Correspondence to: Thaddeus A. Bargiello, Department of Neuroscience, Kennedy Center, Albert Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, NY 10461. Fax:718-430-8821 E-mail:bargiell{at}aecom.yu.edu.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Gap junctions are intercellular channels formed by the serial, head to head arrangement of two hemichannels. Each hemichannel is an oligomer of six protein subunits, which in vertebrates are encoded by the connexin gene family. All intercellular channels formed by connexins are sensitive to the relative difference in the membrane potential between coupled cells, the transjunctional voltage (Vj), and gate by the separate action of their component hemichannels (Harris, A.L., D.C. Spray, and M.V. Bennett. 1981. J. Gen. Physiol. 77:95117). We reported previously that the polarity of Vj dependence is opposite for hemichannels formed by two closely related connexins, Cx32 and Cx26, when they are paired to form intercellular channels (Verselis, V.K., C.S. Ginter, and T.A. Bargiello. 1994. Nature. 368:348351). The opposite gating polarity is due to a difference in the charge of the second amino acid. Negative charge substitutions of the neutral asparagine residue present in wild-type Cx32 (Cx32N2E or Cx32N2D) reverse the gating polarity of Cx32 hemichannels from closure at negative Vj to closure at positive Vj. In this paper, we further examine the mechanism of polarity reversal by determining the gating polarity of a chimeric connexin, in which the first extracellular loop (E1) of Cx32 is replaced with that of Cx43 (Cx43E1). The resulting chimera, Cx32*Cx43E1, forms conductive hemichannels when expressed in single Xenopus oocytes and intercellular channels in pairs of oocytes (Pfahnl, A., X.W. Zhou, R. Werner, and G. Dahl. 1997. Pflügers Arch. 433:733779). We demonstrate that the polarity of Vj dependence of Cx32*Cx43E1 hemichannels in intercellular pairings is the same as that of wild-type Cx32 hemichannels and is reversed by the N2E substitution. In records of single intercellular channels, Vj dependence is characterized by gating transitions between fully open and subconductance levels. Comparable transitions are observed in Cx32*Cx43E1 conductive hemichannels at negative membrane potentials and the polarity of these transitions is reversed by the N2E substitution. We conclude that the mechanism of Vj dependence of intercellular channels is conserved in conductive hemichannels and term the process Vj gating. Heteromeric conductive hemichannels comprised of Cx32*Cx43E1 and Cx32N2E*Cx43E1 subunits display bipolar Vj gating, closing to substates at both positive and negative membrane potentials. The number of bipolar hemichannels observed in cells expressing mixtures of the two connexin subunits coincides with the number of hemichannels that are expected to contain a single oppositely charged subunit. We conclude that the movement of the voltage sensor in a single connexin subunit is sufficient to initiate Vj gating. We further suggest that Vj gating results from conformational changes in individual connexin subunits rather than by a concerted change in the conformation of all six subunits.
Key Words: ion channels, voltage dependence, bipolar gating, gating mechanisms, gap junction
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Gap junctions are intercellular channels formed by the interaction of two hemichannels or connexins. Each hemichannel is composed of six protein subunits, connexins, which surround a central aqueous pore with a radius of 7 Å (
7 Å resolution (
All intercellular channels formed by the 16 members of the vertebrate connexin gene family are sensitive to the difference in membrane potential between coupled cells, the transjunctional voltage (Vj), while some also display weak sensitivity to the absolute membrane potential (Vm) (see
We have reported that the polarity of Vj dependence is opposite in hemichannels formed by Cx32 and Cx26 when they are paired to form intercellular channels (
While negative charge substitutions up to and including the 10th amino acid residue can reverse the Vj-gating polarity of Cx32 hemichannels (Purnick, Oh, Abrams, Verselis, and Bargiello, manuscript submitted for publication), a negative charge substitution of the 11th amino acid residue (Cx32S11D) cannot. Taken together, these results indicate that the first 10 amino acid residues can reside within the transjunctional electric field and that charged residues at any of these positions can contribute to the formation of the transjunctional voltage sensor. We have recently shown that the inherent structural flexibility of a glycine residue (G12), conserved in Group I (ß) connexins, allows the formation of a turn, which positions the amino terminus within the channel pore (Purnick, Benjamin, Verselis, Barhgiello, and Dowd, manuscript submitted for publication). It should be noted that there is no evidence for a comparable turn in the NH2 terminus of Group II () connexins, although D3N appears to reverse the gating polarity of Cx46 hemichannels (Verselis, V.K., unpublished observation).
