Heterotypic Gap Junction Channels Research Articles

The rectification of heterotypic Cx46/Cx50 gap junction channels depends on intracellular magnesium.

Gap junction (GJ) intercellular communication is crucial in many physiological and pathological processes. A GJ channel is formed by head-to-head docking of two hexameric hemichannels from two neighboring cells. Heterotypic GJ channels formed by two different homomeric connexin hemichannels often display rectification properties in the current-voltage relationship while the underlying mechanisms are not fully clear. Here we studied heterotypic Cx46/Cx50 GJs at a single GJ channel level. Our data showed unitary Cx46/Cx50 GJ channel conductance (γj) rectification when 5 mmol/L Mg2+ was included in the patch pipette solution, while no γj rectification was observed when no Mg2+ was added. Including 5 mmol/L Mg2+ in pipette solution significantly decreased the γj of homotypic Cx46 GJ with little change in homotypic Cx50 γj. A missense point variant in Cx46 (E43F) reduced the Mg2+-dependent reduction in γj of Cx46 E43F GJ, indicating that E43 might be partially responsible for Mg2+-dependent decrease in γj of Cx46. A comprehensive understanding of Mg2+ modulation of GJ at the individual channel level is useful in understanding factors in modulating GJ-mediated intercellular communication in health and diseases.

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.

Engineered Cx40 Variants Showed Heterotypic Colocalization and Increased GAP Junctional Coupling with Cx43

The gap junction (GJ) channel provides a low resistance passage for rapid action potential propagation in the heart. Both connexin40 (Cx40) and Cx43 are abundantly expressed in and frequently co-localized between atrial myocytes, possibly forming heterotypic GJ channels. However, conflicting results have been obtained on the functional status of heterotypic Cx40/Cx43 GJs. Here we provide experimental evidence that the docking and formation of heterotypic Cx40/Cx43 GJs can be substantially increased by designing Cx40 variants on the extracellular domains (E1 and E2). Specifically, Cx40 D55N and P193Q, substantially increased the probability to form GJ plaque-like structures at the cell-cell interfaces with Cx43. More importantly, the coupling conductance (Gj) of D55N/Cx43 and P193Q/Cx43 GJ channels are significantly increased from the Gj of Cx40/Cx43. Our homology models indicate that the electrostatic interactions and surface structures at the docking interface are key factors preventing Cx40 from docking to Cx43. Improving heterotypic Gj of these atrial connexins might be potentially useful in improving the coupling and synchronization of the atrial myocardium. Supported by HSFC of Canada.

Engineered Cx40 variants increased docking and function of heterotypic Cx40/Cx43 gap junction channels

Gap junction (GJ) channels provide low resistance passages for rapid action potential propagation in the heart. Both connexin40 (Cx40) and Cx43 are abundantly expressed in and frequently co-localized between atrial myocytes, possibly forming heterotypic GJ channels. However, conflicting results have been obtained on the functional status of heterotypic Cx40/Cx43 GJs. Here we provide experimental evidence that the docking and formation of heterotypic Cx40/Cx43 GJs can be substantially increased by designed Cx40 variants on the extracellular domains (E1 and E2). Specifically, Cx40 D55N and P193Q, substantially increased the probability to form GJ plaque-like structures at the cell–cell interfaces with Cx43 in model cells. More importantly the coupling conductance (Gj) of D55N/Cx43 and P193Q/Cx43 GJ channels are significantly increased from the Gj of Cx40/Cx43 in N2A cells. Our homology models indicate the electrostatic interactions and surface structures at the docking interface are key factors preventing Cx40 from docking to Cx43. Improving heterotypic Gj of these atrial connexins might be potentially useful in improving the coupling and synchronization of atrial myocardium.

