Chemical gating of gap junction channels

Chemical gating of gap junction channels is a complex phenomenon that may involve intra- and intermolecular interactions among connexin domains and a cytosolic molecule (calmodulin?) that may function as channel plug. This article focuses on the methodology we have employed for studying the molecular basis of chemical gating by lowered cytosolic pH. Our approach has combined molecular genetics and biophysics, using exposure to 100% CO(2) for assaying chemical gating efficiency. Chimeras of connexin 32 (Cx32) and connexin 38 (Cx38) and Cx32 mutants modified at residues of the cytoplasmic loop, the initial C-terminus domain, or both have been expressed in Xenopus oocytes, and channel expression and gating have been tested electrophysiologically by double voltage clamp. In addition, various channel forms, including homotypic, heterotypic, and heteromeric channel combinations, have been evaluated for chemical gating sensitivity.     

Magnesium gating of cardiac gap junction channels

We aimed to study kinetics of modulation by intracellular Mg²⁺ of cardiac gap junction (Mg²⁺ gate). Paired myocytes of guinea-pig ventricle were superfused with solutions containing various concentrations of Mg²⁺. In order to rapidly apply Mg²⁺ to one aspect of the gap junction, the non-junctional membrane of one of the pair was perforated at nearly the connecting site by pulses of nitrogen laser beam. The gap junction conductance (G_j) was measured by clamping the membrane potential of the other cell using two-electrode voltage clamp method. The laser perforation immediately increased G_j, followed by slow G_j change with time constant of 3.5 s at 10 mM Mg²⁺. Mg²⁺ more than 1.0 mM attenuated dose-dependently the gap junction conductance and lower Mg²⁺ (0.6 mM) increased G_j with a Hill coefficient of 3.4 and a half-maximum effective concentration of 0.6 mM. The time course of G_j changes was fitted by single exponential function, and the relationship between the reciprocal of time constant and Mg²⁺ concentration was almost linear. Based on the experimental data, a mathematical model of Mg²⁺ gate with one open state and three closed states well reproduced experimental results. One-dimensional cable model of thirty ventricular myocytes connected to the Mg²⁺ gate model suggested a pivotal role of the Mg²⁺ gate of gap junction under pathological conditions.     

Voltage-dependent gating of single gap junction channels in an insect cell line.

De novo formation of cell pairs was used to examine the gating properties of single gap junction channels. Two separate cells of an insect cell line (clone C6/36, derived from the mosquito Aedes albopictus) were pushed against each other to provoke formation of gap junction channels. A dual voltage-clamp method was used to control the voltage gradient between the cells (Vj) and measure the intercellular current (Ij). The first sign of channel activity was apparent 4.7 min after cell contact. Steady-state coupling reached after 30 min revealed a conductance of 8.7 nS. Channel formation involved no leak between the intra- and extracellular space. The first opening of a newly formed channel was slow (25-28 ms). Each preparation passed through a phase with only one operational gap junction channel. This period was exploited to examine the single channel properties. We found that single channels exhibit several conductance states with different conductances gamma j; a fully open state (gamma j(main state)), several substates (gamma j(substates)), a residual state (gamma j(residual)) and a closed state (gamma j(closed)). The gamma j(main state) was 375 pS, and gamma j(residual) ranged from 30 to 90 pS. The transitions between adjacent substates were 1/7-1/4 of gamma j(main state). Vj had no effect on gamma j(main state), but slightly affected gamma j (residual). The lj transitions involving gamma j(closed) were slow (15-60 ms), whereas those not involving gamma j(closed) were fast (< 2 ms). An increase in Vj led to a decrease in open channel probability. Depolarization of the membrane potential (Vm) increased the incidence of slow transitions leading to gamma j(closed). We conclude that insect gap junctions possess two gates, a fast gate controlled by Vj and giving rise to gamma j(substates) and gamma j(residual), and a slow gate sensitive to Vm and able to close the channel completely.     

