Mutations in M2 alter the selectivity of the mouse nicotinic acetylcholine receptor for organic and alkali metal cations

We measured the permeability ratios (PX/PNa) of 3 wild-type, 1 hybrid, 2 subunit-deficient, and 22 mutant nicotinic receptors expressed in Xenopus oocytes for alkali metal and organic cations using shifts in the bi-ionic reversal potential of the macroscopic current. Mutations at three positions (2', 6', 10') in M2 affected ion selectivity. Mutations at position 2' (alpha Thr244, beta Gly255, gamma Thr253, delta Ser258) near the intracellular end of M2 changed the organic cation permeability ratios as much as twofold and reduced PCs/PNa and PK/PNa by 16-18%. Mutations at positions 6' and 10' increased the glycine ethyl ester/Na+ and glycine methyl ester/Na+ permeability ratios. Two subunit alterations also affected selectivity: omission of the delta subunit reduced PCs/PNa by 16%, and substitution of Xenopus delta for mouse delta increased Pguanidinium/PNa more than twofold and reduced PCs/PNa by 34% and PLi/PNa by 20%. The wild-type mouse receptor displayed a surprising interaction with the primary ammonium cations; relative permeability peaked at a chain length equal to four carbons. Analysis of the organic permeability ratios for the wild-type mouse receptor shows that (a) the diameter of the narrowest part of the pore is 8.4 A; (b) the mouse receptor departs significantly from size selectivity for monovalent organic cations; and (c) lowering the temperature reduces Pguanidinium/PNa by 38% and Pbutylammonium/PNa more than twofold. The results reinforce present views that positions -1' and 2' are the narrowest part of the pore and suggest that positions 6' and 10' align some permeant organic cations in the pore in an interaction similar to that with channel blocker, QX-222

Functional characterization of a glutamate/aspartate transporter from the mosquito Aedes aegypti

Glutamate elicits a variety of effects in insects, including inhibitory and excitatory signals at both neuromuscular junctions and brain. Insect glutamatergic neurotransmission has been studied in great depth especially from the standpoint of the receptor-mediated effects, but the molecular mechanisms involved in the termination of the numerous glutamatergic signals have only recently begun to receive attention. In vertebrates, glutamatergic signals are terminated by Na^+/K^+-dependent high-affinity excitatory amino acid transporters (EAAT), which have been cloned and characterized extensively. Cloning and characterization of a few insect homologues have followed, but functional information for these homologues is still limited. Here we report a study conducted on a cloned mosquito EAAT homologue isolated from the vector of the dengue virus, Aedes aegypti. The deduced amino acid sequence of the protein, AeaEAAT, exhibits 40–50% identity with mammalian EAATs, and 45–50% identity to other insect EAATs characterized thus far. It transports l-glutamate as well as l- and d-aspartate with high affinity in the micromolar range, and demonstrates a substrate-elicited anion conductance when heterologously expressed in Xenopus laevis oocytes, as found with mammalian homologues. Analysis of the spatial distribution of the protein demonstrates high expression levels in the adult thorax, which is mostly observed in the thoracic ganglia. Together, the work presented here provides a thorough examination of the role played by glutamate transport in Ae. aegypti

Tris+/Na+ permeability ratios of nicotinic acetylcholine receptors are reduced by mutations near the intracellular end of the M2 region