Functional formation of heterotypic gap junction channels by connexins-40 and -43

Connexin40 (Cx40) and connexin43 (Cx43) are co-expressed in the cardiovascular system, yet their ability to form functional heterotypic Cx43/Cx40 gap junctions remains controversial. We paired Cx43 or Cx40 stably-transfected N2a cells to examine the formation and biophysical properties of heterotypic Cx43/Cx40 gap junction channels. Dual whole cell patch clamp recordings demonstrated that Cx43 and Cx40 form functional heterotypic gap junctions with asymmetric transjunctional voltage (Vj) dependent gating properties. The heterotypic Cx43/Cx40 gap junctions exhibited less Vj gating when the Cx40 cell was positive and pronounced gating when negative. Endogenous N2a cell connexin expression levels were 1,000-fold lower than exogenously expressed Cx40 and Cx43 levels, measured by real-time PCR and Western blotting methods, suggestive of heterotypic gap junction formation by exogenous Cx40 and Cx43. Imposing a [KCl] gradient across the heterotypic gap junction modestly diminished the asymmetry of the macroscopic normalized junctional conductance – voltage (Gj-Vj) curve when [KCl] was reduced by 50% on the Cx43 side and greatly exacerbated the Vj gating asymmetries when lowered on the Cx40 side. Pairing wild-type (wt) Cx43 with the Cx40 E9,13K mutant protein produced a nearly symmetrical heterotypic Gj-Vj curve. These studies conclusively demonstrate the ability of Cx40 and Cx43 to form rectifying heterotypic gap junctions, owing primarily to alternate amino-terminal (NT) domain acidic and basic amino acid differences that may play a significant role in the physiology and/or pathology of the cardiovascular tissues including cardiac conduction properties and myoendothelial intercellular communication.

Mix and match: Investigating heteromeric and heterotypic gap junction channels in model systems and native tissues

This review is based in part on a roundtable discussion session: "Physiological roles for heterotypic/heteromeric channels" at the 2013 International Gap Junction Conference (IGJC 2013) in Charleston, South Carolina. It is well recognized that multiple connexins can specifically co-assemble to form mixed gap junction channels with unique properties as a means to regulate intercellular communication. Compatibility determinants for both heteromeric and heterotypic gap junction channel formation have been identified and associated with specific connexin amino acid motifs. Hetero-oligomerization is also a regulated process; differences in connexin quality control and monomer stability are likely to play integral roles to control interactions between compatible connexins. Gap junctions in oligodendrocyte:astrocyte communication and in the cardiovascular system have emerged as key systems where heterotypic and heteromeric channels have unique physiologic roles. There are several methodologies to study heteromeric and heterotypic channels that are best applied to either heterologous expression systems, native tissues or both. There remains a need to use and develop different experimental approaches in order to understand the prevalence and roles for mixed gap junction channels in human physiology.

Heterotypic connexin50/connexin50 mutant gap junction channels reveal interactions between two hemichannels during transjunctional voltage‐dependent gating

To investigate transjunctional voltage (Vj)-dependent gating mechanisms of connexin50 (Cx50) gap junction (GJ) channels and to elucidate the relative contribution of each hemichannel of a heterotypic GJ channel to Vj-dependent gating, we performed dual voltage-clamp recordings on heterotypic GJ channels formed by Cx50 and a mutant, Cx50N9R or a chimera, Cx50-Cx36N. Our results provide evidence that the two component hemichannels interact with each other during Vj-dependent gating. Cx50/Cx50N9R heterotypic GJ channels exhibited asymmetrical Vj-dependent gating which cannot be ascribed to the function of an individual hemichannel for a certain polarity of voltage; instead it can only be ascribed to the combined effects of both hemichannels. Single GJ channel open dwell-time analyses showed that homotypic Cx50 channels adopted short-lived and long-lived open states. Heterotypic combinations of Cx50/Cx50N9R gave rise to shorter mean dwell-times when Cx50-expressing cells received relatively positive Vj, and longer mean dwell-times when positive Vj was applied at the Cx50N9R side. In contrast, Cx50/Cx50-Cx36N heterotypic channels showed asymmetrical Vj-dependent gating, which appears to be caused by enhanced and reduced Vj-gating sensitivity of Cx50-Cx36N and Cx50 hemichannels, respectively. Unitary conductance of the main open state of both types of heterotypic GJ channel cannot be simply predicted by assuming a Vj redistribution across the two hemichannels arranged in series in heterotypic GJ channels. Our data also reveal reasons for the invisibility of fast Vj-gating transitions from open to substate in homotypic Cx50N9R and Cx50-Cx36N channels.

