Pitfalls when examining gap junction hemichannels: interference from volume-regulated anion channels

Human HeLa cells transfected with mouse connexin45 were used to explore the experimental conditions suitable to measure currents carried by gap junction hemichannels. Experiments were performed with a voltage-clamp technique and whole-cell recording. Lowering [Ca2+]o from 2mM to 20nM evoked an extra current, I m, putatively carried by Cx45 hemichannels. However, the variability of I m (size, voltage sensitivity, kinetics) suggested the involvement of other channels. The finding that growth medium in the incubator increased the osmolarity with time implied that volume-regulated anion channels (VRAC) may participate. This assumption was reinforced by the following observations. On the one hand, keeping [Ca2+]o normal while the osmolarity of the extracellular solution was reduced from 310 to 290mOsm yielded a current characteristic of VRAC; I VRAC activated/deactivated at negative/positive voltage, giving rise to the conductance functions g VRAC,inst=f(V m) (inst: instantaneous; V m: membrane potential) and g VRAC,ss=f(V m) (ss: steady state). Moreover, it was reversibly inhibited by mibefradil, a Cl-channel blocker (binding constant K d=38μM, Hill coefficient n=12), but not by the gap junction channel blocker 18α-glycyrrhetinic acid. On the other hand, minimizing the osmotic imbalance while [Ca2+]o was reduced led to a current typical for Cx45 hemichannels; I hc activated/deactivated at positive/negative voltage. Furthermore, it was reversibly inhibited by 18α-glycyrrhetinic acid or palmitoleic acid, but not by mibefradil. Computations based on g VRAC,ss=f(V m) and g hc,ss=f(V m) indicated that the concomitant operation of both currents results in a bell-shaped conductance-voltage relationship. The functional implications of the data presented are discussed. Conceivably, VRAC and hemichannels are involved in a common signaling pathwa

Regulation of Cx45 hemichannels mediated by extracellular and intracellular calcium

Connexin45 (Cx45) hemichannels (HCs) open in the absence of Ca2+ and close in its presence. To elucidate the underlying mechanisms, we examined the role of extra- and intracellular Ca2+ on the electrical properties of HCs. Experiments were performed on HeLa cells expressing Cx45 using electrical (voltage clamp) and optical (Ca2+ imaging) methods. HCs exhibit a time- and voltage-dependent current (I hc), activating with depolarization and inactivating with hyperpolarization. Elevation of [Ca2+]o from 20nM to 2μM reversibly decreases I hc, decelerates its rate of activation, and accelerates its deactivation. Our data suggest that [Ca2+]o modifies the channel properties by adhering to anionic sites in the channel lumen and/or its outer vestibule. In this way, it blocks the channel pore and reversibly lowers I hc and modifies its kinetics. Rapid lowering of [Ca2+]o from 2mM to 20nM, achieved early during a depolarizing pulse, led to an outward I hc that developed with virtually no delay and grew exponentially in time paralleled by unaffected [Ca2+]i. A step increase of [Ca2+]i evoked by photorelease of Ca2+ early during a depolarizing pulse led to a transient decrease of I hc superimposed on a growing outward I hc; a step decrease of [Ca2+]i elicited by photoactivation of a Ca2+ scavenger provoked a transient increase in I hc. Hence, it is tempting to assume that Ca2+ exerts a direct effect on Cx45 hemichannel

On-Track Testing as a Validation Method of Computational Fluid Dynamic Simulations of a Formula SAE Vehicle

This thesis is about the validation of a computational fluid dynamics simulation of a ground vehicle by means of a low-budget coast-down test. The vehicle is built to the standards of the 2014 Formula SAE rules. It is equipped with large wings in the front and rear of the car; the vertical loads on the tires are measured by specifically calibrated shock potentiometers. The coast-down test was performed on a runway of a local airport and is used to determine vehicle specific coefficients such as drag, downforce, aerodynamic balance, and rolling resistance for different aerodynamic setups. The test results are then compared to the respective simulated results. The drag deviates about 5% from the simulated to the measured results. The downforce numbers show a deviation up to 18% respectively. Moreover, a sensitivity analysis of inlet velocities, ride heights, and pitch angles was performed with the help of the computational simulation

Cardiac connexins Cx43 and Cx45: formation of diverse gap junction channels with diverse electrical properties