Tris+/Na+ permeability ratios were measured from shifts in the biionic reversal potentials of the macroscopic ACh-induced currents for 3 wild- type (WT), 1 hybrid, 2 subunit-deficient, and 25 mutant nicotinic receptors expressed in Xenopus oocytes. At two positions near the putative intracellular end of M2, 2' (alpha Thr244, beta Gly255, gamma Thr253, delta Ser258) and -1', point mutations reduced the relative Tris+ permeability of the mouse receptor as much as threefold. Comparable mutations at several other positions had no effects on relative Tris+ permeability. Mutations in delta had a greater effect on relative Tris+ permeability than did comparable mutations in gamma; omission of the mouse delta subunit (delta 0 receptor) or replacement of mouse delta with Xenopus delta dramatically reduced relative Tris+ permeability. The WT mouse muscle receptor (alpha beta gamma delta) had a higher relative permeability to Tris+ than the wild-type Torpedo receptor. Analysis of the data show that (a) changes in the Tris+/Na+ permeability ratio produced by mutations correlate better with the hydrophobicity of the amino acid residues in M2 than with their volume; and (b) the mole-fraction dependence of the reversal potential in mixed Na+/Tris+ solutions is approximately consistent with the Goldman- Hodgkin-Katz voltage equation. The results suggest that the main ion selectivity filter for large monovalent cations in the ACh receptor channel is the region delimited by positions -1' and 2' near the intracellular end of the M2 helix

The transporter-like protein inebriated mediates hyperosmotic stimuli through intracellular signaling

We cloned the inebriated homologue MasIne from Manduca sexta and expressed it in Xenopus laevis oocytes. MasIne is homologous to neurotransmitter transporters but no transport was observed with a number of putative substrates. Oocytes expressing MasIne respond to hyperosmotic stimulation by releasing intracellular Ca(2+), as revealed by activation of the endogenous Ca(2+)-activated Cl(-) current. This Ca(2+) release requires the N-terminal 108 amino acid residues of MasIne and occurs via the inositol trisphosphate pathway. Fusion of the N terminus to the rat gamma-aminobutyric acid transporter (rGAT1) also renders rGAT1 responsive to hyperosmotic stimulation. Immunohistochemical analyses show that MasIne and Drosophila Ine have similar tissue distribution patterns, suggesting functional identity. Inebriated is expressed in tissues and cells actively involved in K(+) transport, which suggests that it may have a role in ion transport, particularly of K(+). We propose that stimulation of MasIne releases intracellular Ca(2+) in native tissues, activating Ca(2+)-dependent K(+) channels, and leading to K(+) transport

An Engineered Glutamate-gated Chloride (GluCl) Channel for Sensitive, Consistent Neuronal Silencing by Ivermectin

A modified invertebrate glutamate-gated Cl− channel (GluCl αβ) was previously employed to allow pharmacologically induced silencing of electrical activity in CNS neurons upon exposure to the anthelmintic drug ivermectin (IVM). Usefulness of the previous receptor was limited by 1) the high concentration of IVM necessary to elicit a consistent silencing phenotype, raising concern about potential side effects, and 2) the variable extent of neuronal spike suppression, due to variations in the co-expression levels of the fluorescent protein-tagged α and β subunits. To address these issues, mutant receptors generated via rational protein engineering strategies were examined for improvement. Introduction of a gain-of-function mutation (L9′F) in the second transmembrane domain of the α subunit appears to facilitate β subunit incorporation and substantially increase heteromeric GluCl αβ sensitivity to IVM. Removal of an arginine-based endoplasmic reticulum retention motif (RSR mutated to AAA) from the intracellular loop of the β subunit further promotes heteromeric expression at the plasma membrane possibly by preventing endoplasmic reticulum-associated degradation of the β subunit rather than simply reducing endoplasmic reticulum retention. A monomeric XFP (mXFP) mutation that prevents fluorescent protein dimerization complements the mutant channel effects. Expression of the newly engineered GluCl opt α-mXFP L9′F + opt β-mXFP Y182F RSR_AAA receptor in dissociated neuronal cultures markedly increases conductance and reduces variability in spike suppression at 1 nm IVM. This receptor, named “GluClv2.0,” is an improved tool for IVM-induced silencing

Successful Cessation Programs that Reduce Comorbidity May Explain Surprisingly Low Smoking Rates Among Hospitalized COVID-19 Patients

