Gating Of Gap Junction Channels Research Articles

Calcium Role in Gap Junction Channel Gating: Direct Electrostatic or Calmodulin-Mediated?

The chemical gating of gap junction channels is mediated by cytosolic calcium (Ca2+i) at concentrations ([Ca2+]i) ranging from high nanomolar (nM) to low micromolar (µM) range. Since the proteins of gap junctions, connexins/innexins, lack high-affinity Ca2+-binding sites, most likely gating is mediated by a Ca2+-binding protein, calmodulin (CaM) being the best candidate. Indeed, the role of Ca2+-CaM in gating is well supported by studies that have tested CaM blockers, CaM expression inhibition, testing of CaM mutants, co-localization of CaM and connexins, existence of CaM-binding sites in connexins/innexins, and expression of connexins (Cx) mutants, among others. Based on these data, since 2000, we have published a Ca2+-CaM-cork gating model. Despite convincing evidence for the Ca2+-CaM role in gating, a recent study has proposed an alternative gating model that would involve a direct electrostatic Ca2+-connexin interaction. However, this study, which tested the effect of unphysiologically high [Ca2+]i on the structure of isolated junctions, reported that neither changes in the channel's pore diameter nor connexin conformational changes are present, in spite of exposure of isolated gap junctions to [Ca2+]i as high at the 20 mM. In conclusion, data generated in the past four decades by multiple experimental approaches have clearly demonstrated the direct role of Ca2+-CaM in gap junction channel gating.

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 Hydrophobic Residues in Amino Terminal Domains of Cx46 and Cx50 Are Important for Their Gap Junction Channel Ion Permeation and Gating.

Lens gap junctions (GJs) formed by Cx46 and Cx50 are important to keep lens transparency. Functional studies on Cx46 and Cx50 GJs showed that the Vj-gating, single channel conductance (γj), gating polarity, and/or channel open stability could be modified by the charged residues in the amino terminal (NT) domain. The role of hydrophobic residues in the NT on GJ properties is not clear. Crystal and cryo-EM GJ structures have been resolved, but the NT domain structure has either not been resolved or has showed very different orientations depending on the component connexins and possibly other experimental conditions, making it difficult to understand the structural basis of the NT in Vj-gating and γj. Here, we generated missense variants in Cx46 and Cx50 NT domains and studied their properties by recombinant expression and dual whole-cell patch clamp experiments on connexin-deficient N2A cells. The NT variants (Cx46 L10I, N13E, A14V, Q15N, and Cx50 I10L, E13N, V14A, N15Q) were all able to form functional GJs with similar coupling%, except Cx46 N13E, which showed a significantly reduced coupling%. The GJs of Cx46 N13E, A14V and Cx50 E13N, N15Q showed a reduced coupling conductance. Vj-gating of all the variant GJs were similar to the corresponding wild-type GJs except Cx46 L10I. The γj of Cx46 N13E, A14V, Cx50 E13N, and N15Q GJs was reduced to 51%, 82%, 87%, and 74%, respectively, as compared to their wild-type γjs. Structural models of Cx46 L10I and A14V predicted steric clashes between these residues and the TM2 residues, which might be partially responsible for our observed changes in GJ properties. To verify the importance of hydrophobic interactions, we generated a variant, Cx50 S89T, which also shows a steric clash and failed to form a functional GJ. Our experimental results and structure models indicate that hydrophobic interactions between the NT and TM2 domain are important for their Vj-gating, γj, and channel open stability in these and possibly other GJs.

Anesthetics and Cell-Cell Communication: Potential Ca2+-Calmodulin Role in Gap Junction Channel Gating by Heptanol, Halothane and Isoflurane.

Cell–cell communication via gap junction channels is known to be inhibited by the anesthetics heptanol, halothane and isoflurane; however, despite numerous studies, the mechanism of gap junction channel gating by anesthetics is still poorly understood. In the early nineties, we reported that gating by anesthetics is strongly potentiated by caffeine and theophylline and inhibited by 4-Aminopyridine. Neither Ca2+ channel blockers nor 3-isobutyl-1-methylxanthine (IBMX), forskolin, CPT-cAMP, 8Br-cGMP, adenosine, phorbol ester or H7 had significant effects on gating by anesthetics. In our publication, we concluded that neither cytosolic Ca2+i nor pHi were involved, and suggested a direct effect of anesthetics on gap junction channel proteins. However, while a direct effect cannot be excluded, based on the potentiating effect of caffeine and theophylline added to anesthetics and data published over the past three decades, we are now reconsidering our earlier interpretation and propose an alternative hypothesis that uncoupling by heptanol, halothane and isoflurane may actually result from a rise in cytosolic Ca2+ concentration ([Ca2+]i) and consequential activation of calmodulin linked to gap junction proteins.

