Dynamic model for ventricular junctional conductance during the cardiac action potential

The ventricular action potential was applied to paired neonatal murine ventricular myocytes in the dual whole cell configuration. During peak action potential voltages >100 mV, junctional conductance (g(j)) declined by 50%. This transjunctional voltage (V(j))-dependent inactivation exhibited two time constants that became progressively faster with increasing V(j). G(j) returned to initial peak values during action potential repolarization and even exceeded peak g(j) values during the final 5% of repolarization. This facilitation of g(j) was observed <30 mV during linearly decreasing V(j) ramps. The same behavior was observed in ensemble averages of individual gap junction channels with unitary conductances of 100 pS or lower. Immunohistochemical fluorescent micrographs and immunoblots detect prominent amounts of connexin (Cx)43 and lesser amounts of Cx40 and Cx45 proteins in cultured ventricular myocytes. The time dependence of the g(j) curves and channel conductances are consistent with the properties of predominantly homomeric Cx43 gap junction channels. A mathematical model depicting two inactivation and two recovery phases accurately predicts the ventricular g(j) curves at different rates of stimulation and repolarization. Functional differences are apparent between ventricular myocytes and Cx43-transfected N2a cell gap junctions that may result from posttranslational modification. These observations suggest that gap junctions may play a role in the development of conduction block and the genesis and propagation of triggered arrhythmias under conditions of slowed conduction (<10 cm/s).     

Gating characteristics of a steeply voltage-dependent gap junction channel in rat Schwann cells

The gating properties of macroscopic and microscopic gap junctional currents were compared by applying the dual whole cell patch clamp technique to pairs of neonatal rat Schwann cells. In response to transjunctional voltage pulses (Vj), macroscopic gap junctional currents decayed exponentially with time constants ranging from < 1 to < 10 s before reaching steady-state levels. The relationship between normalized steady-state junctional conductance (Gss) and (Vj) was well described by a Boltzmann relationship with e-fold decay per 10.4 mV, representing an equivalent gating charge of 2.4. At Vj > 60 mV, Gss was virtually zero, a property that is unique among the gap junctions characterized to date. Determination of opening and closing rate constants for this process indicated that the voltage dependence of macroscopic conductance was governed predominantly by the closing rate constant. In 78% of the experiments, a single population of unitary junctional currents was detected corresponding to an unitary channel conductance of approximately 40 pS. The presence of only a limited number of junctional channels with identical unitary conductances made it possible to analyze their kinetics at the single channel level. Gating at the single channel level was further studied using a stochastic model to determine the open probability (Po) of individual channels in a multiple channel preparation. Po decreased with increasing Vj following a Boltzmann relationship similar to that describing the macroscopic Gss voltage dependence. These results indicate that, for Vj of a single polarity, the gating of the 40 pS gap junction channels expressed by Schwann cells can be described by a first order kinetic model of channel transitions between open and closed states.     

Gating of mammalian cardiac gap junction channels by transjunctional voltage.

Numerous two-cell voltage-clamp studies have concluded that the electrical conductance of mammalian cardiac gap junctions is not modulated by the transjunctional voltage (Vj) profile, although gap junction channels between low conductance pairs of neonatal rat ventricular myocytes are reported to exhibit Vj-dependent behavior. In this study, the dependence of macroscopic gap junctional conductance (gj) on transjunctional voltage was quantitatively examined in paired 3-d neonatal hamster ventricular myocytes using the double whole-cell patch-clamp technique. Immunolocalization with a site-specific antiserum directed against amino acids 252-271 of rat connexin43, a 43-kD gap junction protein as predicted from its cDNA sequence, specifically stained zones of contact between cultured myocytes. Instantaneous current-voltage (Ij-Vj) relationships of neonatal hamster myocyte pairs were linear over the entire voltage range examined (0 less than or equal to Vj less than or equal to +/- 100 mV). However, the steady-state Ij-Vj relationship was nonlinear for Vj greater than +/- 50 mV. Both inactivation and recovery processes followed single exponential time courses (tau inactivation = 100-1,000 ms, tau recovery approximately equal to 300 ms). However, Ij recovered rapidly upon polarity reversal. The normalized steady-state junctional conductance-voltage relationship (Gss-Vj) was a bell-shaped curve that could be adequately described by a two-state Boltzmann equation with a minimum Gj of 0.32-0.34, a half-inactivation voltage of -69 and +61 mV and an effective valence of 2.4-2.8. Recordings of gap junction channel currents (ij) yielded linear ij-Vj relationships with slope conductances of approximately 20-30 and 45-50 pS. A kinetic model, based on the Boltzmann relationship and the polarity reversal data, suggests that the opening (alpha) and closing (beta) rate constants have nearly identical voltage sensitivities with a Vo of +/- 62 mV. The data presented in this study are not consistent with the contingent gating scheme (for two identical gates in series) proposed for other more Vj-dependent gap junctions and alternatively suggest that each gate responds to the applied Vj independently of the state (open or closed) of the other gate.     