Stochastic 16-State Model of Voltage Gating of Gap-Junction Channels Enclosing Fast and Slow Gates

Gap-junction (GJ) channels formed of connexin (Cx) proteins provide a direct pathway for electrical and metabolic cell-cell interaction. Each hemichannel in the GJ channel contains fast and slow gates that are sensitive to transjunctional voltage (Vj). We developed a stochastic 16-state model (S16SM) that details the operation of two fast and two slow gates in series to describe the gating properties of homotypic and heterotypic GJ channels. The operation of each gate depends on the fraction of Vj that falls across the gate (VG), which varies depending on the states of three other gates in series, as well as on parameters of the fast and slow gates characterizing 1), the steepness of each gate's open probability on VG; 2), the voltage at which the open probability of each gate equals 0.5; 3), the gating polarity; and 4), the unitary conductances of the gates and their rectification depending on VG. S16SM allows for the simulation of junctional current dynamics and the dependence of steady-state junctional conductance (gj,ss) on Vj. We combined global coordinate optimization algorithms with S16SM to evaluate the gating parameters of fast and slow gates from experimentally measured gj,ss-Vj dependencies in cells expressing different Cx isoforms and forming homotypic and/or heterotypic GJ channels.

Novel Transjunctional-Voltage Dependent Gating and Unitary Conductance Properties of the Heterotypic Gap Junction Channels Formed by Cx50 and Cx50 Chimera/Mutant

Gap junctions (GJ) are intercellular channels that connecting the cytoplasm of two cells. GJ channel conductance (Gj) is regulated by transjunctional voltage (Vj-gating), which is the voltage difference between the interiors of the cell pair. Previously, to elucidate the molecular mechanisms underlying the Cx50 GJ channel Vj-gating, domain swapping (e.g. replacing the amino-terminus of Cx50 by that of Cx36, named Cx50-Cx36N) and site-directed mutagenesis (e.g. Cx50N9R), along with dual whole-cell voltage-clamp experiments were carried out in neuroblastoma cells (N2A) (Xin et al., 2010). Here we performed studies on cell pairs forming heterotypic GJ channels between Cx50 and Cx50 chimera/mutant at macroscopic, as well as single channel level. Cx50/Cx50Cx36N heterotypic channels showed asymmetrical Vj-gating properties with respect to Vj = 0. The macroscopic Gj showed substantial reduction (by 82% at Vj = 100mV) when depolarizing Cx50-expressing cell, whereas a moderate reduction (by 61% at Vj = −100mV) when hyperpolarizing Cx50-expressing cell. This observation is in agreement with previous finding that Cx50 hemichannel gates when the cell is depolarized. Single Cx50/Cx50Cx36N heterotypic channel displayed a main conductance of ∼120pS at low Vj pulses (± 20mV), but the conductance rectified with increasing Vj pulses. Cx50/Cx50N9R heterotypic GJ channel also exhibited asymmetrical Vj-gating properties at macroscopic level, and single channel recordings indicated a main conductance of ∼150pS at Vj = 20mV. These results demonstrate that the Vj-gating properties and unitary conductance of Cx50 hemichannels were evidently modified when docking with Cx50-Cx36N or Cx50N9R mutants. These novel properties of heterotypic channels provide insights into the Vj-gating mechanisms of Cx50 GJ channels.

Asparagine 175 of Connexin32 Is a Critical Residue for Docking and Forming Functional Heterotypic Gap Junction Channels with Connexin26