HeLa cells expressing rat connexin43 (Cx43) and/or mouse Cx45 were studied with the dual voltage-clamp technique. Different types of cell pairs were established and their gap junction properties determined, i.e. the dependence of the instantaneous and steady-state conductances (g j,inst, g j,ss) on the transjunctional voltage (V j) and the kinetics of inactivation of the gap junction current (I j). Pairs of singly transfected cells showed homogeneous behaviour at both V j polarities. Homotypic Cx43-Cx43 and Cx45-Cx45 cell pairs yielded distinct symmetrical functions g j,inst=f(V j) and g j,ss=f(V j). Heterotypic Cx43-Cx45 preparations exhibited asymmetric functions g j,inst=f(V j) and g j,ss=f(V j) suggesting that connexons Cx43 and Cx45 gate with positive and negative V j, respectively. Preparations containing a singly (Cx43 or Cx45) or doubly (Cx43/45) transfected cell showed quasi-homogeneous behaviour at one V j polarity and heterogeneous behaviour at the other polarity. The former yielded Boltzmann parameters intermediate between those of Cx43-Cx43, Cx45-Cx45 and Cx43-Cx45 preparations; the latter could not be explained by homotypic and heterotypic combinations of homomeric connexons. Each pair of doubly transfected cells (Cx43/Cx45) yielded unique functions g j,inst=f(V j) and g j,ss=f(V j). This can not be explained by combinations of homomeric connexons. We conclude that Cx43 and Cx45 form homomeric-homotypic, homomeric-heterotypic channels as well as heteromeric-homotypic and heteromeric-heterotypic channels. This has implications for the impulse propagation in specific areas of the hear

Gap Junction Channels and Cardiac Impulse Propagation

The role of gap junction channels on cardiac impulse propagation is complex. This review focuses on the differential expression of connexins in the heart and the biophysical properties of gap junction channels under normal and disease conditions. Structural determinants of impulse propagation have been gained from biochemical and immunocytochemical studies performed on tissue extracts and intact cardiac tissue. These have defined the distinctive connexin coexpression patterns and relative levels in different cardiac tissues. Functional determinants of impulse propagation have emerged from electrophysiological experiments carried out on cell pairs. The static properties (channel number and conductance) limit the current flow between adjacent cardiomyocytes and thus set the basic conduction velocity. The dynamic properties (voltage-sensitive gating and kinetics of channels) are responsible for a modulation of the conduction velocity during propagated action potentials. The effect is moderate and depends on the type of Cx and channel. For homomeric-homotypic channels, the influence is small to medium; for homomeric-heterotypic channels, it is medium to strong. Since no data are currently available on heteromeric channels, their influence on impulse propagation is speculative. The modulation by gap junction channels is most prominent in tissues at the boundaries between cardiac tissues such as sinoatrial node-atrial muscle, atrioventricular node-His bundle, His bundle-bundle branch and Purkinje fibers-ventricular muscle. The data predict facilitation of orthodromic propagatio

The electrical behaviour of rat connexin46 gap junction channels expressed in transfected HeLa cells

Pairs of human HeLa cells expressing rat connexin46 were used to study the electrical properties of gap junction channels with the dual voltage-clamp method. The steady-state conductance (g j,ss) had a bell-shaped dependence on transjunctional voltage (V j). The parameters of the Boltzmann fit were: V j,0=42mV, g j,min=0.12, z=2.5 (pipette solution: K+ aspartate−; 27°C). The Boltzmann parameters were sensitive to the ionic composition of the pipette solution (KCl, K+ aspartate−, TEA+ Cl−, TEA+ aspartate−). The Vj-dependent inactivation of the junctional current I j was approximated by single exponentials (exceptions: two exponentials with KCl at V j≥75mV and K+ aspartate− at V j=125mV). The time constant of inactivation (τi) decreased with increasing V j and was sensitive to the pipette solution. The larger the ions, the slower the inactivation. Recovery from inactivation followed a single exponential. The time constant of recovery (τr) increased with increasing V j. Single-channel currents showed a main state, several substates and a residual state. The corresponding conductances γj,main and γj,residual decreased slightly with increasing V j; extrapolation to V j=0mV yielded values of 152 and 28pS, respectively (K+ aspartate−; 37°C). The values of γj,main and γj,residual were dependent on pipette solution. The ratio γj,main/γj,residual increased with increasing ionic size, suggesting that the residual state impairs ion permeation more severely than the main state. The γj,main data suggest that the ionic selectivity of Cx46 channels may be controlled primarily by ionic size. Compared with hemichannel results, docking of connexons may modify the channel structure and thereby affect the ionic selectivity of gap junction channels. The open channel probability at steady state (P o) decreased with increasing V j. The parameters of the Boltzmann fit were: V j,0=41mV, z=2.2 (K+ aspartate−; 27°C

