Sweetening K-channels: what sugar taught us about permeation and gating.

Because they enable for the modification of both viscosity and osmolarity, sugars have been used as a biophysical probe of voltage-gated K-channels for a while. Viscosity variations made it possible to measure the pore sizes in large and small conductance K-channels using techniques similar to those used in the 1980s to study the gramicidin A channel. These analyses led to the finding that the size of the internal mouth appears to be the primary cause of the conductance differences between Shaker-like channels and large conductance BK-channels. As an osmotic agent, adding sugar unilaterally causes streaming potentials that indicate H2O/K+ cotransport across the BK-channel pore. Osmotic experiments on Shaker K-channels suggest that the pore gate operation and the slow inactivation displace comparable amounts of water. Functionally isolated voltage sensors allow estimation of individual osmotic work for each voltage sensing charge during voltage-activation, reporting dramatic internal and external remodeling of the Voltage Sensing Domain´s solvent exposed surfaces. Remarkably, each charge of the VSD appears to take a unique trajectory. Thus, manipulation of viscosity and osmolarity, together with 3D structures, brings in solid grounds to harmonize function and structure in membrane proteins such as K-channels and, in a wider scope, other structurally dynamic proteins.

A scenario for the origin of life: Volume regulation by bacteriorhodopsin required extremely voltage sensitive Na-channels and very selective K-channels.

The osmotic activity produced by internal, non-permeable, anionic nucleic acids and metabolites causes a persistent and life-threatening cell swelling, or cellular edema, produced by the Gibbs-Donnan effect. This evolutionary-critical osmotic challenge must have been resolved by LUCA or its ancestors, but we lack a cell-physiology look into the biophysical constraints to the solutions. Like mycoplasma, early cells conceivably preserved their volume with Cl- , Na+ , and K+ -channels, Na+ /H+ -exchangers, and a light-dependent bacteriorhodopsin-like H+ -pump. Here, I simulated protocells having these ionic-permeabilities and inhabiting an oceanic pond before the Great-Oxygenation-Event. Protocells showed better volume control and stable resting potentials at lower external pH and higher temperatures, favoring a certain type of extremophile life. Prevention of Na+ -influx at night, with low bacteriorhodopsin activity, required deep shutdown of highly voltage-sensitive Na+ -channels and extremely selective K+ -channels, two conserved features essential for modern neuronal encoding. The Gibbs-Donnan effect universality implies that extraterrestrial cells, if they exist, may reveal similar volume-controlling mechanisms.

Educación y determinismo social: algunas reflexiones a partir del concepto de Movilidad Educacional Intergeneracional (MEI) / Education and social determinism: some reflections about the concept of Intergenerational Educational Mobility

Resumen La movilidad educacional intergeneracional (MEI), entendida como la asociación del nivel educacional alcanzado por los padres con relación al nivel educacional alcanzado por hijos, se presenta como un concepto que ha alcanzado relevancia en la investigación social durante el último tiempo. Diferentes estudios lo han utilizado para caracterizar los procesos de expansión de los sistemas educacionales contemporáneos, así como para comprender la evolución del fenómeno de la desigualdad educacional. Sin embargo, se ha utilizado mayormente como un indicador dentro los análisis. En este artículo proponemos una reflexión del concepto de movilidad educacional intergeneracional en tanto concepto teórico-sociológico que permite reflexionar sobre los procesos de determinismo social, en la línea de los trabajos que prolongan conceptos como el de habitus. Para lograr este propósito, este artículo se desarrolla de la siguiente manera. En primer lugar, se presenta el concepto de movilidad educacional intergeneracional y su influencia en tanto indicador de igualdad y referente de la movilidad social. En segundo lugar, se relaciona esta movilidad social con los conceptos de habitus y campo académico, además de poner en evidencia las trayectorias que rompen con el esquema de reproducción social; el llamado milagro sociológico. En tercer lugar, se examinan los elementos de aculturación y asimilación que se ponen en marcha en el proceso de migración y movilidad social. Por último, presentamos la relación entre los distintos factores de facilitación y obstaculización con los conceptos tratados.

