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* Department of Neuroscience and Department of Anesthesiology of Albert Einstein College of Medicine, Bronx, New York; and Institut für Genetik, Abteilung Molekulargenetik, Universität Bonn, Bonn, Germany
Correspondence: Address reprint requests to Dr. Feliksas F. Bukauskas, 1300 Morris Park Ave., Albert Einstein College of Medicine, Bronx, NY 10461. Tel.: 718-430-4130; Fax: 718-430-8944; E-mail: fbukausk{at}aecom.yu.edu.
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ABSTRACT |
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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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONSUPPLEMENTARY MATERIALACKNOWLEDGEMENTSREFERENCES |
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,2). Each GJ channel is composed of two hemichannels (connexons), which in turn are composed of six Cx subunits. Thus far, at least 20 distinct Cx isoforms have been cloned (3). Cell-cell communication can be organized through homotypic (same Cx isotype in both hemichannels), heterotypic (hemichannels differ in Cx isotypes) or/and heteromeric (different Cx isotypes in at least one hemichannel) channels that vary highly in conductance, perm-selectivity, and gating properties (2).
Cx40, Cx43, and Cx45 are expressed in different tissues, but most abundantly in cardiovascular and central nervous systems. Cx40, Cx43, and Cx45 have been regarded as the three major cardiac connexins (3,4). mCx30.2 has been recently identified as the fourth cardiac connexin in rodents; its putative human ortholog is Cx31.9 (5). Given the specific expression patterns of these connexins, their capacity to form heterotypic and heteromeric junctions will significantly influence the nature of intercellular communication and the spread of excitation within and between different regions of the heart (6–9). Blood vessels express Cx40, Cx43, Cx45, and Cx37, with Cx40 most abundantly expressed in endothelial cells and Cx43 in smooth muscle cells (10). In the CNS, astrocytes abundantly express Cx43, endothelial cells of the blood-brain barrier express Cx40 and Cx43 (11), and neurons of the olivocerebellar system express Cx45 (12). Accordingly, a number of studies have reported malformation and dysfunction not only of the heart, but also of the whole cardiovascular system of mice in which Cx40, Cx43, or Cx45 were knocked out (13–16). Only Cx40 knockout mice survive during the postnatal period and they exhibit severe dysfunction of the cardiovascular system, such as impaired sinoatrial-nodal function, slowed conduction velocity in atria and the AV node, impaired conduction in bundle branches, structural abnormalities of endothelial and smooth muscle layers in the blood vessels, deficient conduction of vasodilator stimuli along vessel walls, elevated mean arterial pressure and blood pressure regulation abnormalities (17–20).
Several studies reported formation of functional heterotypic junctions between cells expressing Cx45 with those expressing Cx40 and Cx43 (21–25). Initial electrophysiological studies in Xenopus oocytes (10) and HeLa transfectants (26) reported that Cx40 and Cx43 do not form functional heterotypic channels. These findings were confirmed subsequently in pairs of Xenopus oocytes expressing Cx40 and Cx43 (27). Furthermore, when HeLa cells expressing Cx43 were cocultured with those expressing Cx40, immunohistochemical evidence for formation of heterotypic junctional plaques (JPs) was lacking (26) suggesting that Cx40 and Cx43 hemichannels are unable to dock or to cluster into visible JPs. Those studies were stimulated by findings that Cx40 and Cx43 are expressed in different cell types in the cardiovascular system that come into direct contact. Cx43 is the major connexin expressed in working myocardium of the heart and smooth muscle cells of the blood vessels, whereas Cx40 is mainly expressed in the atrium, atrioventricular node, AV bundle, and endothelial cells. These findings suggested that intercellular communication can be spatially regulated by the selective expression of different connexins. An inability to form heterotypic junctions would preclude communication between cells residing in close apposition, perhaps enabling them to perform different function, e.g., endothelial and smooth muscle cells or cells of the conduction system and working myocardium of the heart. Subsequent electrophysiological studies in transfected mammalian cells reported that Cx40 and Cx43 can form functional heterotypic channels although junctional conductance was low (21–23). More recently, concerns have been raised that the reported coupling between Cx40- and Cx43-expressing cells may have been caused by formation of heterotypic/heteromeric junctions with an endogenous connexin rather than by Cx40/Cx43 heterotypic junctions (28) and low efficiency of formation of heteromeric junctions was reported due to inability of Cx40 and Cx43 to dock (29).
Here, we examined formation and gating properties of heterotypic junctions in HeLa cells stably transfected with Cx40, Cx43, or Cx45 in native and GFP-fused forms. We demonstrate that heterologous Cx40/Cx43 HeLa cell pairs lack JPs composed of Cx40 and Cx43 and exhibit low levels of coupling explained by the formation of heterotypic junctions with endogenously expressed Cx45, i.e., Cx40/Cx45 and Cx43/Cx45 junctions. We show that in response to transjunctional voltage (Vj), Cx40/Cx45 heterotypic GJ channels exhibit mainly gating transitions between the fully open state (52 pS) and the residual state (10 pS), giving rise to 42 pS gating transitions. The residual state of Cx45 hemichannels rectifies and we demonstrate signal transfer asymmetry in Cx40/Cx45 junctions that can be modulated from unidirectional to bidirectional by small changes in the difference of the holding potentials of coupled cells. We show that both fast and slow gates of Cx40 exhibit a negative gating polarity.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONSUPPLEMENTARY MATERIALACKNOWLEDGEMENTSREFERENCES |
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,30). To study heterotypic junctions, two types of cells expressing different connexins were mixed in equal quantities and seeded at a density of 104 cells/cm2 on coverslips placed in culture dishes.