Calmodulin-Connexin Partnership in Gap Junction Channel Regulation-Calmodulin-Cork Gating Model.

In the past four decades numerous findings have indicated that gap junction channel gating is mediated by intracellular calcium concentrations ([Ca2+i]) in the high nanomolar range via calmodulin (CaM). We have proposed a CaM-based gating model based on evidence for a direct CaM role in gating. This model is based on the following: CaM inhibitors and the inhibition of CaM expression to prevent chemical gating. A CaM mutant with higher Ca2+ sensitivity greatly increases gating sensitivity. CaM co-localizes with connexins. Connexins have high-affinity CaM-binding sites. Connexin mutants paired to wild type connexins have a higher gating sensitivity, which is eliminated by the inhibition of CaM expression. Repeated trans-junctional voltage (Vj) pulses progressively close channels by the chemical/slow gate (CaM’s N-lobe). At the single channel level, the gate closes and opens slowly with on-off fluctuations. Internally perfused crayfish axons lose gating competency but recover it by the addition of Ca-CaM to the internal perfusion solution. X-ray diffraction data demonstrate that isolated gap junctions are gated at the cytoplasmic end by a particle of the size of a CaM lobe. We have proposed two types of CaM-driven gating: “Ca-CaM-Cork” and “CaM-Cork”. In the first, the gating involves Ca2+-induced CaM activation. In the second, the gating occurs without a [Ca2+]i rise.

Gap Junction Channelopathies and Calmodulinopathies. Do Disease-Causing Calmodulin Mutants Affect Direct Cell-Cell Communication?

The cloning of connexins cDNA opened the way to the field of gap junction channelopathies. Thus far, at least 35 genetic diseases, resulting from mutations of 11 different connexin genes, are known to cause numerous structural and functional defects in the central and peripheral nervous system as well as in the heart, skin, eyes, teeth, ears, bone, hair, nails and lymphatic system. While all of these diseases are due to connexin mutations, minimal attention has been paid to the potential diseases of cell–cell communication caused by mutations of Cx-associated molecules. An important Cx accessory protein is calmodulin (CaM), which is the major regulator of gap junction channel gating and a molecule relevant to gap junction formation. Recently, diseases caused by CaM mutations (calmodulinopathies) have been identified, but thus far calmodulinopathy studies have not considered the potential effect of CaM mutations on gap junction function. The major goal of this review is to raise awareness on the likely role of CaM mutations in defects of gap junction mediated cell communication. Our studies have demonstrated that certain CaM mutants affect gap junction channel gating or expression, so it would not be surprising to learn that CaM mutations known to cause diseases also affect cell communication mediated by gap junction channels.

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.

Calmodulin-Cork Model of Gap Junction Channel Gating-One Molecule, Two Mechanisms.

The Calmodulin-Cork gating model is based on evidence for the direct role of calmodulin (CaM) in channel gating. Indeed, chemical gating of cell-to-cell channels is sensitive to nanomolar cytosolic calcium concentrations [Ca2+]i. Calmodulin inhibitors and inhibition of CaM expression prevent chemical gating. CaMCC, a CaM mutant with higher Ca2+-sensitivity greatly increases chemical gating sensitivity (in CaMCC the NH2-terminal EF-hand pair (res. 9–76) is replaced by the COOH-terminal pair (res. 82–148). Calmodulin colocalizes with connexins. Connexins have high-affinity CaM binding sites. Several connexin mutants paired to wild-type connexins have a high gating sensitivity that is eliminated by inhibition of CaM expression. Repeated transjunctional voltage (Vj) pulses slowly and progressively close a large number of channels by the chemical/slow gate (CaM lobe). At the single-channel level, the chemical/slow gate closes and opens slowly with on-off fluctuations. The model proposes two types of CaM-driven gating: “Ca-CaM-Cork” and “CaM-Cork”. In the first, gating involves Ca2+-induced CaM-activation. In the second, gating takes place without [Ca2+]i rise. The Ca-CaM-Cork gating is only reversed by a return of [Ca2+]i to resting values, while the CaM-Cork gating is reversed by Vj positive at the gated side.