Gap junctions in the rabbit sinoatrial node

In comparison to the cellular basis of pacemaking, the electrical interactions mediating synchronization and conduction in the sinoatrial node are poorly understood. Therefore, we have taken a combined immunohistochemical and electrophysiological approach to characterize gap junctions in the nodal area. We report that the pacemaker myocytes in the center of the rabbit sinoatrial node express the gap junction proteins connexin (Cx)40 and Cx46. In the periphery of the node, strands of pacemaker myocytes expressing Cx43 intermingle with strands expressing Cx40 and Cx46. Biophysical properties of gap junctions in isolated pairs of pacemaker myocytes were recorded under dual voltage clamp with the use of the perforated-patch method. Macroscopic junctional conductance ranged between 0.6 and 25 nS with a mean value of 7.5 nS. The junctional conductance did not show a pronounced sensitivity to the transjunctional potential difference. Single-channel recordings from pairs of pacemaker myocytes revealed populations of single-channel conductances at 133, 202, and 241 pS. With these single-channel conductances, the observed average macroscopic junctional conductance, 7.5 nS, would require only 30-60 open gap junction channels.     

Voltage-dependent blockade of connexin40 gap junctions by spermine

The effects of spermine and spermidine, endogenous polyamines that block many forms of ion channels, were investigated in homotypic connexin (Cx)-40 gap junctions expressed in N2A cells. Spermine blocked up to 95% of I(j) through homotypic Cx40 gap junctions in a concentration- and transjunctional voltage (V(j))-dependent manner. V(j) was varied from 5 to 50 mV in 5-mV steps and the dissociation constants (K(m)) were determined from spermine concentrations ranging from 10 micro M to 2 mM. The K(m) values ranged from 4.9 mM to 107 micro M for 8.6 < or = V(j) < or = 37.7 mV, within the physiological range of intracellular spermine for V(j) > or = 20 mV. The K(m) values for spermidine were > or = 5 mM. Estimates of the electrical distance (delta) for spermine (z = +4) and spermidine (z = +3) were 0.96 and 0.76 respectively. Cx40 single channel conductance was 129 pS in the presence of 2-mM spermine and channel open probability was significantly reduced in a V(j)-dependent manner. Similar concentrations of spermine did not block I(j) through homotypic Cx43 gap junctions, indicating that spermine selectively blocks Cx40 gap junctions. This is contrary to our previous findings that large tetraalkylammonium ions, also known to block several forms of ion channels, block junctional currents (I(j)) through homotypic connexin Cx40 and Cx43 gap junctions.     

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.     

Exchange of gating properties between rat cx46 and chicken cx45.6

Cx46 and Cx50 are coexpressed in lens fiber cells where they form fiber-fiber gap junctions. Recent studies have shown that both proteins play a critical role in maintaining lens transparency. Although both Cx46 and Cx50 (or its chicken ortholog, Cx45.6) show a high degree of sequence homology, they exhibit marked differences in gap junctional channel gating, unitary gap junctional channel conductance, and hemichannel gating. To better understand which regions of the protein are responsible for these functional differences, we have constructed a series of chimeric Cx46-Cx45.6 gap junctional proteins in which a single transmembrane or intracellular domain of Cx45.6 was replaced with the corresponding domain of Cx46, expressed them in Xenopus oocyte pairs or N2A cells, and examined the resulting gap junctional conductances. Our results showed that four out of six of the chimeras induced high levels of gap junctional coupling. Of these chimeras, only Cx45.6-46NT showed significant changes in voltage-dependent gating properties. Exchanging the N-terminus had multiple effects. It slowed the inactivation kinetics of the macroscopic junctional currents so that they resembled those of Cx46, reduced the voltage sensitivity of the steady-state junctional conductance, and decreased the conductance of single gap junctional channels. Additional point mutations identified a uniquely occurring arginine in the N-terminus of Cx46 as the main determinant for the change in voltage-dependent gating.     

