Kinetic properties of a voltage-dependent junctional conductance

We have proposed that the gap junctions between amphibian blastomeres are comprised of voltage-sensitive channels. The kinetic properties of the junctional conductance are here studied under voltage clamp. When the transjunctional voltage is stepped to a new voltage of the same polarity, the junctional conductance changes as a single exponential to a steady-state level. The time constant of the conductance change is determined by the existing transjunctional voltage and is independent of the previous voltage. For each voltage polarity, the relations between voltage, time constant, and steady-state conductance are well modeled by a reversible two-state reaction scheme in which the calculated rate constants for the transitions between the states are exponential functions of voltage. The calculated rate constant for the transition to the low-conductance state is approximately twice as voltage dependent as that for the transition to the high-conductance state. When the transjunctional voltage polarity is reversed, the junctional conductance undergoes a transient recovery. The polarity reversal data are well modeled by a reaction scheme in which the junctional channel has two gates, each with opposite voltage sensitivity, and in which an open gate may close only if the gate in series with it is open. A simple explanation for this contingent gating is a mechanism in which each gate senses only the local voltage drop within the channel.     

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.     

Development changes in regulation of embryonic chick heart gap junctions

Summary Embryonic chick myocyte pairs were isolated from ventricular tissue of 4-day, 14-day, and 18-day heart for the purpose of examining the relationship between macroscopic junctional conductance and transjunctional voltage during cardiac development. The double whole-cell patch-clamp technique was employed to directly measure junctional conductance over a transjunctional voltage range of ±100 mV. At all ages, the instantaneous junctional current (or conductance=current/voltage) varied linearly with respect to transjunctional voltage. This initial response was followed by a time- and voltage-dependent decline in junctional current to new steady-state values. For every experiment, the steady-state junctional conductance was normalized to the instantaneous value obtained at each potential and the data was pooled according to developmental age. The mean steadystate junctional conductance-voltage relationship for each age group was fit using a two-state Boltzmann distribution described previously for other voltage-dependent gap junctions. From this model, it was revealed that half-inactivation voltage for the transjunctional voltage-sensitive conductance shifted towards larger potentials by 10 mV, the equivalent gating charge increased by approximately 1 electron, and the minimal voltage-insensitive conductance exactly doubled (increased from 18 to 36%) between 4 and 18 days of development. Decay time constants were similar at all ages examined as rate increased with increasing transjunctional potential. This data provides the first direct experimental evidence for developmental changes in the regulation of intercellular communication within a given tissue. This information is consistent with the hypothesis that developmental expression of multiple gap junction proteins (connexins) may confer different regulatory mechanisms on intercellular communication pathways within a given cell or tissue.     

Action potential modulation of connexin40 gap junctional conductance

Connexin40 (Cx40) is abundantly expressed in the atrial myocardium, ventricular conduction system, and vascular endothelial and smooth muscle cells of the mammalian cardiovascular system. Rapid conduction through cardiac tissues depends on electrotonic transfer of the action potential between neighboring cells. To determine whether transjunctional voltages (Vj) elicited by an action potential can modulate conductance of Cx40 gap junctions, simulated myocardial action potentials were applied as voltage-clamp waveforms to Cx40 gap junctions expressed in mouse neuro2A (N2A) cells. Junctional currents resembled the action potential morphology but declined by >50% from peak to near-constant plateau values. Kinetics of Cx40 voltage gating were examined at peak voltages > or =100 mV, and decay time constants changed e-fold per 17.6 mV for Vj > +/-40 mV. Junctional conductance recovered during phase 3 repolarization and early diastole to initial values. These phasic changes in junctional conductance were due to rapid decay kinetics, increasing to tens of milliseconds at peak Vj of 130 mV, and the increase in the steady-state conductance curve as Vj returned toward 0 mV. Time-dependent conductance curves for Cx40 were modeled with one inactivation and two recovery Vj-dependent components. There was a temporal correlation between development of conduction delay or block and the inactivation phase of junctional conductance. Likewise, recovery of junctional conductance was coincident with recovery from refractoriness, suggesting that gap junctions may play a role in the genesis and propagation of cardiac arrhythmias.     

Functional gap junctions in the early sea urchin embryo are localized to the vegetal pole.