Influence of V5/6-His Tag on the Properties of Gap Junction Channels Composed of Connexin43, Connexin40 or Connexin45

HeLa cells expressing wild-type connexin43, connexin40 or connexin45 and connexins fused with a V5/6-His tag to the carboxyl terminus (CT) domain (Cx43-tag, Cx40-tag, Cx45-tag) were used to study connexin expression and the electrical properties of gap junction channels. Immunoblots and immunolabeling indicated that tagged connexins are synthesized and targeted to gap junctions in a similar manner to their wild-type counterparts. Voltage-clamp experiments on cell pairs revealed that tagged connexins form functional channels. Comparison of multichannel and single-channel conductances indicates that tagging reduces the number of operational channels, implying interference with hemichannel trafficking, docking and/or channel opening. Tagging provoked connexin-specific effects on multichannel and single-channel properties. The Cx43-tag was most affected and the Cx45-tag, least. The modifications included (1) V j-sensitive gating of I j (V j, gap junction voltage; I j, gap junction current), (2) contribution and (3) kinetics of I j deactivation and (4) single-channel conductance. The first three reflect alterations of fast V j gating. Hence, they may be caused by structural and/or electrical changes on the CT that interact with domains of the amino terminus and cytoplasmic loop. The fourth reflects alterations of the ion-conducting pathway. Conceivably, mutations at sites remote from the channel pore, e.g., 6-His-tagged CT, affect protein conformation and thus modify channel properties indirectly. Hence, V5/6-His tagging of connexins is a useful tool for expression studies in vivo. However, it should not be ignored that it introduces connexin-dependent changes in both expression level and electrophysiological propertie

Phytotoxic effects of metabolites from Alternaria euphorbiicola against its host plant Euphorbia heterophylla

A bioassay-guided fractionation of culture filtrates of the fungus Alternaria euphorbiicola, a pathogen of the weed Euphorbia heterophylla, led to the isolation of anhydromevalonolactone (1), tyrosol (2), (R)-( - )-mevalonolactone (3), and cycloglycylproline (4). When tested on the punctured leaves of the host plant, these compounds produced bleached lesions with dark brown margins at concentrations as low as 80 µM. When tested on the leaves of other relevant weeds, only cycloglycylproline showed selective activity against E. heterophylla. This is the first report on the isolation of phytotoxins from A. euphorbiicola and on the phytotoxicity of anhydromevalonolactone, (R)-( - )-mevalonolactone, and cycloglycylproline

NOVAS OPÇÕES DE SUBSTRATOS PARA O CULTIVO DE Cyrtopodium cardiochilum (ORCHIDACEAE)

O presente trabalho teve como objetivo avaliar o efeito de diferentes substratos no cultivo da espécie Cyrtopodium cardiochilum (Orchidaceae). As mudas foram cultivadas em vasos de polietileno, em casa de vegetação com tela de sombreamento de polipropileno de 60% de luminosidade. Os tratamentos constituíram-se de brita de gnaisse (granulometria variando de 8 15 mm), argila expandida (15 22 mm de diâmetro), seixo rolado (8 15 mm), casca de coco em cubos (coxim) e vermiculita. Foram analisadas também as combinações de brita e argila expandida, brita e seixo, argila expandida e seixo, na proporção 1:1 (v/v); brita, argila expandida e seixo na proporção 1:1:1 (v/v/v); e brita, argila expandida, seixo e vermiculita na proporção 1:1:1:1 (v/v/v/v). Após seis meses de instalação do experimento foram avaliadas as seguintes variáveis: altura das plantas, diâmetro dos pseudobulbos, número de folhas, produção de massa de matéria seca total (MST), da parte aérea (MSPA) e do sistema radicular (MSR). Utilizou-se o delineamento experimental em blocos casualizados, composto por nove tratamentos e oito repetições. Não houve diferença entre os tratamentos para: altura de planta, número de folhas, diâmetro dos pseudobulbos e MSR. Para a MSPA e MST observou-se diferença entre os substratos, com destaque para o tratamento argila expandida + brita de gnaisse. Conclui-se que todos os substratos testados podem ser utilizados no cultivo de C. cardiochilum, destacando-se o substrato argila expandida + brita de gnaisse na proporção de 1:1 (v/v)