Electrophysiological measurements
For simultaneous electrophysiological and fluorescence recording, cells were grown on 22 x 22 mm No. 0 coverslips and transferred to an experimental chamber (31) mounted on the stage of Olympus IX70 inverted microscope (Olympus America, Melville, NY) equipped with a fluorescence imaging system. The chamber was perfused with a modified Krebs-Ringer solution containing (in mM): 140 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 5 HEPES, 5 glucose, 2 pyruvate (pH = 7.4). Patch pipettes were filled with pipette solution containing (in mM): 10 Na Asparpate, 130 KCl, 0.26 CaCl2, 1 MgCl2, 3 MgATP, 5 HEPES (pH 7.2), 2 EGTA ([Ca2+] = 5 x 10–8 M).
Junctional conductance, gj, was measured using the dual whole-cell patch clamp system (32,33). Briefly, each cell of a pair was voltage clamped to the same holding potential (V1 = V2) with a separate patch clamp amplifiers. By stepping the voltage in one cell and keeping the other constant (V2), junctional current (Ij) was measured as the change in current in the unstepped cell, I2, in response to the applied transjunctional voltage (Vj = V2 – V1). We generally maintained the holding potential in the unstepped cell close to the resting potential to reduce the noise in Ij that is generated by nonjunctional current. Thus, gj is obtained by dividing the change in I2 by Vj. With low levels of coupling, unitary junctional currents can be recorded as discrete quantal changes in the unstepped cell that are accompanied by equal and opposite quantal changes in the stepped cell. Voltages and currents were digitized using a MIO-16x A/D converter (National Instruments, Austin, TX) and acquired and analyzed using custom-made software designed by E. B. Trexler and V. K. Verselis.
In all cocultures of cells expressing different combinations of Cx isoforms at least one type of cell expressed a Cx fused with GFP to allow visualization of Cx distribution, particularly the size and number of junctional plaques. All cell lines expressing wild-type Cxs before coculturing were prelabeled with DAPI or DiI (Invitrogen, Carlsbad, California). Isolated heterotypic pairs were selected by identifying pairs of cells in which cells were expressing GFPs of different color or one expressed GFP and the other was labeled with DAPI or DiI. Isolated heterologous pairs were selected for recording between 24 and 60 h after platting.
Fluorescence imaging
Fluorescence images were acquired and analyzed using a Hamamatsu cooled digital camera mounted on an Olympus IX70 microscope and UltraVIEW software for image acquisition and analysis (PerkinElmer Life Sciences, Boston, MA). Appropriate excitation and emission filters (Chroma Technology, Brattleboro, VT) were used to image CFP, EGFP, DAPI, and DiI.
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RESULTS |
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). Double immunogold labeling of HeLa parental cells and HeLa transfectants expressing Cx40 and Cx43 also shows dispersed expression of GJ channel like particles formed of Cx45 (35). This finding correlates with electrophysiological data showing that among 29 HeLa parental cell pairs only three were coupled with an average gj of 0.04 nS (21). Fig. 1 F, which is pseudocolor overlap of EGFP (red) and CFP (cyan) signals, shows a junctional plaque in the region of contact between cells expressing Cx43-EGFP and Cx45-CFP. This cell pair exhibited electrical coupling with gj = 17 nS. Similarly, we have observed junctional plaques between cells expressing Cx40 and Cx45 (not shown).
Electrophysiological examination of heterologous Cx40/Cx43 cell pairs showed that a majority were coupled, but that coupling was typically weak with mean gj values for heterologous Cx40-CFP/Cx43-EGFP and Cx40/Cx43-EGFP combinations of 0.95 ± 0.59 nS (n = 25) and 1.06 ± 0.81 nS (n = 10), respectively. Only three cell pairs lacked coupling. All functionally coupled cell pairs exhibited gating asymmetry, which varied substantially among cell pairs. Examination of 35 homologous cell pairs formed of HeLa parental (untransfected) cells showed measurable coupling in only 5 cases, with gj values in each case below 0.1 nS. When HeLa parental cells were paired with HeLa cells expressing either Cx40-CFP or Cx43-EGFP, all cell pairs were coupled, but gj was low, 0.3 and 0.5 nS, respectively. Thus, gj measured between cells expressing Cx40 and Cx43 is close to the sum of gjs measured in Cx43/parental are Cx40/parental heterologous cell pairs, suggesting that heterotypic channels in which endogenous Cx45 pairs with Cx40 and Cx43 form in an independent fashion. The data obtained from different pairing combinations are summarized in Table 1. These results indicate that coupling between HeLa parental cells is rare and low when it is present, likely because of low levels of Cx45 expression and consequently inefficient JP formation. Coupling and junction formation involving Cx45 remains low, but increases such that coupling levels of 0.5 nS are achieved when at least one cell abundantly expresses Cx40 or Cx43.
