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 and are regulated by various chemical factors. Voltage gating of GJs may play a physiological role, particularly in excitable cells which can exhibit large transients in membrane potential during the generation of an action potential. 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 transfected with Cx43 and Cx45, isoforms that are expressed in cardiac tissue, data sets were fit 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 connexins. 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 not just as a modeling tool, but also to provide a means of testing GJ channel gating behavior. SignificanceGap junction (GJ) channels gate in response to transjunctional voltage which provides the capacity for dynamic regulation of intercellular coupling. Kinetic properties of GJs in modeling studies have been infrequently addressed and we present a computational model of voltage gating that can account for both kinetic and steady-state changes in junctional conductance, gj. Although GJs possess two gating mechanisms, our analysis indicates that changes in gj for each voltage polarity can be adequately described by a kinetic scheme describing a single mechanism in each of the hemichannels, suggesting functional dominance of one mechanism over a substantial voltage range. This property allowed for model simplification that can be applied for efficient simulation of sizeable cell clusters and analyses of electrophysiological data.

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.

Introduction Gap junction (GJ) channels, formed of connexin (Cx) proteins, conduit electrical excitation in the heart. It is well established that GJ conductance (gj) depends on transjunctional voltage (Vj), but little is still known about its dynamics during the spread of cardiac excitation. Transitional tissue of atrioventricular (AV) node expresses Cx43 and Cx45, which could form heterotypic GJs, which are highly sensitive to Vj. Purpose Evaluation of heterotypic gj dynamics of Cx43/Cx45 channels during the propagation of excitation in cardiac tissue. Methods The CE-4SM model of cardiac tissue, consisting of discrete cells connected through dynamic GJs, was developed by combining Fenton-Karma equations, which define cardiac excitability (CE), and a 4-state model (4SM) of GJ channel gating, which describes the dependence of gj on Vj and its kinetics. Global optimization methods were applied for 4SM parameter evaluation to fit gj timecourses obtained from electrophysiological recordings. Experiments were performed using dual-whole-cell patch clamp in Novikoff cells, endogenously expressing Cx43, and Hela cells transfected with Cx45. Results We developed a combined cardiac excitability model (CE-4SM), that allowed us to study an interaction between the spread of cardiac excitation and GJ channel voltage. Modeling results shows that during propagation of excitation, the phase difference between each action potential (AP) in adjacent cells, generates two subsequent Vj spikes. First spike causes small decrease of gj, while the second one, a minor increase. These impulses can accumulate into a large overall decay until the impulse rate-dependent steady-state is reached. This phenomenon is caused by variation of delays between APs and consequentially, different durations and amplitudes of Vj spikes developed across GJ channels. Interestingly, such gj decrease was capable of causing drift of spiral wave rotor or termination of fibrillation-like arrhythmia during our modeling experiments. Also, we performed in-vitro experiments to evaluate 4SM model parameters and validate simulation results that showed gj decay at a high pulse rate, which could occur during reentry-like tachycardia. Conclusions Voltage gating of Cx43/Cx45 GJ channels could act as a control circuit, that lowers gj between the transitional cells of AV node at high impulse rate. Presumably, this could help to reduce the conduction or even act as a block of high-rate impulses passing through AV node. Also, such gj decrease could cause a drift of spiral waves and disruption of fibrillation-like processes. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Lithuanian University of Health Sciences, Institute of Cardiology

Širdies ir kraujagyslių sistemos ligos pasaulyje yra vie­nos iš dažniausiai pasitaikančių susirgimų žmonių po­puliacijoje. Žinoma, jog šių ligų atsiradimui įtakos turi įvairūs veiksniai. Vienas iš rečiau aptariamų veiksnių, kuris, manoma, prisideda prie kardiovaskulinės sistemos patofiziologinių pokyčių ir ligų atsiradimo, yra baltymo koneksino, formuojančio šešianarį kompleksą ląstelės membranoje, plyšinių jungčių funkcijų bei jo genų raiš­kos pakitimai. Šio tyrimo tikslas – apžvelgti esamą nau­jausią mokslinę literatūrą, kurioje aprašomos koneksinų funkcijos širdies ir kraujagyslių sistemoje bei išsiaiškinti šių baltymų įtaką kardiovaskulinės sistemos patofizio­loginiams pokyčiams. Literatūros paieška atlikta kom­piuterinėje bibliografinėje mokslinių publikacijų bazėje PubMed, naudojant atitinkamus raktažodžius bei jų de­rinius. Literatūros apžvalgos analizei atrinkti ir išanali­zuoti 28 moksliniai straipsniai anglų kalba, atitinkantys analizuojamą temą. Tyrimo rezultatai atskleidė, kad ko­neksinai, sudarydami plyšines, jungtis užtikrina tinkamą tarpląstelinę sąveiką bei sujungia dviejų ląstelių viduląs­telines terpes. Be to, jie užtikrina greitą impulso sklidimą širdyje ir yra svarbūs sinchronizuotam širdies raumens susitraukimui. Tam tikri koneksinų genų polimorfizmai bei mutacijos siejamos su padidėjusia kardiovaskulinių ligų išsivystymo rizika. Išvados: 1) baltymų koneksinų suformuoti plyšinių jung­čių kanalai užtikrina tinkamą tarpląstelinę sąveiką ir yra svarbūs daugeliui ląstelių funkcijų; 2) nepakitę koneksinų kanalai būtini normaliam organų bei jų sistemų funkcio­navimui; 3) koneksinų raiškos pokyčiai, mutacijos bei po­limorfizmai susiję su širdies ir kraujagyslių ligų geneze.

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 and are regulated by various chemical factors. Voltage gating of GJs may play a physiological role, particularly in excitable cells which can exhibit large transients in membrane potential during the generation of an action potential. 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 transfected with Cx43 and Cx45, isoforms that are expressed in cardiac tissue, data sets were fit 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 connexins. 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 not just as a modeling tool, but also to provide a means of testing GJ channel gating behavior. SignificanceGap junction (GJ) channels gate in response to transjunctional voltage which provides the capacity for dynamic regulation of intercellular coupling. Kinetic properties of GJs in modeling studies have been infrequently addressed and we present a computational model of voltage gating that can account for both kinetic and steady-state changes in junctional conductance, gj. Although GJs possess two gating mechanisms, our analysis indicates that changes in gj for each voltage polarity can be adequately described by a kinetic scheme describing a single mechanism in each of the hemichannels, suggesting functional dominance of one mechanism over a substantial voltage range. This property allowed for model simplification that can be applied for efficient simulation of sizeable cell clusters and analyses of electrophysiological data.