Abstract Intergenerational educational mobility, understood as the association of the educational level attained by parents in relation to the educational level attained by their children, is a concept that has gained relevance in social research in recent times. Different studies have used it to characterize the expansion processes of contemporary educational systems, as well as to understand the evolution of the phenomenon of educational inequality. However, it has been used mostly as an indicator within the analysis. In this article we propose a reflection on the concept of intergenerational educational mobility as a theoretical-sociological concept that allows us to reflect on the processes of social determinism, along the lines of works that extend concepts such as habitus. To achieve this purpose, this article is developed as follows. First, the concept of intergenerational educational mobility and its influence as an indicator of equality and a referent of social mobility is presented. Secondly, this social mobility is related to the concepts of habitus and academic field, in addition to highlighting the trajectories that break with the scheme of social reproduction; the so-called sociological miracle. Thirdly, we examine the elements of acculturation and assimilation that are set in motion in the process of migration and social mobility. Finally, we present the relationship between the various facilitating and hindering factors with the concepts discussed.

Neural Network Model for Predicting Student Failure in the Academic Leveling Course of Escuela Politécnica Nacional.

The purpose of this study is to train an artificial neural network model for predicting student failure in the academic leveling course of the Escuela Politécnica Nacional of Ecuador, based on academic and socioeconomic information. For this, 1308 higher education students participated, 69.0% of whom failed the academic leveling course; besides, 93.7% of the students self-identified as mestizo, 83.9% came from the province of Pichincha, and 92.4% belonged to general population. As a first approximation, a neural network model was trained with twelve variables containing students' academic and socioeconomic information. Then, a dimensionality reduction process was performed from which a new neural network was modeled. This dimension reduced model was trained with the variables application score, vulnerability index, regime, gender, and population segment, which were the five variables that explained more than 80% of the first model. The classification accuracy of the dimension reduced model was 0.745, while precision and recall were 0.883 and 0.778, respectively. The area under ROC curve was 0.791. This model could be used as a guide to lead intervention policies so that the failure rate in the academic leveling course would decrease.

Binding of κ-Conotoxin-PVIIA to Open and Closed Shaker K-Channels Are Differentially Affected by the Ionic Strength.

κ-Conotoxin-PVIIA (κ-PVIIA) is a potassium-channel blocking peptide from the venom of the fish-hunting snail, Conus purpurascens, which is essential for quick prey's excitotoxic immobilization. Binding of one κ-PVIIA to Shaker K-channels occludes the K+-conduction pore without additional conformational effects. Because this 27-residue toxin is +4-charged at neutral pH, we asked if electrostatic interactions play a role in binding. With Voltage-Clamp electrophysiology, we tested how ionic strength (IS) affects κ-PVIIA blockade to Shaker. When IS varied from ~0.06 to ~0.16 M, the dissociation constant for open and closed channels increased by ~5- and ~16-fold, respectively. While the association rates decreased equally, by ~4-fold, in open and closed channels, the dissociation rates increased 4-5-fold in closed channels but was IS-insensitive in open channels. To explain this differential IS-dependency, we propose that the bound κ-PVIIA wobbles, so that in open channels the intracellular environment, via ion-conduction pore, buffers the imposed IS-changes in the toxin-channel interface. A Brønsted-Bjerrum analysis on the rates predicts that if, instead of fish, the snail preyed on organisms with seawater-like lymph ionic composition, a severely harmless toxin, with >100-fold diminished affinity, would result. Thus, considerations of the native ionic environment are essential for conotoxins evaluation as pharmacological leads.

Trans-toxin ion-sensitivity of charybdotoxin-blocked potassium-channels reveals unbinding transitional states.