A recent, non-peer-reviewed meta-analysis suggests that smoking may reduce the risk of hospitalization with COVID-19 because the prevalence of smoking among hospitalized COVID-19 is less than that of the general population. However, there are alternative explanations for this phenomena based on (1) the failure to report, or accurately record, smoking history during emergency hospital admissions and (2) a pre-disposition to avoid smoking among COVID-19 patients with tobacco-related comorbidities (a type of “reverse” causation). For example, urine testing of hospitalized patients in Australia for cotinine showed that smokers were under-counted by 37% because incoming patients failed to inform staff about their smoking behavior. Face-to-face interviews can introduce bias into the responses to attitudinal and behavioral questions not present in the self-completion interviews typically used to measure smoking prevalence in the general population. Subjects in face-to-face interviews may be unwilling to admit socially undesirable behavior and attitudes under direct questioning. Reverse causation may also contribute to the difference between smoking prevalence in the COVID-19 and general population. Patients hospitalized with COVID-19 may be simply less prone to use tobacco than the general population. A potentially robust “reverse causation” hypothesis for reduced prevalence of smokers in the COVID-19 population is the enrichment of patients in that population with serious comorbidities that motivates them to quit smoking. We judge that this “smoking cessation” mechanism may account for a significant fraction of the reduced prevalence of smokers in the COVID-19 population. Testing this hypothesis will require a focused research program

Regions of β4·β2 subunit chimeras that contribute to the agonist selectivity of neuronal nicotinic receptors

AbstractFifteen chimeric nicotinic receptors β subunits were constructed consisting of N-terminal neuronal β4 sequences and C-terminal β2 sequences. Responses to cytisine, nicotine, or tetramethylammonium were compared to acetylcholine responses for these subunits expressed in Xenopus oocytes with α3 subunits. The results show that (i) two residues in the extracellular domain of chimeric β4·β2 subunits (108β2F/β4V, 110β2S/β4T) account for much of the relative cytisine sensitivity; and (ii) four extracellular residues of chimeric β4·β2 subunits (112β2A/β4V, 113β2V/β4I and 115β2S/β4R, 116β2Y/β4S) account for most of the relative tetramethylammonium sensitivity. The data did not permit localization of nicotine sensitivity to any particular region

Ewald Sums for One Dimension

We derive analytic solutions for the potential and field in a one-dimensional system of masses or charges with periodic boundary conditions, in other words Ewald sums for one dimension. We also provide a set of tools for exploring the system evolution and show that it's possible to construct an efficient algorithm for carrying out simulations. In the cosmological setting we show that two approaches for satisfying periodic boundary conditions, one overly specified and the other completely general, provide a nearly identical clustering evolution until the number of clusters becomes small, at which time the influence of any size-dependent boundary cannot be ignored. Finally we compare the results with other recent work with the hope of providing clarification over differences these issues have induced. We explain that modern formulations of physics require a well defined potential which is not available if the forces are screened directly.Comment: 2 figures added references expanded discussion of algorithm corrected figures added discussion of screened forc

Planning an Escape: Considerations for the Development of Applied Escape Rooms

Background. Teams are essential to a wide array of applications and organizations often utilize varying interventions to improve the effectiveness of their teams. Due to their collaborative and modifiable characteristics, escape rooms are being increasingly utilized as an avenue to both deliver team interventions and to function as testbeds in research. Escape rooms are complex, interdependent activities which warrant careful planning to be effectively implemented. Despite the growing literature base concerning escape rooms, there is still limited practical guidance to inform the development of an escape room. Aim. The purpose of this article is to provide seven considerations that are relevant to the development, implementation, and effectiveness of an escape room. Specifically, guidance is provided in determining the objectives, identifying a theme, assigning the roles, establishing participant interdependence, selecting a venue, designing the puzzles, and creating the assessments. Conclusion. The considerations provided in this article can advance the science underlying the use of escape rooms and preclude difficulties associated with their use