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 (V_j) dependent gating properties. The heterotypic Cx43/Cx40 gap junctions exhibited less V_j 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 (G_j-V_j) curve when [KCl] was reduced by 50% on the Cx43 side and greatly exacerbated the V_j 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 G_j-V_j 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.     

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 (V_j) dependent gating properties. The heterotypic Cx43/Cx40 gap junctions exhibited less V_j 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 (G_j-V_j) curve when [KCl] was reduced by 50% on the Cx43 side and greatly exacerbated the V_j 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 G_j-V_j 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.     

Equilibrium properties of a voltage-dependent junctional conductance

The conductance of junctions between amphibian blastomeres is strongly voltage dependent. Isolated pairs of blastomeres from embryos of Ambystoma mexicanum, Xenopus laevis, and Rana pipiens were voltage clamped, and junctional current was measured during transjunctional voltage steps. The steady-state junctional conductance decreases as a steep function of transjunctional voltage of either polarity. A voltage-insensitive conductance less than 5% of the maximum remains at large transjunctional voltages. Equal transjunctional voltages of opposite polarities produce equal conductance changes. The conductance is half maximal at a transjunctional voltage of approximately 15 mV. The junctional conductance is insensitive to the potential between the inside and outside of the cells. The changes in steady-state junctional conductance may be accurately modeled for voltages of each polarity as arising from a reversible two-state system in which voltage linearly affects the energy difference between states. The voltage sensitivity can be accounted for by the movement of about six electron charges through the transjunctional voltage. The changes in junctional conductance are not consistent with a current-controlled or ionic accumulation mechanism. We propose that the intramembrane particles that comprise gap junctions in early amphibian embryos are voltage-sensitive channels.     

Hindered diffusion through an aqueous pore describes invariant dye selectivity of Cx43 junctions

The permselectivity (permeance/conductance) of Cx43-comprised gap junctions is a variable parameter of junctional function. To ascertain whether this variability in junctional permselectivity is explained by heterogeneous charge or size selectivity of the comprising channels, the permeance of individual Cx43 gap junctions to combinations of two dyes differing in either size or charge was determined in four cell types: Rin43, NRKe, HeLa43, and cardiac myocytes. The results show that Cx43 junctions are size- but not charge-selective and that both selectivities are constant parameters of junctional function. The consistency of dye selectivities indicates that the large continuum of measured junctional permselectivities cannot be ascribed to an equivalent continuum of individual channel selectivities. Further, the relative dye permeance sequence of NBD-M-TMA approximately Alexa 350 > Lucifer yellow > Alexa 488 > Alexa 594 (Stokes radii of 4.3 A, 4.4 A, 4.9 A, 5.8 A, and 7.4 A, respectively) and the conductance sequence of KCl > TEACl approximately Kglutamate are well described by hindered diffusion through an aqueous pore with radius approximately 10 A and length 160 A. The permselectivity and dye selectivity data suggest the variable presence in Cx43-comprised junctions of conductive channels that are either dye-impermeable or dye-permeable.     

Molecular analysis of voltage dependence of heterotypic gap junctions formed by connexins 26 and 32.

Heterotypic gap junctions formed by pairing Xenopus oocytes expressing hemichannels formed of Cx32 with those expressing hemichannels formed of Cx26 displayed novel transjunctional voltage (Vj) dependence not predicted by the behavior of these connexins in homotypic configurations. Rectification of initial and steady-state currents was observed. Relative positivity and negativity on the Cx26 side of the junction resulted in increased and decreased initial conductance (gj0), respectively. Only relative positivity on the Cx26 decreased steady-state conductance (gj infinity). This behavior suggested that interactions between hemichannels influences gap junction gating. The role of the first extracellular loop (E1) in these interactions was examined by pairing Cx32 and Cx26 with a chimeric connexin in which Cx32 E1 was replaced with Cx26 E1 (Cx32*26E1). Both junctions rectified with gj0/Vj relations that were less steep than that observed for Cx32/Cx26. Decreases in gj infinity occurred for either polarity Vj in the Cx32/Cx32*26E1 junction. Mutation of two amino acids in Cx26 E1 increased the steepness of both the gj0/Vj and gj infinity/Vj relations. These data demonstrate that fast rectification can arise from mismatched E1 domains and that E1 may contribute to the voltage sensing mechanisms underlying both fast and slow Vj-dependent processes.     