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Cx43 and Cx45
To examine compatibility between Cx43 and Cx45, we cocultured HeLa cells expressing Cx43-EGFP with those expressing Cx45 and found that JPs readily formed between heterologous cells, consistent with previous reports (24,25). We selected and examined heterologous cell pairs that contained at least one clearly identifiable and sizeable JP comprising a Cx43-EGFP/Cx45 heterotypic junction. Fig. 2 A shows the steady-state Gj-Vj dependence, which superimposes results of two types of measurements. Data points shown as open circles were collected from 24 Cx43-EGFP/Cx45 cell pairs by measuring gj at the end of 30 s Vj steps. For each cell pair, gj was normalized to its value at Vj = 0 to give normalized conductance, Gj. Gj-Vj dependence measured by applying long (150 s) Vj ramps (see solid line; averaged from three experiments) shows similar but slightly steeper Gj-Vj dependence for both Vj polarities; the difference suggests that steady-state may have been estimated better with the Vj ramps. As previously described in (25), albeit over a smaller range of Vj, Cx43-EGFP/Cx45 junctions exhibit a highly asymmetric steady-state Gj-Vj relation. Gj increases to a peak and decreases to near zero for modest voltages relatively negative and positive, respectively, on the Cx43-EGFP side. This steep change in Gj has been shown to result from gating of the Cx45 hemichannel via its slow Vj gating mechanism (25). The reduction in Gj after reaching a peak for Vjs relatively negative on the Cx43-EGFP side was shown to result from closure of the slow gate in Cx43-EGFP hemichannels, which is less steeply sensitive than in Cx45. The modest increase in Gj after a reduction to near zero for Vjs relatively positive on the Cx43-EGFP side is ascribable to an interaction between the slow and fast Vj gates that operate in series in Cx45 hemichannels (25). Vj acts on both gates, but closure of one gate would cause a larger fraction of Vj to drop across it thereby changing Vj across the other gate. In effect, the open probability of one gate is dependent on the state of the other, originally described by Harris et al. (36) to explain interaction between gates in separate hemichannels. The increasing conductance at large Vjs can be explained by an increasing pool of Cx45 hemichannels entering the residual (subconductance) state (25).
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,38). Fig. 2 B shows an example of Vj and Ij records and the calculated gj from a single Cx43-EGFP/Cx45 cell pair in which small (–20 to +20 mV) and brief (0.8 s) Vj ramps as well as long-duration Vj steps of different amplitudes were applied to the Cx43-EGFP cell. Initially, gj was 15 nS and increased 20% in response to a negative Vj step of –40 mV. For a positive Vj step of +40 mV, gj decreased rapidly to near zero, reaching steady state at gj = 0.7 nS. Stepwise increases in Vj (time interval, 100–160 s) resulted in an increase in gj of nearly 4-fold. All Cx43-EGFP/Cx45 cell pairs examined (mean gj = 13.3 ± 3.5 nS, n = 35) demonstrated this same behavior, which is atypical for most homotypic and heterotypic GJs, but is observed in Cx45 homotypic GJs (25) as well as in heterotypic junctions containing Cx45 on one side, such as Cx31/Cx45 (39) and Cx40/Cx45 (see below) junctions. This allowed us to compare I/V relations of channels that are largely in the open state to those that are gated to the residual state by Vj. Fig. 2 C shows superposed I/V plots measured during different time intervals when repeated ramps were superimposed onto Vj steps of different amplitudes (a–d) and during recovery from gating induced by these Vj steps (e); see corresponding lines and squares in Fig. 2, B and C, respectively. The I/V relationship measured in response to Vj ramps applied around Vj = 0 are essentially linear (see square e). In contrast, the I/V relationship measured in response to Vj ramps applied on top of Vj steps positive on the Cx43-EGFP side is nonlinear. Thus, both data sets shown in the inset of Fig. 2 A and in Fig. 2 C support the view that at positive Vjs Cx45 hemichannels largely reside in the residual state, which rectifies. We conclude that it must be the Cx45 hemichannel that is gated to the residual substate as this form of gating in the Cx43-EGFP hemichannel is abolished by fusion with EGFP (40).
Cx40 and Cx45
To examine gating of Cx40/Cx45 junctions, we cocultured HeLa cells expressing Cx40-CFP with those expressing Cx45 and found that JPs readily formed between heterologous cells and that heterologous cell pairs exhibited electrical cell-cell coupling with a mean value for gj of 4.5 ± 3.1 nS (n = 28). The dependence of Gj on Vj was evaluated in response to long Vj ramps as shown in Fig. 3 A. Cx40-CFP/Cx45 heterotypic junctions exhibited an asymmetric steady-state Gj-Vj relation similar to that of Cx43-EGFP/Cx45 junctions, i.e., Gj decreased to near zero at relatively negative voltages on the Cx45 side and then increased modestly at larger Vjs, exceeding +60 mV (see Fig. 3 B and its inset). Fig. 3 C summarizes data obtained from five cell pairs. Based on the ratio of Gj at Vj = 0 mV to that at Vj = –40 mV, it would appear that only 30–40% of the channels are open at Vj = 0.