Correlative studies of gating in Cx46 and Cx50 hemichannels and gap junction channels

Transjunctional voltage (V(j)) gating of gap junction (GJ) channels formed of connexins has been proposed to occur by gating of the component hemichannels. We took advantage of the ability of Cx46 and Cx50 to function as unapposed hemichannels to identify gating properties intrinsic to hemichannels and how they contribute to gating of GJ channels. We show that Cx46 and Cx50 hemichannels contain two distinct gating mechanisms that generate reductions in conductance for both membrane polarities. At positive voltages, gating is similar in Cx46 and Cx50 hemichannels, primarily showing increased transitioning to long-lived substates. At negative voltages, Cx46 currents deactivate completely and the underlying single hemichannels exhibit transitions to a fully closed state. In contrast, Cx50 currents do not deactivate completely at negative voltages and the underlying single hemichannels predominantly exhibit transitions to various substates. Transitions to a fully closed state occur, but are infrequent. In the respective GJ channels, both forms of gating contribute to the reduction in conductance by V(j). However, examination of gating of mutant hemichannels and GJ channels in which the Asp at position 3 was replaced with Asn (D3N) showed that the positive hemichannel gate predominantly closes Cx50 GJs, whereas the negative hemichannel gate predominantly closes Cx46 GJs in response to V(j). We also report, for the first time, single Cx50 hemichannels in oocytes to be inwardly rectifying, high conductance channels (gamma = 470 pS). The antimalarial drug mefloquine, which selectively blocks Cx50 and not Cx46 GJs, shows the same selectivity in Cx50 and Cx46 hemichannels indicating that the actions of such uncoupling agents, like voltage gating, are intrinsic hemichannel properties.     

Two distinct gating mechanisms in gap junction channels: CO2-sensitive and voltage-sensitive.

The chemical gating of single-gap junction channels was studied by the dual whole-cell voltage-clamp method in HeLa cells transfected with connexin43 (HeLa43) and in fibroblasts from sciatic nerves. Junctional current (Ij), single-channel conductance, and Ij kinetics were studied in cell pairs during CO2 uncoupling and recoupling at small transjunctional voltages (Vj < 35 mV: Vj gating absent) and at high Vj (Vj > 40 mV: Vj gating strongly activated). In the absence of Vj gating, CO2 exclusively caused Ij slow transitions from open to closed channel states (mean transition time: approximately 10 ms), corresponding to a single-channel conductance of approximately 120 pS. At Vj > 40 mV, Vj gating induced fast Ij flickering between open, gamma j(main state), and residual, gamma j(residual), states (transition time: approximately 2 ms). The ratio gamma j(main state)/gamma j(residual) was approximately 4-5. No obvious correlation between Ij fast flickering and CO2 treatment was noticed. At high Vj, in addition to slow Ij transitions between open and closed states, CO2 induced slow transitions between residual and closed states. During recoupling, each channel reopened by a slow transition (mean transition time: approximately 10 ms) from closed to open state (rarely from closed to residual state). Fast Ij flickering between open and residual states followed. The data are in agreement with the hypothesis that gap junction channels possess two gating mechanisms, and indicate that CO2 induces channel gating exclusively by the slow gating mechanism.     

Connexin-based gap junction hemichannels: gating mechanisms

Connexins (Cxs) form hemichannels and gap junction channels. Each gap junction channel is composed of two hemichannels, also termed connexons, one from each of the coupled cells. Hemichannels are hexamers assembled in the ER, the Golgi, or a post Golgi compartment. They are transported to the cell surface in vesicles and inserted by vesicle fusion, and then dock with a hemichannel in an apposed membrane to form a cell-cell channel. It was thought that hemichannels should remain closed until docking with another hemichannel because of the leak they would provide if their permeability and conductance were like those of their corresponding cell-cell channels. Now it is clear that hemichannels formed by a number of different connexins can open in at least some cells with a finite if low probability, and that their opening can be modulated under various physiological and pathological conditions. Hemichannels open in different kinds of cells in culture with conductance and permeability properties predictable from those of the corresponding gap junction channels. Cx43 hemichannels are preferentially closed in cultured cells under resting conditions, but their open probability can be increased by the application of positive voltages and by changes in protein phosphorylation and/or redox state. In addition, increased activity can result from the recruitment of hemichannels to the plasma membrane as seen in metabolically inhibited astrocytes. Mutations of connexins that increase hemichannel open probability may explain cellular degeneration in several hereditary diseases. Taken together, the data indicate that hemichannels are gated by multiple mechanisms that independently or cooperatively affect their open probability under physiological as well as pathological conditions.     