Gap junction channels formed by coexpressed connexin40 and connexin43

Many cardiovascular cells coexpress multiple connexins (Cx), leading to the potential formation of mixed (heteromeric) gap junction hemichannels whose biophysical properties may differ from homomeric channels containing only one connexin type. We examined the potential interaction of connexin Cx43 and Cx40 in HeLa cells sequentially stably transfected with these two connexins. Immunoblots verified the production of comparable amounts of both connexins, cross-linking showed that both connexins formed oligomers, and immunofluorescence showed extensive colocalization. Moreover, Cx40 copurified with (His)(6)-tagged Cx43 by affinity chromatography of detergent-solubilized connexons, demonstrating the presence of both connexins in some hemichannels. The dual whole cell patch-clamp method was used to compare the gating properties of gap junctions in HeLa Cx43/Cx40 cells with homotypic (Cx40-Cx40 and Cx43-Cx43) and heterotypic (Cx40-Cx43) gap junctions. Many of the observed single channel conductances resembled those of homotypic or heterotypic channels. The steady-state junctional conductance (g(j,ss)) in coexpressing cell pairs showed a reduced sensitivity to the voltage between cells (V(j)) compared with homotypic gap junctions and/or an asymmetrical V(j) dependence reminiscent of heterotypic gap junctions. These gating properties could be fit using a combination of homotypic and heterotypic channel properties. Thus, whereas our biochemical evidence suggests that Cx40 and Cx43 form heteromeric connexons, we conclude that they are functionally insignificant with regard to voltage-dependent gating.     

Gap junction channels: distinct voltage-sensitive and -insensitive conductance states.

All mammalian gap junction channels are sensitive to the voltage difference imposed across the junctional membrane, and parameters of voltage sensitivity have been shown to vary according to the gap junction protein that is expressed. For connexin43, the major gap junction protein in the cardiovascular system, in the uterus, and between glial cells in brain, voltage clamp studies have shown that transjunctional voltages (Vj) exceeding +/- 50 mV reduce junctional conductance (gj). However, substantial gj remains at even very large Vj values; this residual voltage-insensitive conductance has been termed gmin. We have explored the mechanism underlying gmin using several cell types in which connexin43 is endogenously expressed as well as in communication-deficient hepatoma cells transfected with cDNA encoding human connexin43. For pairs of transfectants exhibiting series resistance-corrected maximal gj (gmax) values ranging from < 2 to > 90 nS, the ratio gmin/gmax was found to be relatively constant (about 0.4-0.5), indicating that the channels responsible for the voltage-sensitive and -insensitive components of gj are not independent. Single channel studies further revealed that different channel sizes comprise the voltage-sensitive and -insensitive components, and that the open times of the larger, more voltage-sensitive conductance events declined to values near zero at large voltages, despite the high gmin. We conclude that the voltage-insensitive component of gj is ascribable to a voltage-insensitive substate of connexin43 channels rather than to the presence of multiple types of channels in the junctional membrane. These studies thus demonstrate that for certain gap junction channels, closure in response to specific stimuli may be graded, rather than all-or-none.     

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.     

Connexin expression and gap junctional coupling in human cumulus cells: contribution to embryo quality

Gap junctional coupling among cumulus cells is important for oogenesis since its deficiency in mice leads to impaired folliculogenesis. Multiple connexins (Cx), the subunits of gap junction channels, have been found within ovarian follicles in several species but little is known about the connexins in human follicles. The aim of this study was to determine which connexins contribute to gap junctions in human cumulus cells and to explore the possible relationship between connexin expression and pregnancy outcome from in vitro fertilization (IVF). Cumulus cells were obtained from IVF patients undergoing intra-cytoplasmic sperm injection (ICSI). Connexin expression was examined by RT-PCR and confocal microscopy. Cx43 was quantified by immunoblotting and gap junctional coupling was measured by patch-clamp electrophysiology. All but 5 of 20 connexin mRNAs were detected. Of the connexin proteins detected, Cx43 forms numerous gap junction-like plaques but Cx26, Cx30, Cx30.3, Cx32 and Cx40 appeared to be restricted to the cytoplasm. The strength of gap junctional conductance varied between patients and was significantly and positively correlated with Cx43 level, but neither was correlated with patient age. Interestingly, Cx43 level and intercellular conductance were positively correlated with embryo quality as judged by cleavage rate and morphology, and were significantly higher in patients who became pregnant than in those who did not. Thus, despite the presence of multiple connexins, Cx43 is a major contributor to gap junctions in human cumulus cells and its expression level may influence pregnancy outcome after ICSI.     