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) and Cx31/Cx45 (39) junctions, Cx40/Cx45 junctions exhibit signal transfer asymmetry, which can be effectively modulated by the difference in the holding potentials between the cells. In the example shown (Fig. 3 D), a Cx45-expressing cell (cell 1) was voltage clamped to –50 mV, and repeated, brief (90 ms) steps ±90 mV in amplitude were applied. The Cx40-expressing cell (cell 2) was maintained in current clamp mode, which permits recording of the coupling potential in cell 2 evoked by voltage steps applied to cell 1. At the start of the recording, depolarizing voltage steps applied to the Cx45 cell were effectively transferred, evident by the voltage responses in cell 2, whereas transfer of hyperpolarizing voltage steps was essentially blocked. Upon progressive depolarization of cell 1, indicated by the vertical arrows, the same repeated steps elicited responses in cell 2 that progressively become more symmetric (compare responses in the period from 0 to 40 s to those from 70 to 80 s). An inset showing recordings in an expanded time scale better illustrates the time course of the changes associated with reversal of the polarity of the voltage steps applied to cell 1. The change in the difference between holding potentials of the two cells, 
Vh = V1 – V2, is plotted in the bottom trace; values were calculated between the repeated pulses. Thus, Cx40/Cx45 like Cx43/Cx45 (25), Cx31/Cx45 (39), or Cx47/Cx45 (Feliksas F. Bukauskas, unpublished) channels demonstrate electrical signal transfer asymmetry, which can be modulated from unidirectional to bidirectional by relatively small changes in Vh.
Cx40 and Cx43
As shown previously in Fig. 1, we examined compatibility between Cx40 and Cx43 using several combinations of cocultured HeLa cells. Examination of 35 cells pairs formed between HeLa cells expressing Cx40 or Cx40-CFP and those expressing Cx43-EGFP showed no cases in which there were well-resolvable JPs, i.e., with sizes exceeding 1 µm in diameter. Although small fluorescent puncta were observed in regions where heterologous cells were in contact, in no case did these puncta show colocalization of both EGFP and CFP fluorescence (Fig. 1) indicating that they represented small heterotypic junctions formed between Cx40-CFP or Cx43-EGFP and an endogenously expressed connexin, presumably Cx45. All cell pairs with small puncta that were examined electrophysiologically demonstrated low levels of coupling. Fig. 4 A shows a frequency histogram assembled from Cx40-CFP/Cx43-EGFP heterologous cell pairs. A peak in the Gaussian distribution occurs at gj = 1 nS. All junctions exhibited asymmetry in gating (Fig. 4 B) with slightly higher sensitivity when the Cx43-EGFP side was made relatively positive.
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) and HeLaCx40-CFP and neither of 11 randomly selected cell pairs demonstrated functional coupling (data not shown). Our earlier findings demonstrating junction formation and functional coupling between Novikoff and HeLa cells expressing Cx43-EGFP (40), indicate that Novikoff and HeLa cells can form junctions if the Cxs are compatible.
Unitary gating events of heterotypic gap junction channels
Unitary gating events of Cx43/Cx45 channels were described previously (25,42). To characterize gating events associated with Cx40/Cx45 channels, we examined heterologous cell pairs in which one cell expressed Cx40 or Cx40-CFP and the other cell expressed Cx45. Cells expressing wild-type Cx40 or Cx45 were identified by prelabeling with DiI or DAPI. Unitary gating events associated with Cx40(DAPI)/Cx45 heterotypic channels are shown in Fig. 5 A. Application of a positive voltage step to the HeLaCx40 cell caused very rapid, full uncoupling, consistent with closure of the highly voltage-sensitive Cx45 hemichannel. Application of a negative voltage step to the same cell produced closure that was less voltage-sensitive. Most of the unitary gating events, shown in two insets, were 42 pS in magnitude; see gating transitions indicated by asterisks in both insets. Although infrequent, we also observed transitions 48–58 pS in magnitude (indicated by dashed arrow in the left inset). Fig. 5 B shows a histogram of the amplitudes of unitary gating transitions, which were calculated from three Cx40/Cx45 and five Cx40-CFP/Cx45 cell pairs. A fit of the frequency histogram to Gaussian functions (solid lines) resulted in two peaks at 42 and 52 pS. Each event was defined as a transition between different conductance states, where the residence time in each state was at least 20 ms. Fig. 5 C show Vj and Ij records from a Cx40-CFP/Cx45 cell pair when a Vj ramp (from –112 to 112 mV) was applied to the Cx45 cell and only one channel was operational during recovery from CO2 application. The Ij trace shows fast channel closure when the Cx45 side is relatively negative and reopening upon reversing the polarity of Vj. The I/V plot from this record is shown on the right. The steepness of the linear regression line derived from the points that correspond to the open state (see dashed line) indicated a conductance of 53 pS. This conductance is close to that predicted by the series arrangement of Cx45 and Cx40 hemichannels assuming that their conductances are twice that of the corresponding cell-cell channels, i.e., 64 and 300 pS (64 x 300/(64 + 300) = 52.7 pS) (25,43). Thus, we attribute the 52 pS peak in the histogram to gating events between the fully open (main) state and the closed state and the 42 pS peak to gating events between the main open state and substate(s); the latter transitions dominate at the Vj that was applied. It has been suggested that the unitary conductance of Cx40/Cx45 channels is 40 pS (44), which likely corresponds to the transitions between the main open state and substate(s). Both macroscopic (see Fig. 3 C) and single channel records (see Fig. 5 A) support the view that Cx40 gates at negativity on its cytoplasmic side. Most of the gating transitions were between the open state and substate(s) suggesting that macroscopic Vj gating of Cx40 hemichannels largely reflects the fast Vj gating mechanism. Gating between fully open and closed states is present, but is substantially less (10-fold) frequent. Therefore, we conclude that both gates of Cx40 exhibit negative gating polarity, but the slow gating mechanism is substantially less sensitive to Vj than the fast one.