In silico and in vitro studies have made progress in understanding protein-protein complex formation; however, the molecular mechanisms for their dissociation are unclear. Protein-protein complexes, lasting from microseconds to years, often involve induced-fit, challenging computational or kinetic analysis. Charybdotoxin (CTX), a peptide from the Leiurus scorpion venom, blocks voltage-gated K+-channels in a unique example of binding/unbinding simplicity. CTX plugs the external mouth of K+-channels pore, stopping K+-ion conduction, without inducing conformational changes. Conflicting with a tight binding, we show that external permeant ions enhance CTX-dissociation, implying a path connecting the pore, in the toxin-bound channel, with the external solution. This sensitivity is explained if CTX wobbles between several bound conformations, producing transient events that restore the electrical and ionic trans-pore gradients. Wobbling may originate from a network of contacts in the interaction interface that are in dynamic stochastic equilibria. These partially-bound intermediates could lead to distinct, and potentially manipulable, dissociation pathways.

Effective pore size and radius of capture for K(+) ions in K-channels.

Reconciling protein functional data with crystal structure is arduous because rare conformations or crystallization artifacts occur. Here we present a tool to validate the dimensions of open pore structures of potassium-selective ion channels. We used freely available algorithms to calculate the molecular contour of the pore to determine the effective internal pore radius (r(E)) in several K-channel crystal structures. r(E) was operationally defined as the radius of the biggest sphere able to enter the pore from the cytosolic side. We obtained consistent r(E) estimates for MthK and Kv1.2/2.1 structures, with r(E) = 5.3-5.9 Å and r(E) = 4.5-5.2 Å, respectively. We compared these structural estimates with functional assessments of the internal mouth radii of capture (r(C)) for two electrophysiological counterparts, the large conductance calcium activated K-channel (r(C) = 2.2 Å) and the Shaker Kv-channel (r(C) = 0.8 Å), for MthK and Kv1.2/2.1 structures, respectively. Calculating the difference between r(E) and r(C), produced consistent size radii of 3.1-3.7 Å and 3.6-4.4 Å for hydrated K(+) ions. These hydrated K(+) estimates harmonize with others obtained with diverse experimental and theoretical methods. Thus, these findings validate MthK and the Kv1.2/2.1 structures as templates for open BK and Kv-channels, respectively.

Zebrafish CaV2.1 calcium channels are tailored for fast synchronous neuromuscular transmission.

The CaV2.2 (N-type) and CaV2.1 (P/Q-type) voltage-dependent calcium channels are prevalent throughout the nervous system where they mediate synaptic transmission, but the basis for the selective presence at individual synapses still remains an open question. The CaV2.1 channels have been proposed to respond more effectively to brief action potentials (APs), an idea supported by computational modeling. However, the side-by-side comparison of CaV2.1 and CaV2.2 kinetics in intact neurons failed to reveal differences. As an alternative means for direct functional comparison we expressed zebrafish CaV2.1 and CaV2.2 α-subunits, along with their accessory subunits, in HEK293 cells. HEK cells lack calcium currents, thereby circumventing the need for pharmacological inhibition of mixed calcium channel isoforms present in neurons. HEK cells also have a simplified morphology compared to neurons, which improves voltage control. Our measurements revealed faster kinetics and shallower voltage-dependence of activation and deactivation for CaV2.1. Additionally, recordings of calcium current in response to a command waveform based on the motorneuron AP show, directly, more effective activation of CaV2.1. Analysis of calcium currents associated with the AP waveform indicate an approximately fourfold greater open probability (PO) for CaV2.1. The efficient activation of CaV2.1 channels during APs may contribute to the highly reliable transmission at zebrafish neuromuscular junctions.

K⁺ conduction and Mg²âº blockade in a shaker Kv-channel single point mutant with an unusually high conductance.

