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

A model for trust building: The state, market, and cultural norms in Sino-Chilean economic exchange

The Free Trade Agreement (FTA) between the People’s Republic of China (PRC) and Chile came into effect in 2006. The complementarities of both nations’ economies, their shared profile as export–oriented countries, the shared institutional framework and their diplomatic history forecasted a promising future of cooperation. Yet, expectations have hardly been fulfilled. Based on the analytical framework provided by trust theories –a culturally-shaped variable– and on the empirical support given by an extensive fieldwork, this dissertation explains why Sino-Chilean economic cooperation has not flourished as expected. For this, it explores the sources of international –between states– and transnational –between business and corporations– trust. The findings show the pivotal role trust has played in their economic foreign policy and how they have used it as a strategy to fulfill their national interests. Although the results demonstrate that both states have engaged in a trust-building process, the trust constructed is nevertheless narrow and weak, since it is bounded to particular objectives, especially from the strongest party. At a transnational level, the dissertation analyzes the main sources that are framing trust-building, which embrace market, cultural and institutional variables. Based on this, it is argued that trust entails a paradox: although it is a necessary condition for mutual growth and a source of economic benefits, building trust can be costly to acquire and maintain, given the structure in which is embedded. Finally, the research tackles the process of transnational trust building carried out by the main actors. Through the examination of the case study, this research illustrates the nuances of the trust-building process, portraying trust as a complex but necessary variable for economic development, and by doing this, offering a novel view on the role culture plays on growth

Estructuralismo e internalismo: hacia un pluralismo ontológico y epistemológico

Este trabajo se estructura del siguiente modo: en primer lugar, expondremos las nociones centrales del realismo internalista y del estructuralismo. Posteriormente, la búsqueda de la física teórica contemporánea de una 'teoría del todo' que permita no sólo unificar la física, sino también explicar la diversidad de los fenómenos de la naturaleza, será el punto de partida para ver que ambas perspectivas filosóficas acuerdan en rechazar consistentemente dicha búsqueda

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

A G protein-gated K channel is activated via beta 2-adrenergic receptors and G beta gamma subunits in Xenopus oocytes

In many tissues, inwardly rectifying K channels are coupled to seven- helix receptors via the Gi/Go family of heterotrimeric G proteins. This activation proceeds at least partially via G beta gamma subunits. These experiments test the hypothesis that G beta gamma subunits activate the channel even if released from other classes of heterotrimeric G proteins. The G protein-gated K channel from rat atrium, KGA/GIRK1, was expressed in Xenopus oocytes with various receptors and G proteins. The beta 2-adrenergic receptor (beta 2AR), a Gs-linked receptor, activated large KGA currents when the alpha subunit, G alpha s, was also overexpressed. Although G alpha s augmented the coupling between beta 2AR and KGA, G alpha s also inhibited the basal, agonist-independent activity of KGA. KGA currents stimulated via beta 2AR activated, deactivated, and desensitized more slowly than currents stimulated via Gi/Go-linked receptors. There was partial occlusion between currents stimulated via beta 2AR and the m2 muscarinic receptor (a Gi/Go-linked receptor), indicating some convergence in the mechanism of activation by these two receptors. Although stimulation of beta 2AR also activates adenylyl cyclase and protein kinase A, activation of KGA via beta 2AR is not mediated by this second messenger pathway, because direct elevation of intracellular cAMP levels had no effect on KGA currents. Experiments with other coexpressed G protein alpha and beta gamma subunits showed that (a) a constitutively active G alpha s mutant did not suppress basal KGA currents and was only partially as effective as wild type G alpha s in coupling beta 2AR to KGA, and (b) beta gamma subunits increased basal KGA currents. These results reinforce present concepts that beta gamma subunits activate KGA, and also suggest that beta gamma subunits may provide a link between KGA and receptors not previously known to couple to inward rectifiers

Dissecting the primary to specialist referrals using graph neural networks: exploring the relationship between physician referral patterns, primary care access and healthcare spending

The healthcare referral system affects all points of the healthcare ecosystem – access to care, patient satisfaction, physician utilization and healthcare costs. The state of these variables plays a critical role in determining healthcare efficiency. In this paper we dissect the medical referrals from primary to secondary care in Florida in 2015 and tackle them from three perspectives – influence of physician experience in referral choice, relationship between physician referral choice and Medicare spending, and pattern detection given different referral windows. To accomplish our goal of identifying patterns in primary to secondary referral mechanisms, we use Graph Neural Networks (GNN) unsupervised model to learn the vectoral representation of our physician nodes and their properties in the network. This work provides new discoveries on factors that influence the referral patterns and can be used to make better decisions when aiming to improve the efficiency of referrals

La comida en la pampa durante el auge salitrero en Chile: una visión desde la zooarqueología histórica

Durante cerca de 60 años (1880-1940 d C.), el salitre fue el principal sustento económico de Chile. Pese a su importancia dentro de la historia reciente del país, se han realizado muy pocos estudios arqueológicos orientados a contrastar y complementar la abundante información histórica disponible. Este trabajo apunta hacia esa dirección a través del análisis faunístico de siete asentamientos salitreros ubicados en el desierto de Atacama (Norte de Chile). Se discute la diversidad taxonómica de la muestra, la frecuencia de partes esqueletales y las modificaciones culturales, que presentan una estrecha similitud con los datos extraídos de la documentación escrita.For nearly 60 years (1880-1940 A.D.), saltpetre was the main source of economic income in Chile. Despite its importance in the country’s recent history, very scarce archaeological research was oriented to contrast and complement the abundant historical information on hand. This work aims in this direction, through the analysis of faunal remains of seven mining settlements located in the Atacama Desert (northern Chile). We discuss the taxonomic diversity of the sample, the frequency of skeletal parts and cultural modifications, whose, in sum, show a close resemblance with the available historiographic data

Low Molecular Weight mRNA Encodes a Protein That Controls Serotonin 5-HT_(1c) and Acetylcholine M_1 Receptor Sensitivity in Xenopus Oocytes

Serotonin 5-HT_(1c) and acetylcholine M_1 receptors activate phosphoinositidase, resulting in an increased formation of IP_3 and 1,2 diacylglycerol. In Xenopus oocytes injected with mRNA encoding either of these receptors, Ca^(2+) released from intracellular stores in response to IP3 then opens Ca^(2+)-gated Cl^-channels. In the present experiments, oocytes expressing a transcript from a cloned mouse serotonin 5-HT_(1c) receptor were exposed to identical 15-s pulses of agonist, administered 2 min apart; the second current response was two to three times that of the first. However, in those oocytes coinjected with the 5-HT_(1c) receptor transcript and a low molecular weight fraction (0.3-1.5 kb) of rat brain mRNA, the second current response was ~50% of the first. Thus, the low molecular weight RNA encodes a protein (or proteins) that causes desensitization. Experiments using fura-2 or a Ca^(2+)-free superfusate indicated that desensitization of the 5-HT_(1c) receptor response does not result from a sustained elevation of intracellular Ca^(2+) level or require the entry of extracellular Ca^(2+). Photolysis of caged IP_3 demonstrated that an increase in IP_3 and a subsequent rise in Ca^(2+) do not produce desensitization of either the IP_3 or 5-HT_(1c) peak current responses. Furthermore, in oocytes coinjected with the low molecular weight RNA and a transcript from the rat M_1 acetylcholine receptor, the M_1 current response was greatly attenuated. Our data suggest that the proteins involved in attenuation of the M_1 current response and desensitization of the 5-HT_(1c) current response may be the same