Connexin 31 Mutations Associated with Skin Disease and Deafness Display a Variety of Phenotypes When Expressed in Xenopus Oocytes

The gap junction protein Connexin31 (Cx31) is expressed in the skin and inner ear and mutations in the gene encoding Cx31, GJB3, are linked to disorders of skin and hearing. Over thirty mutations have been identified and at the protein level many involve single amino acid substitutions. We characterized many of these using the Xenopus oocyte expression system and compared their function to that of wildtype Cx31. Mutations characterized include G12D, R32W, C86S, L135V, I141V and L209F. A variety of phenotypes were observed ranging from mild alterations in gap junction channel gating to induction of cell death. A mild phenotype was associated with the recessive deafness mutant 141V which formed function channels with itself and Cx31. On the other hand a skin disease mutant Cx31L209F displayed a more dramatic phenotype, inducing cell death via a process that was calcium- and cobalt sensitive. Results are discussed in light of human molecular and structural protein studies.

Chemical and Voltage Gating of Gap Junction Channels Expressed in Xenopus Oocytes.

In most tissues, cells in contact with each other directly intercommunicate via cell-to-cell channels aggregated at gap junctions. Direct cell-to-cell communication provides a fundamental mechanism for coordinating many cellular functions in mature and developing organs, as it enables free exchange of small cytosolic molecules. Gap junction channels are regulated by a chemical gating mechanism sensitive to cytosolic calcium concentration [Ca2+]i in the nanomolar range mediated by Ca2+-activated calmodulin (CaM). Evidence for the relevance of chemical regulation of gap junctional communication to cell function in health and disease prompted the development of methodologies aimed at quantitatively monitoring channel gating. A widely used method is the double voltage clamp of Xenopus laevis oocytes. Basically, this method involves pairing at the vegetal pole devitellinized oocytes in a conical well of a culture dish, inserting in each of them a current and a voltage microelectrode, establishing double voltage clamp and measuring junctional conductance (Gj) from voltage and current records.

Functional asymmetry and plasticity of electrical synapses interconnecting neurons through a 36-state model of gap junction channel gating.

We combined the Hodgkin–Huxley equations and a 36-state model of gap junction channel gating to simulate electrical signal transfer through electrical synapses. Differently from most previous studies, our model can account for dynamic modulation of junctional conductance during the spread of electrical signal between coupled neurons. The model of electrical synapse is based on electrical properties of the gap junction channel encompassing two fast and two slow gates triggered by the transjunctional voltage. We quantified the influence of a difference in input resistances of electrically coupled neurons and instantaneous conductance–voltage rectification of gap junctions on an asymmetry of cell-to-cell signaling. We demonstrated that such asymmetry strongly depends on junctional conductance and can lead to the unidirectional transfer of action potentials. The simulation results also revealed that voltage spikes, which develop between neighboring cells during the spread of action potentials, can induce a rapid decay of junctional conductance, thus demonstrating spiking activity-dependent short-term plasticity of electrical synapses. This conclusion was supported by experimental data obtained in HeLa cells transfected with connexin45, which is among connexin isoforms expressed in neurons. Moreover, the model allowed us to replicate the kinetics of junctional conductance under different levels of intracellular concentration of free magnesium ([Mg2+]i), which was experimentally recorded in cells expressing connexin36, a major neuronal connexin. We demonstrated that such [Mg2+]i-dependent long-term plasticity of the electrical synapse can be adequately reproduced through the changes of slow gate parameters of the 36-state model. This suggests that some types of chemical modulation of gap junctions can be executed through the underlying mechanisms of voltage gating. Overall, the developed model accounts for direction-dependent asymmetry, as well as for short- and long-term plasticity of electrical synapses. Our modeling results demonstrate that such complex behavior of the electrical synapse is important in shaping the response of coupled neurons.

Polyamines preserve connexin 43-mediated gap junctional communication during intracellular hypercalcemia and acidosis.

Changes in the regulation, formation, and gating of connexin-based gap junction channels occur in various disorders. It has been shown that H and Ca are involved in the regulation of gap junctional communication. Ischemia-induced intracellular acidification and Ca overload lead to closure of gap junctions and inhibit an exchange by ions and small molecules throughout the network of cells in the heart, brain, and other tissues. In this study, we examined the role of the polyamines in the regulation of connexin 43 (Cx43)-based gap junction channels under elevated intracellular concentrations of hydrogen ([H]i) and calcium ([Ca]i) ions. Experiments, conducted in Novikoff and A172 human glioblastoma cells, which endogenously express Cx43, showed that polyamines prevent downregulation of Cx43-mediated gap junctional communication caused by elevated [Ca]i and [H]i, accompanying ischemic and other pathological conditions. siRNA knockdown of Cx43 significantly reduces gap junctional communication, indicating that Cx43 gap junctions are the targets for spermine regulation.

