50 and macroscopic peak current) and nAChR expression reveals an average of 3.3 and 3.0 amino acids respectively, thus suggesting a helical secondary structure for the γTM4. [ABSTRACT FROM AUTHOR]

Amyloid beta (Aβ) peptides have been implicated in both Alzheimer's disease (AD) and glaucoma and have been shown to be the key etiological factor in these dangerous health complications. On the other hand, it is well known that Aβ peptide can be generated from its precursor protein and massively released from the blood to nearby tissue upon the activation of platelets due to their involvement in innate immunity and inflammation processes. Here we review evidence about the development of AD and glaucoma neuronal damage showing their dependence on platelet count and activation. The correlation between the effect on platelet count and the effectiveness of anti-AD and anti-glaucoma therapies suggest that platelets may be an important player in these diseases.

We traced the cause of a slow-channel syndrome (SCS) in a patient with progressive muscle weakness, repetitive compound muscle action potential and prolonged low amplitude synaptic currents to a V --> F substitution in the M1 domain of the beta subunit (betaV229F) of the muscle acetylcholine receptor (AChR). In vitro expression studies in Xenopus oocytes indicated that the novel mutation betaV229F expressed normal amounts of AChRs and decreased the ACh EC50 by 10-fold compared to wild type. Kinetic analysis indicated that the mutation displayed prolonged mean open duration and repeated openings during activation. Prolonged openings caused by the betaV229F mutation were due to a reduction in the channel closing rate and an increase in the effective channel opening rate. Repeated openings of the channel during activation were caused by a significant reduction in the agonist dissociation constant. In addition, the betaV229F mutation produced an increase in calcium permeability. The kinetic and permeation studies presented in this work are sufficient to explain the consequences of the betaV229F mutation on the miniature endplate currents and thus are direct evidence that the betaV229F mutation is responsible for compromising the safety margin of neuromuscular transmission in the patient.

The functional role of the alphaM3 transmembrane domain of the Torpedo nicotinic acetylcholine receptor (AChR) was characterized by performing tryptophan-scanning mutagenesis at 13 positions within alphaM3, from residue M278 through I290. The expression of the mutants in Xenopus oocytes was measured by [(125)I]-alpha-bungarotoxin binding, and ACh receptor function was evaluated by using a two-electrode voltage clamp. Six mutants (L279W, F280W, I283W, V285W, S288W, and I289W) were expressed at lower levels than the wild type. Most of these residues have been proposed to face the interior of the protein. The I286W mutant was expressed at 2.4-fold higher levels than the wild type, and the two lipid-exposed mutations, F284W and S287W, were expressed at similar levels as wild type. Binding assays indicated that the alphaM3 domain can accommodate bulky groups in almost all positions. Three mutations, M282W, V285W, and I289W, caused a loss of receptor function, suggesting that the tryptophan side chains alter the conformational changes required for channel assembly or ion channel function. This loss of function suggests that these positions may be involved in helix-helix contacts that are critical for channel gating. The lipid-exposed mutation F284W enhances the receptor macroscopic response at low ACh concentrations and decreases the EC(50). Taken together, our results suggest that alphaM3 contributes to the gating machinery of the nicotinic ACh receptor and that alphaM3 is comprised of a mixture of two types of helical structures.