Hemichannels formed by some connexins including Cx46, Cx56, a Cx32 chimera designated Cx32*Cx43E1, and perhaps Cx32 form voltage-dependent conductive hemichannels when they are unapposed by a hemichannel expressed in a second cell (
In this paper, we explore further the mechanistic and structural correlates of voltage-dependent gating of channels formed by Cx32 by determining the stoichiometry of polarity reversal by the N2E substitution. As these experiments are difficult, if not impossible, to accomplish with intercellular channels, we examined the gating polarity of conductive heteromeric hemichannels formed by expressing mixtures of Cx32*Cx43E1 and Cx32N2E*Cx43E1 in Xenopus oocytes. We demonstrate that heteromeric conductive hemichannels, containing subunits with either positive or negative charges in their NH2 termini, exhibit bipolar Vj gating. The frequency of bipolar hemichannels observed correlates with the fraction of hemichannels that are expected to contain at least one oppositely charged connexin subunit. Therefore, the movement of the voltage sensor in a single connexin subunit is sufficient to initiate Vj gating. We propose that channel closure also results from a conformational change in individual connexin subunits rather than by a concerted change in the conformation of all six subunits.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Construction of Chimeric Connexins, RNA Synthesis, and Oocyte Injection
A Cx32 chimera, Cx32*Cx43E1, in which the first extracellular loop (E1, residues E41R75) of Cx32 is replaced by the corresponding segment of Cx43 (residues E42R76) was made using oligonucleotide primers with the polymerase chain reaction. For Cx32N2E*Cx43E1, the DNA segment containing the N2E substitution was excised by restriction enzymes and subcloned into Cx32*Cx43E1. Both chimeric connexins were cloned into the plasmid vector, pGem-7zf(+) (Promega) and sequenced in entirety. RNA was synthesized from linearized plasmid templates using a "mMESSAGE mMACHINE" T7 Kit (Ambion) according to the manufacturer's protocol. For the subunit stoichiometry experiments, DNA concentrations were determined spectrophotometrically and mixed in appropriate ratios before RNA synthesis.
Approximately 50 nl of 1 ng/nl RNA was coinjected into a Xenopus oocyte with 0.3 pmol/nl of an antisense phosphorothioate oligonucleotide complimentary to Xenopus Cx38. This antisense oligonucleotide blocks all endogenous coupling between oocyte pairs attributable to Cx38 within 72 h (
Electrophysiological Recordings
Macroscopic recordings from pairs of Xenopus oocytes were performed and data were analyzed as described by
Voltage-clamp recordings of macroscopic currents from single Xenopus oocytes were obtained with a two-electrode voltage clamp (GeneClamp 500; Axon Instruments, Inc.). Electrodes were filled with 2 M KCl. Data were acquired using pClamp 6.0 software and a Digidata 1200 interface (Axon Instruments, Inc.).