The gap junction channel is formed by proper docking of two hemichannels. Depending on the connexin(s) in the hemichannels, homotypic and heterotypic gap junction channels can be formed. Previous studies suggest that the extracellular loop 2 (E2) is an important molecular domain for heterotypic compatibility. Based on the crystal structure of the Cx26 gap junction channel and homology models of heterotypic channels, we analyzed docking selectivity for several hemichannel pairs and found that the hydrogen bonds between E2 domains are conserved in a group of heterotypically compatible hemichannels, including Cx26 and Cx32 hemichannels. According to our model analysis, Cx32N175Y mutant destroys three hydrogen bonds in the E2-E2 interactions due to steric hindrance at the heterotypic docking interface, which makes it unlikely to dock with the Cx26 hemichannel properly. Our experimental data showed that Cx26-red fluorescent protein (RFP) and Cx32-GFP were able to traffic to cell-cell interfaces forming gap junction plaques and functional channels in transfected HeLa/N2A cells. However, Cx32N175Y-GFP exhibited mostly intracellular distribution and was occasionally observed in cell-cell junctions. Double patch clamp analysis demonstrated that Cx32N175Y did not form functional homotypic channels, and dye uptake assay indicated that Cx32N175Y could form hemichannels on the cell surface similar to wild-type Cx32. When Cx32N175Y-GFP- and Cx26-RFP-transfected cells were co-cultured, no colocalization was found at the cell-cell junctions between Cx32N175Y-GFP- and Cx26-RFP-expressing cells; also, no functional Cx32N175Y-GFP/Cx26-RFP heterotypic channels were identified. Both our modeling and experimental data suggest that Asn(175) of Cx32 is a critical residue for heterotypic docking and functional gap junction channel formation between the Cx32 and Cx26 hemichannels.

A Stochastic Model of Voltage-Gating of Connexin-Based Gap Junction Channels Containing Fast and Slow Gates

Connexins (Cxs), a family of membrane proteins, form gap junction (GJ) channels that provide a direct pathway for electrical and metabolic cell-cell interaction. Each hemichannel in the GJ channel contains fast and slow gates sensitive to transjunctional voltage (Vj). The fast gate operates between open and residual states with conductances of γF,o and γF,res, while the slow gate operates between open (γS,o) and fully closed (γS,closed=0) states; all γs rectify but at different degree. We developed a stochastic 16-state model (S16SM), which extends earlier reported S4SM (Paulauskas et al., 2009) and accounts operation of four gates in series, instead of two, to describe the gating properties of homotypic and heterotypic GJ channels.

Asparagine175 of Cx32 is a Critical Residue for Docking and Forming Functional Heterotypic Gap Junction Channels with Cx26

Gap junctions result from the docking of two hemichannels. Depend on the connexin(s) in the hemichannels, homotypic and heterotypic gap junction channels can be formed. Previous chimera studies with domain exchange between different connexins indicated that the extracellular loop2 (E2) is important molecular domain to heterotypic compatibility. Based on the high resolution structure of Cx26 gap junction channel and homology models of heterotypic channels, we analyzed docking selectivity for several hemichannel pairs, and found that the hydrogen bonds between E2 domains are conserved in several heterotypically compatible hemichannels, e.g. between Cx26 and Cx32. According to our model analysis, Cx32 mutation, Cx32N175Y, destroys three hydrogen bonds in the E2-E2 interactions at the heterotypic docking interface. Our model predicts that the Cx32N175Y hemichannel is unlikely to dock with Cx26 hemichannel properly due to steric hindrance at the docking interface. Experimentally, we tagged GFP and RFP to the carboxyl terminals of Cx32 and Cx26 to generate Cx32GFP (Cx32N175YGFP) and Cx26RFP, respectively. Our data showed that tagged Cx26 and Cx32 were able to traffic to cell interfaces forming gap junction plaque-like structures in transfected HeLa/N2A cells. However, Cx32N175YGFP exhibited mostly intracellular distribution and rarely seen in cell-cell interfaces. Double patch clamp analysis demonstrated that this Cx32 mutant did not form functional homotypic channels. When wild-type or mutant Cx32GFP expressing cells were cocultured with Cx26RFP expressing cells, Cx32GFP and Cx26RFP were frequently colocalized at the cell-cell interfaces and formed functional Cx32/Cx26 heterotypic channels. No colocalization was found at the cell-cell interfaces between Cx32N175Y and Cx26 expressing cells, also no functional Cx32N175Y/Cx26 heterotypic channels were identified. Our modeling and experimental data indicate that N175 of Cx32 is a critical residue for heterotypic docking between Cx32 and Cx26 hemichannels.