Using the whole-cell voltage-clamp technique we have studied electrical coupling and dye coupling between pairs of blastomeres in 16- to 128-cell-stage sea urchin embryos. Electrical coupling was established between macromeres and micromeres at the 16-cell stage with a junctional conductance (G(j)) of 26 nS that decreased to 12 nS before the next cleavage division. G(j) between descendants of macromeres and micromeres was 12 nS falling to 8 nS in the latter half of the cell cycle. Intercellular current intensity was independent of transjunctional voltage, nondirectional, and sensitive to 1-octanol and therefore appears to be gated through gap junction channels. There was no significant coupling between other pairs of blastomeres. Lucifer yellow did not spread between these electrically coupled cell pairs and in fact significant dye coupling between nonsister cells was observed only at the 128-cell stage. Since 1-octanol inhibited electrical communication between blastomeres at the 16- to 64-cell stage and also induced defects in formation of the archenteron, it is possible that gap junctions play a role in embryonic induction.     

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.     

Gap-junctional permeability in early and cleavage-arrested ascidian embryos.

Using the whole-cell voltage clamp technique, we have studied junctional conductance (Gj), and Lucifer Yellow (LY) coupling in 2-cell and 32-cell ascidian embryos. Gj ranges from 17.5 to 35.3 nS in the 2-cell embryo where there is no passage of LY, and from 3.5 to 12.2 nS in the later embryo where LY dye spread is extensive. In both cases, Gj is independent of the transjunctional potential (Vj). Manually apposed 2-cell or 32-cell embryos established a junctional conductance of up to 10 nS within 30 min of contact. Furthermore, since we did not observe any significant number of cytoplasmic bridges at the EM and Gj is sensitive to octanol, it is probable that blastomeres in the 2-cell and 32-cell embryos are in communication by gap junctions. In order to compare Gj in the two stages and to circumvent problems of cell size, movement and spatial location, we used cytochalasin B to arrest cleavage. Gj in cleavage-arrested 2-cell embryos ranged from 25.0 to 38.0 nS and remained constant over a period of 2.5 h. LY injected into a blastomere of these arrested embryos did not spread to the neighbour cell until they attained the developmental age of a 32- to 64-cell control embryo. Our experiments indicate a change in selectivity of gap junctions at the 32-cell stage that is not reflected by a macroscopic change in ionic permeability.     

Connexin32 gap junction channels in stably transfected cells. Equilibrium and kinetic properties.

Communication-deficient cells (the SKHep1 cell line) were stably transfected with a plasmid containing cDNA which encodes the major gap junction protein of rat liver, connexin32. Application of the dual whole-cell voltage clamp technique with patch electrodes to pairs of transfected SKHep1 cells revealed strong sensitivity of junctional conductance (gj) to transjunctional voltages (Vjs) of either polarity, with the ratio of minimal to maximal gj (gmin/gmax) being approximately 0.1 at the highest Vjs. Steady-state gj values as a function of voltages of either polarity were well fit by the Boltzmann equation. V0, the voltage at which gj was reduced by 50%, was approximately 25-30 mV; A, the Boltzmann parameter describing voltage dependence, was approximately 0.06 (corresponding to an energy difference between states of approximately 1 kCal/mol and to approximately 2 gating charges moving through the field). The kinetics of the transjunctional voltage dependence were slow (tau greater than 5 s at 20-40 mV, tau = 2 s at and beyond 70 mV). Voltage sensitivity of the opening rate constant (alpha) was approximately 30% lower than that of the closing rate constant (beta) over the Vj range 0-70 mV; at higher voltages, voltage sensitivity of alpha and beta saturated. The kinetic response of gj to a paradigm in which gj was first rendered low by a prepulse of opposite polarity indicated that the voltage sensors are likely to be arranged in series. Transitions between open and closed states in response to transjunctional voltages of either polarity are single order processes; transitions from one closed state to the other involve passage through the open state.     

Voltage-clamp studies of gap junctions between uterine muscle cells during term and preterm labor.

Gap junctions between myometrial cells increase dramatically during the final stages of pregnancy. To study the functional consequences, we have applied the double-whole-cell voltage-clamp technique to freshly isolated pairs of cells from rat circular and longitudinal myometrium. Junctional conductance was greater between circular muscle-cell pairs from rats delivering either at term (32 +/- 16 nS, mean +/- SD, n = 128) or preterm (26 +/- 17 nS, n = 33) compared with normal preterm (4.7 +/- 7.6 nS, n = 114) and postpartum (6.5 +/- 10 nS, n = 16); cell pairs from the longitudinal layer showed similar differences. The macroscopic gap junction currents decayed slowly from an instantaneous, constant-conductance level to a steady-state level described by quasisymmetrical Boltzmann functions of transjunctional voltage. In half of circular-layer cell pairs, the voltage dependence of myometrial gap junction conductance is more apparent at smaller transjunctional voltages (< 30 mV) than for other tissues expressing mainly connexin-43. This unusual degree of voltage dependence, although slow, operates over time intervals that are physiologically relevant for uterine muscle. Using weakly coupled pairs, we observed two unitary conductance states: 85 pS (85-90% of events) and 25 pS. These measurements of junctional conductance support the hypothesis that heightened electrical coupling between the smooth muscle cells of the uterine wall emerges late in pregnancy, in preparation for the massive, coordinate contractions of labor.     

