The role of the Cx43/Cx45 gap junction voltage gating on wave propagation and arrhythmogenic activity in cardiac tissue.

Gap junctions (GJs) formed of connexin (Cx) protein are the main conduits of electrical signals in the heart. Studies indicate that the transitional zone of the atrioventricular (AV) node contains heterotypic Cx43/Cx45 GJ channels which are highly sensitive to transjunctional voltage (Vj). To investigate the putative role of Vj gating of Cx43/Cx45 channels, we performed electrophysiological recordings in cell cultures and developed a novel mathematical/computational model which, for the first time, combines GJ channel Vj gating with a model of membrane excitability to simulate a spread of electrical pulses in 2D. Our simulation and electrophysiological data show that Vj transients during the spread of cardiac excitation can significantly affect the junctional conductance (gj) of Cx43/Cx45 GJs in a direction- and frequency-dependent manner. Subsequent simulation data indicate that such pulse-rate-dependent regulation of gj may have a physiological role in delaying impulse propagation through the AV node. We have also considered the putative role of the Cx43/Cx45 channel gating during pathological impulse propagation. Our simulation data show that Vj gating-induced changes in gj can cause the drift and subsequent termination of spiral waves of excitation. As a result, the development of fibrillation-like processes was significantly reduced in 2D clusters, which contained Vj-sensitive Cx43/Cx45 channels.

Modeling and analysis of voltage gating of gap junction channels at a single-channel level.

The advancement of single-channel-level recording via the patch-clamp technique has provided a powerful means of assessing the detailed behaviors of various types of ion channels in native and exogenously expressed cellular environments. However, such recordings of gap junction (GJ) channels are hampered by unique challenges that are related to their unusual intercellular configuration and natural clustering into densely packed plaques. Thus, the methods for reliable cross-correlation of data recorded at macroscopic and single-channel levels are lacking in studies of GJs. To address this issue, we combined our previously published four-state model (4SM) of GJ channel gating by voltage with maximum likelihood estimation (MLE)-based analyses of electrophysiological recordings of GJ channel currents. First, we consider evaluation of single-channel characteristics and the methods for efficient stochastic simulation of single GJ channels from the kinetic scheme described by 4SM using data obtained from macroscopic recordings. We then present an MLE-based methodology for extraction of information about transition rates for GJ channels and, ultimately, gating parameters defined in 4SM from recordings with visible unitary events. The validity of the proposed methodology is illustrated using stochastic simulations of single GJ channels and is extended to electrophysiological data recorded in cells expressing connexin 43 tagged with enhanced green fluorescent protein.

The Amino Terminal Domain and Modulation of Connexin36 Gap Junction Channels by Intracellular Magnesium Ions.

Electrical synapses between neurons in the mammalian CNS are predominantly formed of the connexin36 (Cx36) gap junction (GJ) channel protein. Unique among GJs formed of a number of other members of the Cx gene family, Cx36 GJs possess a high sensitivity to intracellular Mg2+ that can robustly act to modulate the strength of electrical synaptic transmission. Although a putative Mg2+ binding site was previously identified to reside in the aqueous pore in the first extracellular (E1) loop domain, the involvement of the N-terminal (NT) domain in the atypical response of Cx36 GJs to pH was shown to depend on intracellular levels of Mg2+. In this study, we examined the impact of amino acid substitutions in the NT domain on Mg2+ modulation of Cx36 GJs, focusing on positions predicted to line the pore funnel, which constitutes the cytoplasmic entrance of the channel pore. We find that charge substitutions at the 8th, 13th, and 18th positions had pronounced effects on Mg2+ sensitivity, particularly at position 13 at which an A13K substitution completely abolished sensitivity to Mg2+. To assess potential mechanisms of Mg2+ action, we constructed and tested a series of mathematical models that took into account gating of the component hemichannels in a Cx36 GJ channel as well as Mg2+ binding to each hemichannel in open and/or closed states. Simultaneous model fitting of measurements of junctional conductance, gj, and transjunctional Mg2+ fluxes using a fluorescent Mg2+ indicator suggested that the most viable mechanism for Cx36 regulation by Mg2+ entails the binding of Mg2+ to and subsequent stabilization of the closed state in each hemichannel. Reduced permeability to Mg2+ was also evident, particularly for the A13K substitution, but homology modeling of all charge-substituted NT variants showed only a moderate correlation between a reduction in the negative electrostatic potential and a reduction in the permeability to Mg2+ ions. Given the reported role of the E1 domain in Mg2+ binding together with the impact of NT substitutions on gating and the apparent state-dependence of Mg2+ binding, this study suggests that the NT domain can be an integral part of Mg2+ modulation of Cx36 GJs likely through the coupling of conformational changes between NT and E1 domains.

Four-State Model for Simulating Kinetic and Steady-State Voltage-Dependent Gating of Gap Junctions.

Gap junction (GJ) channels, formed of connexin (Cx) proteins, provide a direct pathway for metabolic and electrical cell-to-cell communication. These specialized channels are not just passive conduits for the passage of ions and metabolites but have been shown to gate robustly in response to transjunctional voltage, Vj, the voltage difference between two coupled cells. Voltage gating of GJs could play a physiological role, particularly in excitable cells, which can generate large transients in membrane potential during the propagation of action potentials. We present a mathematical/computational model of GJ channel voltage gating to assess properties of GJ channels that takes into account contingent gating of two series hemichannels and the distribution of Vj across each hemichannel. From electrophysiological recordings in cell cultures expressing Cx43 or Cx45, the principal isoforms expressed in cardiac tissue, various data sets were fitted simultaneously using global optimization. The results showed that the model is capable of describing both steady-state and kinetic properties of homotypic and heterotypic GJ channels composed of these Cxs. Moreover, mathematical analyses showed that the model can be simplified to a reversible two-state system and solved analytically using a rapid equilibrium assumption. Given that excitable cells are arranged in interconnected networks, the equilibrium assumption allows for a substantial reduction in computation time, which is useful in simulations of large clusters of coupled cells. Overall, this model can serve as a tool for the studying of GJ channel gating and its effects on the spread of excitation in networks of electrically coupled cells.