Diversity and properties of connexin gap junction channels

Gap junction channels are composed of two apposing hemichannels (connexons) in the contiguous cells and provide a direct pathway for electrical and metabolic signaling between adjacent cells. The family of connexin genes comprises 20 members in the mouse and 21 genes in the human genome. Connexins are expressed in all tissues except differentiated skeletal muscle, erythrocytes, and mature sperm cells. Various tissues express more than one type of connexins; therefore, homotypic, heterotypic, and heteromeric gap junction channels may form between cells. In this article, we briefly review basic gating and permeability properties of homotypic and heterotypic gap junction channels as well as recent achievements in the research of their regulation by transjunctional voltage, intracellular calcium, pH, and phosphorylation.

Heterotypic gap junction channels as voltage-sensitive valves for intercellular signaling

Gap junction (GJ) channels assembled from connexin (Cx) proteins provide a structural basis for direct electrical and metabolic cell-cell communication. By combining fluorescence imaging and dual whole-cell voltage clamp methods, we demonstrate that in response to transjunctional voltage (Vj) Cx43/Cx45 heterotypic GJs exhibit both Vj-gating and dye transfer asymmetries. The later is affected by ionophoresis of charged fluorescent dyes and voltage-dependent gating. We demonstrate that small differences in resting (holding) potentials of communicating cells can fully block (at relative negativity on Cx45 side) or enhance (at relative positivity on Cx45 side) dye transfer. Similarly, series of high frequency Vj pulses resembling bursts of action potentials (APs) can fully block or increase the transjunctional flux (Jj) of dye depending on whether pulses are generated in the cell expressing Cx43 or Cx45, respectively. Asymmetry of Jj-Vj dependence is enhanced or reduced when ionophoresis and Vj-gating act synergistically or antagonistically, whereas single channel permeability (Pgamma) remains unaffected. This modulation of intercellular signaling by Vj can play a crucial role in many aspects of intercellular communication in the adult, in embryonic development, and in tissue regeneration.

Gating, permselectivity and pH‐dependent modulation of channels formed by connexin57, a major connexin of horizontal cells in the mouse retina

Mouse connexin57 (Cx57) is expressed most abundantly in horizontal cells of the retina, and forms gap junction (GJ) channels, which constitute a structural basis for electrical and metabolic intercellular communication, and unapposed hemichannels (UHCs) that are involved in an exchange of ions and metabolites between the cytoplasm and extracellular milieu. By combining fluorescence imaging and dual whole-cell voltage clamp methods, we showed that HeLa cells expressing Cx57 and C-terminally fused with enhanced green fluorescent protein (Cx57-EGFP) form junctional plaques (JPs) and that only cell pairs exhibiting at least one JP demonstrate cell-to-cell electrical coupling and transfer of negatively and positively charged dyes with molecular mass up to approximately 400 Da. The permeability of the single Cx57 GJ channel to Alexa fluor-350 is approximately 90-fold smaller than the permeability of Cx43, while its single channel conductance (57 pS) is only 2-fold smaller than Cx43 (110 pS). Gating of Cx57-EGFP/Cx45 heterotypic GJ channels reveal that Cx57 exhibit a negative gating polarity, i.e. channels tend to close at negativity on the cytoplasmic side of Cx57. Alkalization of pH(i) from 7.2 to 7.8 increased gap junctional conductance (g(j)) of approximately 100-fold with pK(a) = 7.41. We show that this g(j) increase was caused by an increase of both the open channel probability and the number of functional channels. Function of Cx57 UHCs was evaluated based on the uptake of fluorescent dyes. We found that under control conditions, Cx57 UHCs are closed and open at [Ca(2+)](o) = approximately 0.3 mm or below, demonstrating that a moderate reduction of [Ca(2+)](o) can facilitate the opening of Cx57 UHCs. This was potentiated with intracellular alkalization. In summary, our data show that the open channel probability of Cx57 GJs can be modulated by pH(i) with very high efficiency in the physiologically relevant range and may explain pH-dependent regulation of cell-cell coupling in horizontal cell in the retina.