Steady-state current flow through gap junctions. Effects on intracellular ion concentrations and fluid movement.

Double voltage clamp studies were performed on gap junctions contained in septal membranes of the earthworm median giant axon. The gap junctions exhibited no conductance changes in response to voltages imposed across either the septal membrane or the plasma membrane. However, the trans-septal current displayed a slow (10 s) relaxation in response to transjunctional voltage steps. The experimental evidence suggests that this relaxation is a polarization of the septum due to local accumulation/depletion of permeant ions. A theoretical analysis of this observation suggests that the applied electric field causes accumulation of impermeant anions on one side of the junction and depletion on the other, which leads to a change in concentration of permeant ions to maintain macroscopic electroneutrality. The change in concentration of permeant ions generates a transjunctional equilibrium potential that opposes junctional current flow. These results indicate that currents flowing through gap junctions can have an influence on the distribution of intracellular ions. Moreover, the theoretical analysis suggests that such currents will be accompanied by significant intracellular and intercellular water flow.     

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.     

A voltage-dependent gap junction in Drosophila melanogaster.

Steady-state and kinetic analyses of gap junctional conductance, gi, in salivary glands of Drosophila melanogaster third instar larvae reveal a strong and complex voltage dependence that can be elicited by two types of voltages. Voltages applied between the cells, i.e., transjunctional voltages, Vj, and those applied between the cytoplasm and the extracellular space, inside-outside voltages, Vi,o, markedly alter gj. Alteration of Vi-o while holding Vj = O,i.e., by equal displacement of the voltages in the cells, causes gj to increase to a maximum on hyperpolarization and to decrease to near zero on depolarization. These conductance changes associated with Vi-o are fit by a model in which there are two independent gates in series, one in each series, one in each membrane, where each gate is equally sensitive to Vi-o and exhibits first order kinetics. Vj's generated by applying voltage steps of either polarity to either cell, substantially reduce gj. These conductance changes exhibit complex kinetics that depend on Vi-o as well as Vj. At more positive Vi-o's, the changes in gj have two phases, an early phase consisting of of a decrease in gj for either polarity of Vj and a later phase consisting of an increase in gj on hyperpolarizing either cell and a decrease on depolarizing either cell. At negative Vi-o's in the plateau region of the gj-Vi-o relation, the later slow increase in gj is absent on hyperpolarizing either cell. Also, the early decrease in gj for either polarity of Vj is faster the more positive the Vi-o. The complex time course elicited by applying voltage steps to one cell can be explained as combined actions of Vi-o and Vj, with the early phase ascribable to Vj, but influenced by Vi-o, and the later phase to the changes in Vi-o associated with the generation of Vj. The substantially different kinetics and sensitivity of changes in gj by Vi-o and Vj suggests that the mechanisms of gating by these two voltages are different. Evidently, these gap-junction channels are capable of two distinct, but interactive forms of voltage dependence.     

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.     

Limitations of the dual voltage clamp method in assaying conductance and kinetics of gap junction channels.

The electrical properties of gap junctions in cell pairs are usually studied by means of the dual voltage clamp method. The voltage across the junctional channels, however, cannot be controlled adequately due to an artificial resistance and a natural resistance, both connected in series with the gap junction. The access resistances to the cell interior of the recording pipettes make up the artificial resistance. The natural resistance consists of the cytoplasmic access resistances to the tightly packed gap junction channels in both cells. A mathematical model was constructed to calculate the actual voltage across each gap junction channel. The stochastic open-close kinetics of the individual channels were incorporated into this model. It is concluded that even in the ideal case of complete compensation of pipette series resistance, the number of channels comprised in the gap junction may be largely underestimated. Furthermore, normalized steady-state junctional conductance may be largely overestimated, so that transjunctional voltage dependence is easily masked. The model is used to discuss conclusions drawn from dual voltage clamp experiments and offers alternative explanations for various experimental observations.     