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130 pS. However, we, as well as others (21) who also used HeLa transfectants, did not observe a unitary conductance in this range. Fig. 6 B shows an example of a recording from a Cx43-EGFP/Cx40-CFP heterologous cell pair in which only one channel was active. During a negative voltage step applied to the HeLaCx43-EGFP cell, the channel gates between fully open and closed states with a conductance size of 54 pS. A voltage step of opposite polarity caused very rapid full uncoupling. The conductance and gating characteristics of this channel bear close resemblance to those we reported for Cx43-EGFP/Cx45 channels (25) or for Cx40/Cx45 channels reported here (Fig. 5).
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20 pS to 90 pS, with a majority of the transition sizes falling between 30 pS and 60 pS. A fit of the frequency histogram to multiple Gaussian functions suggest two plausible peaks at 40 pS and 62 pS (solid lines); the dashed line is a composite of the two Gaussian functions. Possible channels within this conductance range are Cx45 homotypic channels as well as Cx40/Cx45 and Cx43/Cx45 heterotypic channels. The conductances in the range of 60–90 pS probably represent a heteromeric channels composed of Cx45 and Cx43 or Cx40. Each of these channels can also show substate gating, with subconductance transitions 75% that of the open state. The presence of substates, as well as the possibility that heteromeric channels form, would lead to smearing of the histogram and poor distinction of peaks associated with each channel type. Heteromeric channels have been shown to form between Cx45 and Cx43 (45). Reported data regarding function of heteromeric channels formed of Cx40 and Cx43 are not in agreement (46,47). |
DISCUSSION |
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). Cx40 is expressed in the atria and the conduction system of ventricles, whereas Cx43 is a major Cx forming GJs between working cardiomyocytes (48). These distinct expression patterns are important for synchronous excitation of cells inside atria and ventricles and for imparting a substantial AV delay that is vital for efficient mechanical function of the heart (9).
It is not well established whether the incompatibility of particular Cx isoforms to form heterotypic junctions resulted from evolutionary directed necessity to perform a specific function. We speculate that this property could affect not only electrical and metabolic communication in the adult tissue but also cell differentiation during development. Typically, most embryonic cells express one or several Cx isoforms, and strong connexin-mediated cell-cell coupling tends to eliminate intercellular gradients of diffusible substances, which include ions, metabolites, small peptides, oligonucleotides and small interfering RNA (siRNA) (2,49,50). Thus, in order for neighboring cell populations to develop independently it may be important to express connexin isoforms that cannot form heterotypic junctions, thereby impairing electrical synchronization, transfer of signaling molecules or metabolic communication between different cell populations while maintaining communication within the same cell types. It is possible that expression of Cx40 by cells forming the His bundle, its branches and terminal network of Purkinje fibers at early stages of development facilitates their differentiation into the conductive system independent from working cardiomyocytes of ventricles expressing mainly Cx43. This functional separation may ultimately lead to anatomical separation, e.g., by a layer of connective tissue in between them. Ultimately, excitation must be transferred from the conduction system to the working myocardium and the appearance of Cx43 expression in parallel to Cx40 in the distal segment of the bundle branches (6) could serve this function at the transitions formed at Purkinje-muscle (P-M) connections (51) discretely distributed on the endocardium surface with 1.5 mm distance between them (52). Presumably Cx43 homotypic GJs are responsible for signal transfer in P-M connections. Thus, expression of incompatible connexins can assist in cell differentiation and also limit interaction between functionally different systems if they are not yet anatomically separated. A similar mechanism may explain development of endothelium, which mainly expresses Cx40, and smooth muscle cell layers, which mainly express Cx43, in the blood vessels.