A Stochastic Four-State Model of Contingent Gating of Gap Junction Channels Containing Two “Fast” Gates Sensitive to Transjunctional Voltage

Connexins, a family of membrane proteins, form gap junction (GJ) channels that provide a direct pathway for electrical and metabolic signaling between cells. We developed a stochastic four-state model describing gating properties of homotypic and heterotypic GJ channels each composed of two hemichannels (connexons). GJ channel contain two “fast” gates (one per hemichannel) oriented opposite in respect to applied transjunctional voltage (Vj). The model uses a formal scheme of peace-linear aggregate and accounts for voltage distribution inside the pore of the channel depending on the state, unitary conductances and gating properties of each hemichannel. We assume that each hemichannel can be in the open state with conductance γh,o and in the residual state with conductance γh,res, and that both γh,o and γh,res rectifies. Gates can exhibit the same or different gating polarities. Gating of each hemichannel is determined by the fraction of Vj that falls across the hemichannel, and takes into account contingent gating when gating of one hemichannel depends on the state of apposed hemichannel. At the single-channel level, the model revealed the relationship between unitary conductances of hemichannels and GJ channels and how this relationship is affected by γh,o and γh,res rectification. Simulation of junctions containing up to several thousands of homotypic or heterotypic GJs has been used to reproduce experimentally measured macroscopic junctional current and Vj-dependent gating of GJs formed from different connexin isoforms. Vj-gating was simulated by imitating several frequently used experimental protocols: 1), consecutive Vj steps rising in amplitude, 2), slowly rising Vj ramps, and 3), series of Vj steps of high frequency. The model was used to predict Vj-gating of heterotypic GJs from characteristics of corresponding homotypic channels. The model allowed us to identify the parameters of Vj-gates under which small changes in the difference of holding potentials between cells forming heterotypic junctions effectively modulates cell-to-cell signaling from bidirectional to unidirectional. The proposed model can also be used to simulate gating properties of unapposed hemichannels.

Human connexin26 and connexin30 form functional heteromeric and heterotypic channels

Mutations in GJB2 and GJB6, the genes that encode the human gap junction proteins connexin26 (Cx26) and connexin30 (Cx30), respectively, cause hearing loss. Cx26 and Cx30 are both expressed in the cochlea, leading to the potential formation of heteromeric hemichannels and heterotypic gap junction channels. To investigate their interactions, we expressed human Cx26 and Cx30 individually or together in HeLa cells. When they were expressed together, Cx26 and Cx30 appeared to interact directly (by their colocalization in gap junction plaques, by coimmunoprecipitation, and by fluorescence resonance energy transfer). Scrape-loading cells that express either Cx26 or Cx30 demonstrated that Cx26 homotypic channels robustly transferred both cationic and anionic tracers, whereas Cx30 homotypic channels transferred cationic but not anionic tracers. Cells expressing both Cx26 and Cx30 also transferred both cationic and anionic tracers by scrape loading, and the rate of calcein (an anionic tracer) transfer was intermediate between their homotypic counterparts by fluorescence recovery after photobleaching. Fluorescence recovery after photobleaching also showed that Cx26 and Cx30 form functional heterotypic channels, allowing the transfer of calcein, which did not pass the homotypic Cx30 channels. Electrophysiological recordings of cell pairs expressing different combinations of Cx26 and/or Cx30 demonstrated unique gating properties of cell pairs expressing both Cx26 and Cx30. These results indicate that Cx26 and Cx30 form functional heteromeric and heterotypic channels, whose biophysical properties and permeabilities are different from their homotypic counterparts.

Permeability of homotypic and heterotypic gap junction channels formed of cardiac connexins mCx30.2, Cx40, Cx43, and Cx45