Modulation of metabolic communication through gap junction channels by transjunctional voltage; synergistic and antagonistic effects of gating and ionophoresis

Gap junction (GJ) channels assembled from connexin (Cx) proteins provide a structural basis for direct electrical and metabolic cell-cell communication. Here, we focus on gating and permeability properties of Cx43/Cx45 heterotypic GJs exhibiting asymmetries of both voltage-gating and transjunctional flux (J(j)) of fluorescent dyes depending on transjunctional voltage (V(j)). Relatively small differences in the resting potential of communicating cells can substantially reduce or enhance this flux at relative negativity or positivity on Cx45 side, respectively. Similarly, series of V(j) pulses resembling bursts of action potentials (APs) reduce J(j) when APs initiate in the cell expressing Cx43 and increase J(j) when APs initiate in the cell expressing Cx45. J(j) of charged fluorescent dyes is affected by ionophoresis and V(j)-gating and the asymmetry of J(j)-V(j) dependence in heterotypic GJs is enhanced or reduced when ionophoresis and V(j)-gating work in a synergistic or antagonistic manner, respectively. Modulation of cell-to-cell transfer of metabolites and signaling molecules by V(j) may occur in excitable as well as non-excitable tissues and may be more expressed in the border between normal and pathological regions where intercellular gradients of membrane potential and concentration of ions are substantially altered. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Assembly of gap junction channels: mechanism, effects of calmodulin antagonists and identification of connexin oligomerization determinants.

The assembly of connexins (Cxs) into gap junction intercellular communication channels was studied. An in vitro cell-free synthesis system showed that formation of the hexameric connexon hemichannels involved dimeric and tetrameric connexin intermediates. Cx32 contains two putative cytoplasmic calmodulin-binding sites, and their role in gap junction channel assembly was investigated. The oligomerization of Cx32 into connexons was reversibly inhibited by a calmodulin-binding synthetic peptide, and by W7, a naphthalene sulfonamide calmodulin antagonist. Removing the calmodulin-binding site located at the carboxyl tail of Cx32 limited connexon formation and resulted in an accumulation of intermediate connexin oligomers. This truncation mutant, Cx32Delta215, when transiently expressed in COS-7 cells, accumulated intracellularly and had failed to target to gap junctions. Immunoprecipitation studies suggested that a C-terminal sequence of Cx32 incorporating the calmodulin-binding site was required for the formation of hetero-oligomers of Cx26 and Cx32 but not for Cx32 homomeric association. A chimera, Cx32TM3CFTR, in which the third transmembrane and proposed channel lining sequence of Cx32 was substituted by a transmembrane sequence of the cystic fibrosis transmembrane conductance regulator, did not oligomerize in vitro and it accumulated intracellularly when expressed in COS-7 cells. The results indicate that amino-acid sequences in the third transmembrane domain and a calmodulin-binding domain in the cytoplasmic tail of Cx32 are likely candidates for regulating connexin oligomerization.     

Self-phosphorylation modulates the gating of rat liver gap junction channels: a nonstationary noise analysis

The effect of phosphorylation on the gating of rat liver gap junction hemichannels (Cx 32) has been investigated. It has been shown that self-phosphorylation of rat liver Cx 32 protein reduces the permeability of proteoliposomes as well the current flowing through multichannels in lipid bilayer membrane (BLM). The low frequency power spectral density analyses of nonstationary noise evolved due to the gating of Cx 32 multichannels demonstrated that self-phosphorylation modulated the channel functioning. A clear change in the power spectrum slopes (alpha) of the nonstationary noise profiles confirmed the modulation of the channel dynamics due to self-regulation.     

Isolation and purification of gap junction channels

This paper reports methods we have developed to solubilize gap junction channels, or connexons, from isolated gap junctions and to purify them in milligram quantities. Two sources of material are used: rat liver gap junctions and gap junctions produced by infecting insect cells with a baculovirus containing the cDNA for human liver beta 1 protein (connexin 32). Complete solubilization is obtained with long chain detergents (lauryl dimethyl amineoxide, dodecyl maltoside) and requires high ionic strength and high pH as well as reducing conditions. The purification involves chromatography on hydroxylapatite and gel filtration on Superose 6. A homogeneous product is indicated by a single band on a silver-stained gel and a homogeneous population of doughnut-shaped particles under the electron microscope. These particles have hexameric symmetry. The purified connexons have a tendency to form aggregates: filaments and sheets. The filaments grow by end-to-end association of connexons and are nonpolar, suggesting that the connexons are paired as in the cell-to-cell channel. The sheets grow by lateral association of the filaments.     