Heterotypic compatibility among Cx40, Cx43, and Cx45
Originally, electrophysiological studies in Xenopus oocytes (10) reported that Cx40 and Cx43 do not form functional heterotypic channels. This conclusion is supported by Gu et al. (27). A similar conclusion was reached using HeLa transfectants in studies that showed a lack of dye transfer (53) and a lack of electrical cell-cell coupling (26); in the latter study, there was also no evidence that Cx40/Cx43 JPs formed when stained with Cx-specific antibodies suggesting that Cx40 and Cx43 hemichannels do not dock or cluster into JPs. However, subsequent studies, performed in HeLa and RIN cell transfectants reported low levels of functional coupling attributed to Cx40/Cx43 heterotypic channels (21,22). Both studies concluded that Cx40/Cx43 channels are functional despite reporting differences in properties such as Vj gating, single channel conductance and open channel current rectification. Cotrell and Burt (22) commented that the differences regarding voltage gating properties could be due to the short duration (0.5 s) of the voltage pulses used in (21) to characterize steady-state gj-Vj dependence and stated that by using this protocol they may have characterized "fast inactivation while changes in slow inactivation were not assessed". It may be that the differences in these studies arise from expression of different intrinsic Cxs in HeLa and RIN cells. Given the potential importance of Cx40 and Cx43 compatibility in the cardiovascular system, the CNS, and other organs, we reexamined this issue, taking advantage of GFP-tagged connexins that allowed us to visualize JP formation and record electrophysiologically from the same cell pairs. The pattern of compatibility using Cxs tagged with color variants of GFP is the same as that reported by Haubrich et al. (26) using wild-type Cxs. In addition, studies performed in Xenopus oocytes demonstrated lack of coupling between oocytes expressing untagged forms of Cx40 and Cx43 (10,27). Thus, GFP tags should not affect the property of hemichannels to dock and form functional GJ channel.
We conclude that Cx40 and Cx43 are incompatible and that the failure to observe functional Cx40/Cx43 heterotypic channels results from an inability of the component hemichannels to dock and/or to cluster into JPs. This conclusion is supported by several observations. In cocultures of HeLa cells expressing Cx40-CFP and Cx43-EGFP we never observed colocalization of CFP and EGFP at identifiable JPs or at any fluorescent puncta in the junctional membrane. What we did observe often were small (<1 µm) fluorescent puncta in the junctional membrane that were always of one color indicating that they were not formed by the pairing of Cx40 with Cx43 (see Fig. 1, D and E). Also, the presence of these puncta was accompanied by low levels of coupling comparable in magnitude with gj measured between HeLa cells expressing Cx40 or Cx43 and HeLa parental cells and by asymmetric Vj gating. In addition, the unitary conductances are significantly below to those predicted (130 pS) for a heterotypic Cx40/Cx43 GJ channel.
Electrophysiological examination of heterologous Cx40-CFP/Cx43-EGFP and Cx40/Cx43-EGFP cell pairs containing small fluorescent puncta revealed a mean gj of 1 nS and asymmetric Vj gating. Given that we propose that this coupling is mediated by interactions with Cx45, the low values of gj are explainable by the low levels of available endogenous Cx45. The asymmetry in voltage dependence is also explainable through combinations of Cx43-EGFP/Cx45 and Cx40-CFP/Cx45 heterotypic channels mixed in with homotypic Cx45 channels. To assess whether the presence of intrinsically expressed Cx45 can account for the experimentally observed data, we fitted the experimentally obtained gj-Vj dependence shown in Fig. 4 B to the sum of Boltzmann equations describing gj-Vj dependencies of Cx40/Cx45, Cx43/Cx45, and Cx45/Cx45 junctions (see Fig. 4 C). For simplicity, we described gj-Vj dependence of any given junction by multiplying two Boltzmann equations, each describing gating of one of the hemichannels, e.g., Gj = {gmin,1+ (1 – gmin,1)/(1 + exp(A1 x (Vj – Vo,1)))} x {gmin,2 + (1 – gmin,2)/(1 + exp(A2 x (–Vj – Vo,2)))}, where gmin, A, and Vo are Boltzmann constants, and subscripts 1 and 2 correspond to each of two hemichannels. For Cx45 homotypic junctions, gmin,1 = gmin,2, A1 = A2, and Vo,1 = Vo,2. The two dashed lines represent the asymmetric Gj-Vj relations of Cx45/Cx43 and Cx45/Cx40 heterotypic junctions derived from the fitting. These two junctions would be situated in opposite orientations. We acknowledge that description of heterotypic junctions by multiplying two Boltzmann equations is approximate even though they closely describe the Gj-Vj relationships shown in Figs. 2 A and 3 C. The solid line represents Cx45 homotypic junctions. Thus, Fig. 4 C illustrates that HeLaCx40/HeLaCx43 cell pairs can exhibit gj-Vj gating with moderate Vj gating sensitivity and asymmetry even when composed of Cx40/Cx45 and Cx43/Cx45 heterotypic junctions, each exhibiting strong Vj gating asymmetry.