We examined the permeabilities of homotypic and heterotypic gap junction (GJ) channels formed of rodent connexins (Cx) 30.2, 40, 43, and 45, which are expressed in the heart and other tissues, using fluorescent dyes differing in net charge and molecular mass. Combining fluorescent imaging and electrophysiological recordings in the same cell pairs, we evaluated the single-channel permeability (P(gamma)). All homotypic channels were permeable to the anionic monovalent dye Alexa Fluor-350 (AF(350)), but mCx30.2 channels exhibited a significantly lower P(gamma) than the others. The anionic divalent dye Lucifer yellow (LY) remained permeant in Cx40, Cx43, and Cx45 channels, but transfer through mCx30.2 channels was not detected. Heterotypic channels generally exhibited P(gamma) values that were intermediate to the corresponding homotypic channels. P(gamma) values of mCx30.2/Cx40, mCx30.2/Cx43, or mCx30.2/Cx45 heterotypic channels for AF(350) were similar and approximately twofold higher than P(gamma) values of mCx30.2 homotypic channels. Permeabilities for cationic dyes were assessed only qualitatively because of their binding to nucleic acids. All homotypic and heterotypic channel configurations were permeable to ethidium bromide and 4,6-diamidino-2-phenylindole. Permeability for propidium iodide was limited only for GJ channels that contain at least one mCx30.2 hemichannel. In summary, we have demonstrated that Cx40, Cx43, and Cx45 are permeant to all examined cationic and anionic dyes, whereas mCx30.2 demonstrates permeation restrictions for molecules with molecular mass over approximately 400 Da. The ratio of single-channel conductance to permeability for AF(350) was approximately 40- to 170-fold higher for mCx30.2 than for Cx40, Cx43, and Cx45, suggesting that mCx30.2 GJs are notably more adapted to perform electrical rather than metabolic cell-cell communication.

Gating Properties of Heterotypic Gap Junction Channels Formed of Connexins 40, 43, and 45

Connexins (Cxs) 40, 43, and 45 are expressed in many different tissues, but most abundantly in the heart, blood vessels, and the nervous system. We examined formation and gating properties of heterotypic gap junction (GJ) channels assembled between cells expressing wild-type Cx40, Cx43, or Cx45 and their fusion forms tagged with color variants of green fluorescent protein. We show that these Cxs, with exception of Cxs 40 and 43, are compatible to form functional heterotypic GJ channels. Cx40 and Cx43 hemichannels are unable or effectively impaired in their ability to dock and/or assemble into junctional plaques. When cells expressing Cx45 contacted those expressing Cx40 or Cx43 they readily formed junctional plaques with cell-cell coupling characterized by asymmetric junctional conductance dependence on transjunctional voltage, V j. Cx40/Cx45 heterotypic GJ channels preferentially exhibit V j-dependent gating transitions between open and residual states with a conductance of ∼42 pS; transitions between fully open and closed states with conductance of ∼52 pS in magnitude occur at substantially lower (∼10-fold) frequency. Cx40/Cx45 junctions demonstrate electrical signal transfer asymmetry that can be modulated between unidirectional and bidirectional by small changes in the difference between holding potentials of the coupled cells. Furthermore, both fast and slow gating mechanisms of Cx40 exhibit a negative gating polarity.

Connexin31 cannot functionally replace connexin43 during cardiac morphogenesis in mice

In the gastrulating mouse embryo, the gap junction protein connexin43 is expressed exclusively in cells derived from the inner cell mass, whereas connexin31 is expressed in cells of the trophoblast lineage. Since connexin43 and connexin31 do not form heterotypic gap junction channels in exogenous expression systems, such as HeLa cells and Xenopus oocytes, previous studies have suggested that the incompatibility of these two connexins could contribute to the separation of connexin43-expressing and connexin31-expressing compartments between embryo and extraembryonic tissues at gastrulation, respectively. Thus, we have generated connexin43 knock-in connexin31 mice, in which the coding region of the connexin43 gene was replaced by that of connexin31. Interbreeding of heterozygous connexin43 knock-in connexin31 mice resulted in homozygous connexin43 knock-in connexin31 mice, but none of them survived to adulthood. As these mice were born at the expected Mendelian frequency, we conclude that the reported incompatibility of connexin43 and connexin31 to form heterotypic gap junction channels does not interfere with normal embryonic development. Neonatal homozygous connexin43 knock-in connexin31 hearts showed malformation in the subpulmonary outlet of the right ventricle, similar to general connexin43-deficient mice. Electrocardiograms of neonatal hearts in homozygous connexin43 knock-in connexin31 mice revealed significantly low voltage of the QRS complex. This is in contrast to previous results from our laboratory which showed that replacement of connexin43 by connexin40 resulted in morphologically and functionally normal hearts. We conclude that connexin31 cannot functionally replace connexin43 during cardiac morphogenesis.