Illuminating Myocyte-Fibroblast Homotypic and Heterotypic Gap Junction Dynamics Using Dynamic Clamp

Fibroblasts play a significant role in the development of electrical and mechanical dysfunction of the heart; however, the underlying mechanisms are only partially understood. One widely studied mechanism suggests that fibroblasts produce excess extracellular matrix, resulting in collagenous septa that slow propagation, cause zig-zag conduction paths, and decouple cardiomyocytes, resulting in a substrate for cardiac arrhythmia. An emerging mechanism suggests that fibroblasts promote arrhythmogenesis through direct electrical interactions with cardiomyocytes via gap junction (GJ) channels. In the heart, three major connexin (Cx) isoforms, Cx40, Cx43, and Cx45, form GJ channels in cell-type-specific combinations. Because each Cx is characterized by a unique time- and transjunctional voltage-dependent profile, we investigated whether the electrophysiological contributions of fibroblasts would vary with the specific composition of the myocyte-fibroblast (M-F) GJ channel. Due to the challenges of systematically modifying Cxs in vitro, we coupled native cardiomyocytes with in silico fibroblast and GJ channel electrophysiology models using the dynamic-clamp technique. We found that there is a reduction in the early peak of the junctional current during the upstroke of the action potential (AP) due to GJ channel gating. However, effects on the cardiomyocyte AP morphology were similar regardless of the specific type of GJ channel (homotypic Cx43 and Cx45, and heterotypic Cx43/Cx45 and Cx45/Cx43). To illuminate effects at the tissue level, we performed multiscale simulations of M-F coupling. First, we developed a cell-specific model of our dynamic-clamp experiments and investigated changes in the underlying membrane currents during M-F coupling. Second, we performed two-dimensional tissue sheet simulations of cardiac fibrosis and incorporated GJ channels in a cell type-specific manner. We determined that although GJ channel gating reduces junctional current, it does not significantly alter conduction velocity during cardiac fibrosis relative to static GJ coupling. These findings shed more light on the complex electrophysiological interplay between cardiac fibroblasts and myocytes.

Stochastic Model of Gap Junctions Exhibiting Rectification and Multiple Closed States of Slow Gates

Gap-junction (GJ) channels formed from connexin (Cx) proteins provide direct pathways for electrical and metabolic cell-cell communication. Earlier, we developed a stochastic 16-state model (S16SM) of voltage gating of the GJ channel containing two pairs of fast and slow gates, each operating between open (o) and closed (c) states. However, experimental data suggest that gates may in fact contain two or more closed states. We developed a model in which the slow gate operates according to a linear reaction scheme, o↔c1↔c2, where c1 and c2 are initial-closed and deep-closed states that both close the channel fully, whereas the fast gate operates between the open state and the closed state and exhibits a residual conductance. Thus, we developed a stochastic 36-state model (S36SM) of GJ channel gating that is sensitive to transjunctional voltage (Vj). To accelerate simulation and eliminate noise in simulated junctional conductance (gj) records, we transformed an S36SM into a Markov chain 36-state model (MC36SM) of GJ channel gating. This model provides an explanation for well-established experimental data, such as delayed gj recovery after Vj gating, hysteresis of gj-Vj dependence, and the low ratio of functional channels to the total number of GJ channels clustered in junctional plaques, and it has the potential to describe chemically mediated gating, which cannot be reflected using an S16SM. The MC36SM, when combined with global optimization algorithms, can be used for automated estimation of gating parameters including probabilities of c1↔c2 transitions from experimental gj-time and gj-Vj dependencies.

Application of stochastic automata networks for creation of continuous time Markov chain models of voltage gating of gap junction channels.

The primary goal of this work was to study advantages of numerical methods used for the creation of continuous time Markov chain models (CTMC) of voltage gating of gap junction (GJ) channels composed of connexin protein. This task was accomplished by describing gating of GJs using the formalism of the stochastic automata networks (SANs), which allowed for very efficient building and storing of infinitesimal generator of the CTMC that allowed to produce matrices of the models containing a distinct block structure. All of that allowed us to develop efficient numerical methods for a steady-state solution of CTMC models. This allowed us to accelerate CPU time, which is necessary to solve CTMC models, ∼20 times.