Examination of unitary events in Cx43-EGFP/Cx40-CFP cells pairs revealed a relatively broad distribution of conductance transitions ranging from 20 pS to 90 pS. This distribution is plausible given the number of potential channel types that can form, such as Cx45 homotypic junctions, Cx40/Cx45 and Cx43/Cx45 heterotypic junctions, and a number of heteromeric forms assembled from Cx43 and Cx45 (45). A simple series arrangement of Cx43 and Cx40 hemichannels would predict a unitary conductance of 130–140 pS. The lack of gating transitions exceeding 90 pS supports our assertion that Cx40/Cx43 heterotypic channels do not form.
Voltage gating and cell-to-cell signaling asymmetry of heterotypic junctions
A property common to GJ channels formed of all Cxs is sensitivity of gj to Vj (54–56). A common feature reported for Vj gating is that steady-state gj does not decline to zero with increasing Vj, but reaches a plateau or residual conductance that varies from 5% to 30% of the maximum gj depending on the Cx isoform. Single channel studies showed that the residual gj is due to incomplete closure of the GJ channels by Vj (57,58). Gating to different levels via distinct fast and slow gating transitions led to the finding that there are two different types of Vj sensitive gates (see Fig. 7 A). One, termed the fast Vj gate, is characterized by transitions between open and residual conductance states, and the other, termed the slow Vj or "loop" gate, is characterized by transitions between open and fully-closed states (reviewed in Verselis et al. (56)). Mutational studies demonstrated that the gating polarity of the fast gating mechanism is governed by charged residues in the N-terminal domain (59,60). Modifications of Cx43, including deletion of the CT domain (24) or attachment of aequorin or EGFP to CT, selectively abolishes fast gating to the residual state (61,62). Taken together, these data demonstrate that there are two molecularly distinct gating mechanisms in each hemichannel.
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), Cx31/Cx45 (39), Cx43/Cx45 (25), and Cx40/Cx45 (see Fig. 3 C). In all cases, there is higher Vj gating sensitivity when the Cx45 side is made relatively negative, which has been shown to result predominantly from closure of the slow gate of the Cx45 hemichannel (25). The fast gate of Cx45 also closes for this polarity, but its voltage sensitivity is shifted to higher Vjs. The fast and slow Vj-sensitive gates of Cx43 GJs also close on relative negativity. Previously, we reported that differences in the unitary conductances of the component hemichannels resulted in a higher Vj gating asymmetry in Cx43/Cx45 junctions than that predicted by the simple docking of two hemichannels exhibiting equal unitary conductances. Most of the Vj applied across a Cx43/Cx45 channel falls across the Cx45 hemichannel, which has 3.5-fold smaller conductance than the Cx43 hemichannel, resulting in increased and decreased Vj gating sensitivities of Cx45 and Cx43 hemichannels, respectively (25).
Fig. 7 B shows the Gj-Vj dependence of Cx40/Cx45 heterotypic junction (from Fig. 3 C; Gj normalized to 1) superposed onto Gj-Vj plots of Cx40 and Cx45 homotypic GJs; Boltzmann parameters for these two plots were derived from earlier reports (43,63). Given that the gating polarity of the Cx45 hemichannel is negative, we attribute Gj decay at negative Vjs (negativity on Cx40 side) to closure of the Cx40 hemichannel and Gj decay in Vj range from –20 to +100 mV to the closure of the Cx45 hemichannel. Thus, the Gj-Vj dependence of Cx45/Cx40 heterotypic junctions allows us to conclude that the gating polarity of the Cx40 hemichannel is negative. In addition, it follows from Fig. 7 B that Vj sensitivity of the Cx40 hemichannel is reduced, whereas Vj sensitivity of the Cx45 hemichannel is enhanced that is in agreement with the view that the difference in the unitary conductances of the component hemichannels influences the Gj-Vj relation.
In earlier studies, based on Vj gating asymmetry in pairs of Xenopus oocytes forming Cx37/Cx40 heterotypic junctions, the gating polarity of Cx40 was predicted to be positive (10,64). In Fig. 7 C, gray and black solid lines show gj-Vj dependences of Cx37 and Cx40 homotypic junctions, respectively, obtained from (10). Based on the correspondence of Gj-Vj dependence of Cx37/Cx40 heterotypic junctions (dashed line in Fig. 7 C) at negative Vjs with that of Cx37, and gj decay for both polarities of Vj, it was concluded that both Cx37 and Cx40 hemichannels close on positivity on their cytoplasmic sides. At that time, the single channel conductances of Cx37 and Cx40 were not known. It turns out that the single channel conductance of Cx37 is very large, 300 pS or more (65), which is 2-fold higher than that of Cx40 (43). Thus, a higher fraction of Vj would drop across the higher-resistance Cx40 hemichannel, resulting in higher and lower apparent sensitivities to Vj for Cx40 and Cx37 hemichannels, respectively. Fig. 7 D shows superposition of the experimental gj-Vj plot of a Cx37/Cx40 heterotypic junction (dashed line; from Bruzzone et al. (10)) and theoretical gj-Vj plot (see solid line with triangles) predicted with data obtained from homotypic Cx40 and Cx37 junctions assuming that voltage drop across the Cx40 hemichannel is twice of that across the Cx37 hemichannel and that the Cxs close at negativity on their cytoplasmic sides. To evaluate correspondence between experimental and theoretical data, we calculated the sum of squared deviations, 