Potassium channels exhibit a large diversity of single-channel conductances. Shaker is a low-conductance K-channel in which Pro475→Asp, a single-point mutation near the internal pore entrance, promotes 6- to 8-fold higher unitary current. To assess the mechanism for this higher conductance, we measured Shaker-P475D single-channel current in a wide range of symmetrical K(+) concentrations and voltages. Below 300 mM K(+), the current-to-voltage relations (i-V) showed inward rectification that disappeared at 1000 mM K(+). Single-channel conductance reached a maximum of ∼190 pS at saturating [K(+)], a value 4- to 5-fold larger than that estimated for the native channel. Intracellular Mg(2+) blocked this variant with ∼100-fold higher affinity. Near zero voltage, blockade was competitively antagonized by K(+); however, at voltages >100 mV, it was enhanced by K(+). This result is consistent with a lock-in effect in a single-file diffusion regime of Mg(2+) and K(+) along the pore. Molecular-dynamics simulations revealed higher K(+) density in the pore, especially near the Asp-475 side chains, as in the high-conductance MthK bacterial channel. The molecular dynamics also showed that K(+) ions bound distally can coexist with other K(+) or Mg(2+) in the cavity, supporting a lock-in mechanism. The maximal K(+) transport rate and higher occupancy could be due to a decrease in the electrostatic energy profile for K(+) throughout the pore, reducing the energy wells and barriers differentially by ∼0.7 and ∼2 kT, respectively.

Hypothyroidism in the adult rat causes incremental changes in brain-derived neurotrophic factor, neuronal and astrocyte apoptosis, gliosis, and deterioration of postsynaptic density.

BACKGROUND: Adult hypothyroidism is a highly prevalent condition that impairs processes, such as learning and memory. Even though tetra-iodothyronine (T(4)) treatment can overcome the hypothyroidism in the majority of cases, it cannot fully recover the patient's learning capacity and memory. In this work, we analyzed the cellular and molecular changes in the adult brain occurring with the development of experimental hypothyroidism. METHODS: Adult male Sprague-Dawley rats were treated with 6-propyl-2-thiouracil (PTU) for 20 days to induce hypothyroidism. Neuronal and astrocyte apoptosis were analyzed in the hippocampus of control and hypothyroid adult rats by confocal microscopy. The content of brain-derived neurotrophic factor (BDNF) was analyzed using enzyme-linked immunosorbent assay (ELISA) and in situ hybridization. The glutamatergic synapse and the postsynaptic density (PSD) were analyzed by electron microscopy. The content of PSD proteins like tyrosine receptor kinase B (TrkB), p75, and N-methyl-D-aspartate receptor (NMDAr) were analyzed by immunoblot. RESULTS: We observed that the hippocampus of hypothyroid adult rats displayed increased apoptosis levels in neurons and astrocyte and reactive gliosis compared with controls. Moreover, we found that the amount of BDNF mRNA was higher in the hippocampus of hypothyroid rats and the content of TrkB, the receptor for BDNF, was reduced at the PSD of the CA3 region of hypothyroid rats, compared with controls. We also observed that the glutamatergic synapses from the stratum radiatum of CA3 from hypothyroid rats, contained thinner PSDs than control rats. This observation was in agreement with a reduced content of NMDAr subunits at the PSD in hypothyroid animals. CONCLUSIONS: Our data suggest that adult hypothyroidism affects the hippocampus by a mechanism that alters the composition of PSD, reduces neuronal and astrocyte survival, and alters the content of the signaling neurotrophic factors, such as BDNF.

K(+) channels: function-structural overview.

Potassium channels are particularly important in determining the shape and duration of the action potential, controlling the membrane potential, modulating hormone secretion, epithelial function and, in the case of those K(+) channels activated by Ca(2+), damping excitatory signals. The multiplicity of roles played by K(+) channels is only possible to their mammoth diversity that includes at present 70 K(+) channels encoding genes in mammals. Today, thanks to the use of cloning, mutagenesis, and the more recent structural studies using x-ray crystallography, we are in a unique position to understand the origins of the enormous diversity of this superfamily of ion channels, the roles they play in different cell types, and the relations that exist between structure and function. With the exception of two-pore K(+) channels that are dimers, voltage-dependent K(+) channels are tetrameric assemblies and share an extremely well conserved pore region, in which the ion-selectivity filter resides. In the present overview, we discuss in the function, localization, and the relations between function and structure of the five different subfamilies of K(+) channels: (a) inward rectifiers, Kir; (b) four transmembrane segments-2 pores, K2P; (c) voltage-gated, Kv; (d) the Slo family; and (e) Ca(2+)-activated SK family, SKCa.

Reduced voltage sensitivity in a K+-channel voltage sensor by electric field remodeling.