The Residues in the First Extracellular Domain Play an Important Role in Transjunctional-Voltage Dependent Gating and Unitary Channel Conductance of Cx50 Gap Junction Channels

Gap junctions (GJ) are intercellular channels that connecting the cytoplasm of neighbor cells. GJ channels formed by Cx50 and Cx36 show drastic disparity in their unitary conductance (γj) and transjunctional voltage-dependent gating (Vj-gating), but the important underlying molecular domains/residues are not clear. Experimental evidence showed that residues in the first extracellular domain (E1) of Cx50 likely line the GJ channel pore and are important factors in determining γj and Vj-gating. We aligned the E1 sequence of Cx50 with that of Cx36 and found 10 different residues (4/10 residues involves a change in charge). We generated a chimera Cx50Cx36E1, in which the E1 of Cx50 was replaced by the E1 of Cx36, and 4 point mutations in E1 of Cx50 (where a charge change occurs, i.e. G46E, D51M, E62N and E68R). Dual patch clamp study on the homotypic GJ channels formed by the chimera or the point mutants in N2A cell pairs indicate that the Cx50Cx36E1 channel showed little change in the Vj-gating properties, but displayed a significantly reduced main single channel conductance (γj). Our studies on the point mutations of Cx50 showed that some of the mutants altered the Vj-gating properties and others changed the γj and/or the probabilities in different conducting states. Our study indicates that Cx50 E1 is an important domain in determining the Vj-gating properties and γj. Charge changes in different residues in the E1 between Cx50 and Cx36 showed different channel properties likely dictated by either their location in the pore structure or the nature of the mutation.

Continuous Time Markov Chain Models of Voltage Gating of Gap Junction Channels

The major goal of this study was to create a continuous time Markov chain (CTMC) models of voltage gating of gap junction (GJ) channels formed of connexin protein. This goal was achieved by using the Piece Linear Aggregate (PLA) formalism to describe the function of GJs and transforming PLA into Markov process. Infinitesimal generator of CTMC was used to automate construction of Markov chain model from description of the system using PLA formalism. Developed Markov chain models were used to simulate gap junctional conductance dependence on transjunctional voltage. The proposed method was implemented to create models of voltage gating of GJ channels containing 4 and 12 gates. CTMC modeling results were compared with the results obtained using a discrete time Markov chain (DTMC) model. It was shown that CTMC modeling requires less CPU time than an analogous DTMC model. DOI: http://dx.doi.org/10.5755/j01.itc.43.2.3198

The Residues in the First Extracellular Domain Play an Important Role in Transjunctional-Voltage Dependent Gating and Unitary Conductance of Cx50 Gap Junction Channels

The Residues in the First Extracellular Domain Play an Important Role in Transjunctional-Voltage Dependent Gating and Unitary Conductance of Cx50 Gap Junction Channels

Injury-triggered Akt phosphorylation of Cx43: a ZO-1-driven molecular switch that regulates gap junction size

The proteins that form vertebrate gap junctions, the connexins, are highly regulated and have short (<2 hour) half-lives. Phosphorylation of connexin43 (Cx43) affects gap junction assembly, channel gating and turnover. After finding dramatic effects on gap junctions with Akt inhibitors, we created an antibody specific for Cx43 phosphorylated on S373, a potential Akt substrate. We found S373 phosphorylation in cells and skin or heart almost exclusively in larger gap-junctional structures that increased dramatically after wounding or hypoxia. We were able to mechanistically show that Akt-dependent phosphorylation of S373 increases gap junction size and communication by completely eliminating the interaction between Cx43 and ZO-1. Thus, phosphorylation on S373 acts as a molecular 'switch' to rapidly increase gap-junctional communication, potentially leading to initiation of activation and migration of keratinocytes or ischemic injury response in the skin and the heart, respectively.

Coregulation of Multiple Signaling Mechanisms in pp60v-Src-Induced Closure of Cx43 Gap Junction Channels

Attenuation in gap junctional coupling has consistently been associated with induction of rapid or synchronous cell division in normal and pathological conditions. In the case of the v-src oncogene, gating of Cx43 gap junction channels has been linked to both direct phosphorylation of tyrosines (Y247 and 265) and phosphorylation of the serine targets of Erk1/2 (S255, 279 and 282) on the cytoplasmic C-terminal domain of Cx43. However, only the latter has been associated with acute, rather than chronic, gating of the channels immediately after v-src expression, a process that is mediated through a "ball-and-chain" mechanism. In this study we show that, while ERK1/2 is necessary for acute closure of gap junction channels, it is not sufficient. Rather, multiple pathways converge to regulate Cx43 coupling in response to expression of v-src, including parallel signaling through PKC and MEK1/2, with additional positive and negative regulatory effects mediated by PI3 kinase, distinguished by the involvement of Akt.