, between two sets of data at all measured Vjs and found that 
. We also calculated a gj-Vj plot (not shown) assuming that both Cxs close on positivity and found that 
. Thus, the assumption that Cx40 and Cx37 possess negative gating polarities provides a 1.7-fold better fit to experimental data. Even though our analysis suggests that gating polarity of Cx37 should be negative, we cannot exclude the possibility that the fast or the slow gate of Cx37 possesses positive gating polarity. Based on the absence of Vj gating in Cx40/Cx43 and Cx40/Cx45 cell pairs at relative negativity on Cx40 side, it was reported that Cx40 gates at positivity on its cytoplasmic side (21); bipolar Vj steps of 0.5 s in duration were used for Vj gating studies. These data do not contradict ours because as follows from Figs. 2 A and 3 C, a substantial fraction of channels are closed at Vj = 0 mV and application of Vj steps positive on Cx45 side should result in an initial increase in gj, and would only show a decay with longer Vj steps. To examine the gj-Vj relation of Cx40/Cx45 GJs, we used voltage steps 30 s in duration and voltage ramps 150 s in duration. We observed substantial reductions in gj at Vjs negative on Cx40 side both at macroscopic (see Fig. 3 C) and single channel levels (see Fig. 5 A). Similarly, it was shown that Vj gating occurs at negativity on the Cx40 side in Cx40/Cx43 cell pairs when Vj steps of 30 s in duration were used (22). Thus, we conclude that Cx40 has a negative gating polarity, like Cx45 and Cx43 hemichannels. Furthermore, our experimental data at the single channel level (Fig. 5) demonstrate gating transitions of 52 pS that we attribute to gating between open and closed states and those of 42 pS that we attribute to gating between open and residual states. Therefore, gating polarities of both slow and fast Vj gating mechanisms of Cx40 are negative.
Thus far, all heterotypic junctions that contain a Cx45 hemichannel on one side (Cx40/Cx45, Fig. 3 B; Cx43/Cx45, Fig. 2 A; Cx31/Cx45, Fig. 3 B in Abrams et al. (39)) show a strong decrease in gj when Vj is stepped from 0 to 40 mV relatively negative on the Cx45 side. A subsequent increase in gj occurs with Vjs exceeding 40 mV. Two mechanisms can contribute to this increase in conductance: 1), interactions between the two series gates in the Cx45 hemichannel (66); and 2), the rectification of the residual state of the Cx45 hemichannel demonstrated in the Fig. 2 C. Rectification of the residual state is a property that has been described in other connexins, such as Cx32 and Cx43, and has been proposed to result from introduction of charge asymmetry in the pore as a result of conformational changes associated with one hemichannel gating (37,38).
Earlier we reported that the strong gj-Vj gating asymmetry of Cx43/Cx45 (25) and Cx31/Cx45 (39) heterotypic junctions produces signal transfer asymmetry that can be increased or decreased by making the cell expressing Cx45 relatively more negative or positive, respectively. In Fig. 3 D, we show that a similar phenomenon occurs in Cx40/Cx45 junctions. Variations of Vh of around zero very effectively modulate the degree of asymmetry, and at Vh 20 mV positive on the Cx40-EGFP side, electrical coupling is almost unidirectional for signals comparable in amplitude to action potentials. Thus, a synapse with Cx45 on the presynaptic side and Cx40 on the postsynaptic side could be highly rectifying. Transmission at synapses using this mechanism would differ from known electrical synapses that are involved in escape systems, where the rectification is very rapid (
= 1 ms (67–69)), and presumably is caused by single channel rectification or very fast gating. It is interesting that in all described rectifying synapses the presynaptic neuron is at more negative resting potential ((70) and reviewed in (71)) resulting in Vh of 15 mV. Therefore, cell-to-cell signaling asymmetry seems to be a common feature of heterotypic junctions containing a Cx45 hemichannel on one side. Such signaling asymmetry may be functionally relevant in the CNS where signal propagation in one direction is preferred; Cx45 expression has been reported in neurons of the olivocerebellar system, in spinal motor neurons and in the ganglion cell and inner nuclear layers of mouse retina (12,72,73). For example, if orthodromic propagation is from the cell expressing Cx45 (presynaptic, if considering an electrical synapse) to the cell expressing Cx40 or Cx43, one would expect antidromic propagation to be drastically reduced (see Fig. 3 D) due to the slow gate of the Cx45 hemichannel, which closes the channels fully. In addition, it may explain, at least in part, differences in the velocity of the spread of excitation in antero- and retrograde directions in sinoatrial (7,8) and AV-nodal regions of the heart.
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONSUPPLEMENTARY MATERIALACKNOWLEDGEMENTSREFERENCES |
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONSUPPLEMENTARY MATERIALACKNOWLEDGEMENTSREFERENCES |
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This work was supported by grants from the National Institutes of Health and the German Research Association, RO1 NS036706 to F.F.B., MH66199 to V.K.V. and Wi 270/25-1,2 to K.W.
Submitted on October 11, 2006; accepted for publication December 4, 2006.
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