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

0106: Influence of the ratio of co-expressed cardiac connexins Cx43 and Cx45 in the formation of gap junction channels and their electrical properties

The cardiac action potential (AP) propagation is regulated to permit the coordinated and rhythmic atrial and ventricular contractions. This regulation requires several factors, especially gap junctions, which ensure a direct pathway for electrical and biochemical signaling. They are clusters of few to hundred intercellular gap junction channels (GJC) made of two hemichannels docked in the membrane of adjacent cells, which are composed of six connexins (Cxs). Their distinct electrical properties are a key factor regulating the propagation of the AP. Four cardiac Cxs, Cx40, Cx43, Cx45 and Cx30.2, exhibit specific patterns of expression that change in the healthy and diseased heart, which leads to different possible configurations of GJC. The aim of this study is to investigate the function of the distinct ratio of co-expressed Cxs in regulating the formation and function of GJC. Electrical properties of GJC (junctional coupling, voltage dependence, unitary conductance) are determined by performing electrical recordings on cell pair by applying the dual voltage-clamp method. Rat Liver Epithelial cells stably transfected to induce accurate Cx43:Cx45 ratios of 0 (single Cx43 expression), 0.5, 1 and 2, are used. The ongoing recordings show distinct electrical properties before and after the induction of Cx45: induction of Cx45 decreases the cell-to-cell coupling and rectifies the voltage dependence of GJC. Preliminary unitary recordings suggest a distinct formation of GJC of mixed Cx43/Cx45 composition in function of the Cx43:Cx45 ratio. Further investigations will provide better understanding on the distinct contributions of Cx43 and Cx45 in the GJC make-up, electrical properties and function of the Cx43/Cx45 expression pattern in regulating the cardiac impulse propagation in the healthy heart, and the pro-arrhythmic behavior in the diseased heart

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

Effects of non-linear GJ channels on the AP propagation : a modelling insight

International audienceBackground: Velocity and pattern of propagation of cardiac AP depends on structural andfunctional properties of the tissue, such as conductivity, dynamics of transmembrane ionicchannels and gap junctions (GJ). Gap junctions are clusters of channels that connect adjacent cells. A gap junction channel(GJC) is made of proteins named ­ connexins. Electrical behavior of GJCs depend on the typeand arrangement of their connexin composition. The dominating connexins in cardiacmyocytes are Cx43, Cx45 and Cx40. Methods and results: In current mathematical models, GJCs are considered to be passive.But, the experimental results, obtained by the dual­voltage clamp technique, show that GJCs display biophysical electrical properties such as voltage gating,i.e. a time and voltage dependence. Here we model Cx43 GJCs. We use the Hodgkin­Huxley formalism to describe GJCsconductance via one gating variable g j = g j (t, V j ). From our experimental results we obtain model parameters: the normalisedsteady state conductance and the time constant to reach the steady state, both voltagedependent. Once we have described the behavior of the single GJC, we write the mathematical model ofthe tissue, where we apply GJ current on specific parts of the cells’ membranes. Numerics and outlook: Some 3D numerical experiments are currently being performed on athin strip of cells, in order to compare the model’s results with the experimental ones. We use a monolayer of 50 × 3 cells, represented by cylinders of 100μm lengthand 10μm radius, with 2μm inter­cellular distance. We model GJCs on the cross sections of the cylinders. Finally, we apply an external stimulus on the border of the domain, and observe the propagation of theAP. Our goal is to make a mathematical model of the heterogeneous GJCs, including Cx45channels, as these have been shown to play a role in arrythmogenesis

Connexin43 ablation in foetal atrial myocytes decreases electrical coupling, partner connexins, and sodium current

Aims Remodelling and regional gradients in expression of connexins (Cx) are thought to contribute to atrial electrical dysfunction and atrial fibrillation. We assessed the effect of interaction between Cx43, Cx40, and Cx45 on atrial cell-to-cell coupling and inward Na current (INa) in engineered pairs of atrial myocytes derived from wild-type mice (Cx43+/+) and mice with genetic ablation of Cx43 (Cx43−/−). Methods and results Cell pairs were engineered by microcontact printing from atrial Cx43+/+ and Cx43−/− murine myocytes (1 day before birth, 3-5 days in culture). Dual and single voltage clamp were used to measure intercellular electrical conductance, gj, and its dependence on transjunctional voltage, Vj, single gap junction channel conductances, and INa. 3D reconstructions of Cx43, Cx40, and Cx45 immunosignals in gap junctions were made from confocal slices. Full genetic Cx43 ablation produced a decrease in immunosignals of Cx40 to 62 ± 10% (mean ± SE; n= 17) and Cx45 to 66 ± 8% (n= 16). Gj decreased from 80 ± 9 nS (Cx43+/+, n= 17) to 24 ± 2 nS (Cx43−/−, n= 35). Single channel analysis showed a shift in the main peak of the channel histogram from 49 ± 1.7 nS (Cx43+/+) to 67 ± 1.8 nS (Cx43−/−) with a second minor peak appearing at 27 ± 1.5 pS. The dependence of gj on Vj decreased with Cx43 ablation. Importantly, peak INa decreased from −350 ± 44 pA/pF (Cx43+/+) to −154 ± 28 pA/pF (Cx43−/−). Conclusions The dependence of Cx40, Cx45, and INa on Cx43 expression indicates a complex interaction between connexins and INa in the atrial intercalated discs that is likely to be of relevance for arrhythmogenesi

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) Vj-sensitive gating of Ij (Vj, gap junction voltage; Ij, gap junction current), (2) contribution and (3) kinetics of Ij deactivation and (4) single-channel conductance. The first three reflect alterations of fast Vj 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 properties

Modelling cardiac electrophysiology with structural heterogeneities and dynamical gap junctions

Bidomain equations are the standard way to model the electric potential in cardiac tissue. We propose the modification of this model for the case of the diseased heart, e.g. fibrosis of the heart tissue. On microscale, we assume to have periodic diffusive inclusions embedded in the healthy tissue modelled by the bidomain equations. We derive the macroscale model using the homogenisation technique. We recover a bidomain model with modified conductivities, that depend on the volume fraction of the diffusive inclusions but also on their geometries.Modèles numériques haute résolution de l'électrophysiologie cardiaqu