Propagation of the nerve impulse relies on the extreme voltage sensitivity of Na(+) and K(+) channels. The transmembrane movement of four arginine residues, located at the fourth transmembrane segment (S4), in each of their four voltage-sensing domains is mostly responsible for the translocation of 12 to 13 e(o) across the transmembrane electric field. Inserting additional positively charged residues between the voltage-sensing arginines in S4 would, in principle, increase voltage sensitivity. Here we show that either positively or negatively charged residues added between the two most external sensing arginines of S4 decreased voltage sensitivity of a Shaker voltage-gated K(+)-channel by up to approximately 50%. The replacement of Val363 with a charged residue displaced inwardly the external boundaries of the electric field by at least 6 A, leaving the most external arginine of S4 constitutively exposed to the extracellular space and permanently excluded from the electric field. Both the physical trajectory of S4 and its electromechanical coupling to open the pore gate seemed unchanged. We propose that the separation between the first two sensing charges at resting is comparable to the thickness of the low dielectric transmembrane barrier they must cross. Thus, at most a single sensing arginine side chain could be found within the field. The conserved hydrophobic nature of the residues located between the voltage-sensing arginines in S4 may shape the electric field geometry for optimal voltage sensitivity in voltage-gated ion channels.

Slow inactivation in Shaker K channels is delayed by intracellular tetraethylammonium.

After removal of the fast N-type inactivation gate, voltage-sensitive Shaker (Shaker IR) K channels are still able to inactivate, albeit slowly, upon sustained depolarization. The classical mechanism proposed for the slow inactivation observed in cell-free membrane patches--the so called C inactivation--is a constriction of the external mouth of the channel pore that prevents K(+) ion conduction. This constriction is antagonized by the external application of the pore blocker tetraethylammonium (TEA). In contrast to C inactivation, here we show that, when recorded in whole Xenopus oocytes, slow inactivation kinetics in Shaker IR K channels is poorly dependent on external TEA but severely delayed by internal TEA. Based on the antagonism with internally or externally added TEA, we used a two-pulse protocol to show that half of the channels inactivate by way of a gate sensitive to internal TEA. Such gate had a recovery time course in the tens of milliseconds range when the interpulse voltage was -90 mV, whereas C-inactivated channels took several seconds to recover. Internal TEA also reduced gating charge conversion associated to slow inactivation, suggesting that the closing of the internal TEA-sensitive inactivation gate could be associated with a significant amount of charge exchange of this type. We interpreted our data assuming that binding of internal TEA antagonized with U-type inactivation (Klemic, K.G., G.E. Kirsch, and S.W. Jones. 2001. Biophys. J. 81:814-826). Our results are consistent with a direct steric interference of internal TEA with an internally located slow inactivation gate as a "foot in the door" mechanism, implying a significant functional overlap between the gate of the internal TEA-sensitive slow inactivation and the primary activation gate. But, because U-type inactivation is reduced by channel opening, trapping the channel in the open conformation by TEA would also yield to an allosteric delay of slow inactivation. These results provide a framework to explain why constitutively C-inactivated channels exhibit gating charge conversion, and why mutations at the internal exit of the pore, such as those associated to episodic ataxia type I in hKv1.1, cause severe changes in inactivation kinetics.

Mutations of nonconserved residues within the calcium channel alpha1-interaction domain inhibit beta-subunit potentiation.