Gating of gap junction channels.

Gap junctional conductance ( gj ) in various species is gated by voltage and intracellular pH (pHi). In amphibian embryos, gj is reduced to half by a 14 mV transjunctional voltage ( Vj ), a change that in fish embryo requires approximately 28 mV. Crayfish septate axon and pairs of dissociated rat myocytes show no voltage dependence of gj over a range of Vj greater than +/- 50 mV. In fish and amphibian blastomeres , gj is steeply decreased by decrease in pHi (n, Hill coefficient: 4.5) and the apparent pKH (7.3) is in the physiological range. In crayfish septate axon the pKH is lower (6.7) and the curve is less steep (n = 2.7). Rises in cytoplasmic Ca can also decrease gj but much higher concentrations are required (greater than 0.1 mM in fish blastomeres). Voltage and pH gates on gap junctions in amphibian embryos appear independent. In squid blastomeres pH gates exhibit some sensitivity to potential, both transjunctional and between inside and outside. A pharmacology of gap junctions is being developed: certain agents block gj directly (aldehydes, alcohols, NEM in crayfish); others block by decreasing pHi (esters that are hydrolyzed by intrinsic esterases, NEM in vertebrates, and, as in the experiments demonstrating the effect of pHi, weak acids). Certain agents block pH sensitivity without affecting voltage dependence (retinoic acid, glutaraldehyde, EEDQ), further indicating separateness of pH and voltage gates. These studies demonstrate a dynamics of gap junctional conductance and variability in gating in a series of possibly homologous membrane channels.     

Selective permeability of gap junction channels

Gap junctions mediate the transfer of small cytoplasmic molecules between adjacent cells. A family of gap junction proteins exist that form channels with unique properties, and differ in their ability to mediate the transfer of specific molecules. Mutations in a number of individual gap junction proteins, called connexins, cause specific human diseases. Therefore, it is important to understand how gap junctions selectively move molecules between cells. Rules that dictate the ability of a molecule to travel through gap junction channels are complex. In addition to molecular weight and size, the ability of a solute to transverse these channels depends on its net charge, shape, and interactions with specific connexins that constitute gap junctions in particular cells. This review presents some data and interpretations pertaining to mechanisms that govern the differential transfer of signals through gap junction channels.     

Structural organization of gap junction channels

Gap junctions were initially described morphologically, and identified as semi-crystalline arrays of channels linking two cells. This suggested that they may represent an amenable target for electron and X-ray crystallographic studies in much the same way that bacteriorhodopsin has. Over 30 years later, however, an atomic resolution structural solution of these unique intercellular pores is still lacking due to many challenges faced in obtaining high expression levels and purification of these structures. A variety of microscopic techniques, as well as NMR structure determination of fragments of the protein, have now provided clearer and correlated views of how these structures are assembled and function as intercellular conduits. As a complement to these structural approaches, a variety of mutagenic studies linking structure and function have now allowed molecular details to be superimposed on these lower resolution structures, so that a clearer image of pore architecture and its modes of regulation are beginning to emerge.     

Structural organization of gap junction channels

Gap junctions were initially described morphologically, and identified as semi-crystalline arrays of channels linking two cells. This suggested that they may represent an amenable target for electron and X-ray crystallographic studies in much the same way that bacteriorhodopsin has. Over 30 years later, however, an atomic resolution structural solution of these unique intercellular pores is still lacking due to many challenges faced in obtaining high expression levels and purification of these structures. A variety of microscopic techniques, as well as NMR structure determination of fragments of the protein, have now provided clearer and correlated views of how these structures are assembled and function as intercellular conduits. As a complement to these structural approaches, a variety of mutagenic studies linking structure and function have now allowed molecular details to be superimposed on these lower resolution structures, so that a clearer image of pore architecture and its modes of regulation are beginning to emerge.