Species-specific voltage-gating properties of connexin-45 junctions expressed in Xenopus oocytes.

Gap junctions composed of connexin-45 (Cx45) homologs from four species, zebrafish, chicken, mouse, and human, were expressed in pairs of Xenopus oocytes. The macroscopic conductance (gj) of all Cx45 junctions was modulated by transjunctional voltage (Vj) and by the inside-outside voltage (Vm), and the modulation was species specific. Although their gating characteristics varied in voltage sensitivity and kinetics, the four Cx45 junctions shared 1) maximum conductance at Vj = 0 and symmetrical gj reduction in response to positive and negative Vj of low amplitude, with little residual conductance; and 2) gj increases in response to simultaneous depolarization of the paired cells. The formation of hybrid channels, comprising Cx45 hemichannels from different species, allowed us to infer that two separate gates exist, one in each hemichannel, and that each Cx45 hemichannel is closed by the negativity of Vj on its cytoplasmic side. Interestingly, the Vm dependence of hybrid channels also suggests the presence of two gates in series, one Vm gate in each hemichannel. Thus the Vj and Vm dependence provides evidence that two independent voltage gates in each Cx45 hemichannel exist, reacting through specific voltage sensors and operating by different mechanisms, properties that have evolved divergently among species.     

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 approximately 42 pS; transitions between fully open and closed states with conductance of approximately 52 pS in magnitude occur at substantially lower ( approximately 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.     

Functional analysis of connexin-26 mutants associated with hereditary recessive deafness

The physiological importance of connexin-26 (Cx26) gap junctions in regulating auditory function is indicated by the finding that autosomal recessive DFNB1 deafness is associated with mutations of the Cx26 gene. To investigate the pathogenic role of Cx26 mutation in recessive hearing loss, four putative DFNB1 Cx26 mutants (V84L, V95M, R127H, and R143W) were stably expressed in N2A cells, a communication-deficient cell line. In N2A cells expressing (R127H) Cx26 gap junctions, macroscopic junctional conductance and ability of transferring neurobiotin between transfected cells were greatly reduced. Despite the formation of defective junctional channels, immunoreactivity of (R127H) Cx26 was mainly localized in the cell membrane and prominent in the region of cell-cell contact. Mutant (V84L), (V95M), or (R143W) Cx26 protein formed gap junctions with a junctional conductance similar to that of wild-type Cx26 junctional channels. (V84L), (V95M), or (R143W) Cx26 gap junctions also permitted neurobiotin transfer between pairs of transfected N2A cells. The present study suggests that (R127H) mutation associated with hereditary sensorineural deafness results in the formation of defective Cx26 gap junctions, which may lead to the malfunction of cochlear gap junctions and hearing loss. Further studies are required to determine the exact mechanism by which mutant (V84L), (V95M), and (R143W) Cx26 proteins, which are capable of forming functional homotypic junctional channels in N2A cells, cause the cochlear dysfunction and sensorineural deafness.     

Calmodulin colocalizes with connexins and plays a direct role in gap junction channel gating

The direct calmodulin (CaM) role in chemical gating was tested with CaM mutants, expressed in oocytes, and CaM-connexin labeling methods. CaMCC, a CaM mutant with greater Ca-sensitivity obtained by replacing the N-terminal EF hand pair with a duplication of the C-terminal pair, drastically increased the chemical gating sensitivity of Cx32 channels and decreased their Vj sensitivity. This only occurred when CaMCC was expressed before Cx32, suggesting that CaMCC, and by extension CaM, interacts with Cx32 before junction formation. Direct CaM-Cx interaction at junctional and cytoplasmic spots was demonstrated by confocal immunofluorescence microscopy in HeLa cells transfected with Cx32 and in cryosectioned mouse liver. This was confirmed in HeLa cells coexpressing Cx32-GFP (green) and CaM-RFP (red) or Cx32-CFP (cyan) and CaM-YFP (yellow) fusion proteins. Significantly, these cells did not form gap junctions. In contrast, HeLa cells expressing only one of the two fusion proteins (Cx32-GFP, Cx32-CFP, CaM-RFP or CaM-YFP) revealed both junctional and non-junctional fluorescent spots. In these cells, CaM-Cx32 colocalization was demonstrated by secondary immunofluorescent labeling of Cx32 in cells expressing CaM-YFP or CaM in cells expressing Cx32-GFP. CaM-Cx colocalization was further demonstrated at rat liver gap junctions by Freeze-fracture Replica Immunogold Labeling (FRIL).