Voltage-dependent calcium channels consist of a pore-forming subunit (Ca(V)alpha(1)) that includes all the molecular determinants of a voltage-gated channel, and several accessory subunits. The ancillary beta-subunit (Ca(V)beta) is a potent activator of voltage-dependent calcium channels, but the mechanisms and structural bases of this regulation remain elusive. Ca(V)beta binds reversibly to a conserved consensus sequence in Ca(V)alpha(1), the alpha(1)-interaction domain (AID), which forms an alpha-helix when complexed with Ca(V)beta. Conserved aromatic residues face to one side of the helix and strongly interact with a hydrophobic pocket on Ca(V)beta. Here, we studied the effect of mutating residues located opposite to the AID-Ca(V)beta contact surface in Ca(V)1.2. Substitution of AID-exposed residues by the corresponding amino acids present in other Ca(V)alpha(1) subunits (E462R, K465N, D469S, and Q473K) hinders Ca(V)beta's ability to increase ionic-current to charge-movement ratio (I/Q) without changing the apparent affinity for Ca(V)beta. At the single channel level, these Ca(V)1.2 mutants coexpressed with Ca(V)beta(2a) visit high open probability mode less frequently than wild-type channels. On the other hand, Ca(V)1.2 carrying either a mutation in the conserved tryptophan residue (W470S, which impairs Ca(V)beta binding), or a deletion of the whole AID sequence, does not exhibit Ca(V)beta-induced increase in I/Q. In addition, we observed a shift in the voltage dependence of activation by +12 mV in the AID-deleted channel in the absence of Ca(V)beta, suggesting a direct participation of these residues in the modulation of channel activation. Our results show that Ca(V)beta-dependent potentiation arises primarily from changes in the modal gating behavior. We envision that Ca(V)beta spatially reorients AID residues that influence the channel gate. These findings provide a new framework for understanding modulation of VDCC gating by Ca(V)beta.

Calcium channel subtypes differentially regulate fusion pore stability and expansion.

Various studies have focused in the relative contribution of different voltage-activated Ca(2+) channels (VACC) to total transmitter release. However, how Ca(2+) entry through a given VACC subtype defines the pattern of individual exocytotic events remains unknown. To address this question, we have used amperometry in bovine chromaffin cells. L, N, and P/Q channels were individually or jointly blocked with furnidipine, omega-conotoxin GVIA, omega-agatoxin IVA, or omega-conotoxin MVIIC. The three channel types contributed similarly to cytosolic Ca(2+) signals induced by 70 mmol/L K(+). However, they exhibited different contributions to the frequency of exocytotic events and they were shown to differently regulate the final steps of the exocytosis. When compared with the other VACC subtypes, Ca(2+) entry through P/Q channels effectively induced exocytosis, it decreased fusion pore stability and accelerated its expansion. Conversely, Ca(2+) entry through N channels was less efficient in inducing exocytotic events, also slowing fusion pore expansion. Finally, Ca(2+) entry through L channels inefficiently induced exocytosis, and the individual blockade of this channel significantly modified fusion pore dynamics. The distance between a given VACC subtype and the release sites could account for the differential effects of the distinct VACC on the fusion pore dynamics.

Slow inactivation in voltage gated potassium channels is insensitive to the binding of pore occluding peptide toxins.

Voltage gated potassium channels open and inactivate in response to changes of the voltage across the membrane. After removal of the fast N-type inactivation, voltage gated Shaker K-channels (Shaker-IR) are still able to inactivate through a poorly understood closure of the ion conduction pore. This, usually slower, inactivation shares with binding of pore occluding peptide toxin two important features: i), both are sensitive to the occupancy of the pore by permeant ions or tetraethylammonium, and ii), both are critically affected by point mutations in the external vestibule. Thus, mutual interference between these two processes is expected. To explore the extent of the conformational change involved in Shaker slow inactivation, we estimated the energetic impact of such interference. We used kappa-conotoxin-PVIIA (kappa-PVIIA) and charybdotoxin (CTX) peptides that occlude the pore of Shaker K-channels with a simple 1:1 stoichiometry and with kinetics 100-fold faster than that of slow inactivation. Because inactivation appears functionally different between outside-out patches and whole oocytes, we also compared the toxin effect on inactivation with these two techniques. Surprisingly, the rate of macroscopic inactivation and the rate of recovery, regardless of the technique used, were toxin insensitive. We also found that the fraction of inactivated channels at equilibrium remained unchanged at saturating kappa-PVIIA. This lack of interference with toxin suggests that during slow inactivation the toxin receptor site remains unaffected, placing a strong geometry-conservative constraint on the possible structural configurations of a slow inactivated K-channel. Such a constraint could be fulfilled by a concerted rotation of the external vestibule.