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.     

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.     

Distinct effects of ectopic expression of Wnt-1, activin B, and bFGF on gap junctional permeability in 32-cell Xenopus embryos.

A polarity in gap junctional permeability normally exists in 32-cell stage Xenopus embryos, in that dorsal cells are relatively more coupled than ventral cells, as measured by transfer of Lucifer yellow dye. The current study extends our analysis of whether gap junctional permeability at this stage can be modulated by secreted factors, and whether the polarity in gap junctional permeability correlates with the effects of ectopic expression of these secreted factors on the subsequent phenotype of the developing embryo. Following ectopic expression of activin B or Wnt-1, but not bFGF, the transfer of Lucifer yellow between ventral animal pole cells is detected in a greater percentage of 32-cell stage embryos. This increased incidence of dye transfer between ventral cells correlates with axial duplications later in development. However, there are differences in the extent of Lucifer yellow transfer between animal and vegetal hemisphere blastomeres which is dependent on whether activin B or Wnt-1 RNA had previously been injected. These results suggest that enhanced gap junctional permeability between ventral cells of 32-cell Xenopus embryos correlates with subsequent defects in the dorsoventral axis, although there are at present no direct data demonstrating a role for gap junctions in establishment or maintenance of this axis. Moreover, while both activin B and bFGF are mesoderm-inducing growth factors, only activin B has effects on gap junctional permeability in 32-cell embryos following ectopic expression, demonstrating an interesting difference in physiological responses to expression of these factors.     

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 junctional communication in the early Xenopus embryo

In the Xenopus embryo, blastomeres are joined by gap junctions that allow the movement of small molecules between neighboring cells. Previous studies using Lucifer yellow (LY) have reported asymmetries in the patterns of junctional communication suggesting involvement in dorso-ventral patterning. To explore that relationship, we systematically compared the transfer of LY and neurobiotin in embryos containing 16-128 cells. In all cases, the junction-permeable tracer was coinjected with a fluorescent dextran that cannot pass through gap junctions. Surprisingly, while LY appeared to transfer in whole-mount embryos, in no case did we observe junctional transfer of LY in fixed and sectioned embryos. The lack of correspondence between data obtained from whole-mounts and from sections results from two synergistic effects. First, uninjected blastomeres in whole-mounts reflect and scatter light originating from the intensely fluorescent injected cell, creating a diffuse background interpretable as dye transfer. Second, the heavier pigmentation in ventral blastomeres masks this scattered signal, giving the impression of an asymmetry in communication. Thus, inspection of whole-mount embryos is an unreliable method for the assessment of dye transfer between embryonic blastomeres. A rigorous and unambiguous demonstration of gap junctional intercellular communication demands both the coinjection of permeant and impermeant tracers followed by the examination of sectioned specimens. Whereas LY transfer was never observed, neurobiotin was consistently transferred in both ventral and dorsal aspects of the embryo, with no apparent asymmetry. Ventralization of embryos by UV irradiation and dorsalization by Xwnt-8 did not alter the patterns of communication. Thus, our results are not compatible with current models for a role of gap junctional communication in dorso-ventral patterning.     

Effect of Membrane Tension on Gap Junctional Conductance of Supporting Cells in Corti's Organ

The effects of turgor pressure-induced membrane tension on junctional coupling of Hensen cell isolates from the inner ear were evaluated by input capacitance or transjunctional conductance measurement techniques. Turgor pressure was altered by changing either pipette pressure or the osmolarities of extracellular solutions. Both positive pipette pressure and extracellular applications of hypotonic solutions, which caused cell size to concomitantly increase, uncoupled the cells as indicated by reduced input capacitance and transjunctional conductance. These changes were, in many cases, reversible and repeatable. Intracellular application of 50 μM H-7, a broad-based protein kinase inhibitor, and 10 mM BAPTA did not block the uncoupling effect of positive turgor pressure on inner ear gap junctions. The transjunctional conductance at a holding potential of −80 mV was 53.6 ± 5.8 nS (mean ± SEM, n = 9) and decreased ∼40% at a turgor pressure of 1.41 ± 0.05 kPa. Considering the coincident kinetics of cell deformation and uncoupling, we speculate that mechanical forces work directly on gap junctions of the inner ear. These results suggest that pathologies that induce imbalances in cochlear osmotic pressure regulation may compromise normal cochlear homeostasis.