In all patch-clamp experiments, the pipette solution was same as a bath solution described above, except CaCl2 and MgCl2 were replaced by 2 mM EDTA and 2 mM EGTA (ND98). Single channel data were acquired using pClamp 7.0 software, an Axopatch 200B integrating patch amplifier, and a Digidata 1200A interface (Axon Instruments, Inc.). Data were acquired at 5 kHz and filtered at 1 kHz with a four-pole low pass Bessel filter.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Polarity of Vj Gating of Cx32*Cx43E1 and Cx32N2E*Cx43E1 Hemichannels Inferred from the Voltage Dependence of Intercellular Channels
A Cx32 chimera, Cx32*Cx43E1, in which the first extracellular loop (E1, residues E41R75) of Cx32 is replaced by the corresponding segment of Cx43 (residues E42R76) forms both conductive hemichannels and intercellular channels in Xenopus oocytes. The amino acid sequence of E1 differs by nine residues in the two connexins, and although four of the differences involve charged amino acid residues, there is no change in the net charge of the E1 loop.
We first examine whether the Cx43E1 substitution changes the gating polarity of Cx32 hemichannels or the ability of the N2E substitution to reverse the gating polarity of Cx32 hemichannels when they are paired to form intercellular channels. The data shown below demonstrates that the Cx43E1 substitution does not reverse the gating polarity of the Cx32 hemichannel, nor does it alter the ability of the N2E substitution to reverse the polarity of Vj dependence.
The conductancevoltage relations of heterotypic Cx32*Cx43E1/Cx32 and Cx32*Cx43E1/Cx26 junctions and homotypic Cx32*Cx43E1 junctions are shown in Fig 1AC. In heterotypic pairings with Cx32, junctional currents decline to steady state levels for either polarity of Vj (Fig 1 A), resembling those of homotypic Cx32 junctions (see
|
In heterotypic pairings with Cx26, junctional conductance decreases to a minimum when the cell expressing Cx26 is relatively positive (Fig 1 B). The voltage dependence of Cx32*Cx43E1/Cx26 junctions is qualitatively similar to that of heterotypic Cx32/Cx26 junctions. We conclude that Cx32*Cx43E1 and Cx26 hemichannels have opposite gating polarities. Thus, the asymmetry in the steady state conductancevoltage relation of the Cx32*Cx43E1/Cx26 junction is due to the closure of both Vj gates when the Cx26 cell is relatively positive and the opening of both Vj gates when the Cx26 cell is made relatively negative.
The conductancevoltage relation of initial currents of the Cx32*Cx43E1/Cx26 heterotypic junction is asymmetric and comparable with that of Cx32/Cx26 heterotypic channels. The voltage dependence of initial conductance reflects the rectification of open-channel currents and is a consequence of asymmetries in the position of charged residues that result from the heterotypic pairing (see
Junctional currents of homotypic Cx32*Cx43E1 intercellular channels decrease for either polarity of Vj, but do so asymmetrically. Currents decrease more when either cell is hyperpolarized to the same voltage (Fig 1 C). In experimental paradigms in which the membrane potentials of both cells are changed equally and simultaneously, junctional conductance decreases when the cells are hyperpolarized and increases when they are depolarized (not shown). This result is consistent with the presence of Vm dependence. Although in this case, "chemical gating" may also contribute to the observed asymmetry in the conductancevoltage relation, as it is possible that the intercellular channel is gated by the voltage-dependent entry of Ca2+ (see
We next examined whether the N2E substitution reverses the gating polarity of Cx32*Cx43E1 hemichannels when they are paired to form intercellular channels. Negative charge substitutions at this position, Cx32N2E and Cx32N2D, reverse the gating polarity of Cx32 hemichannels, from closure at negative Vj to closure at positive Vj (
The conductancevoltage relations of homotypic and heterotypic Cx32N2E*Cx43E1 junctions paired with Cx26 and Cx32 are shown in Fig 1DF. Junctional currents in the Cx32N2E*Cx43E1/Cx32 heterotypic junction decline to steady state levels only when positive Vj is applied to the Cx32N2E*Cx43E1 side of the junction, the Cx32 side of the junction is relatively negative; whereas, in the Cx32N2E*Cx43E1/Cx26 heterotypic junction, steady state currents decline for both polarities of applied Vj. These results are opposite those obtained with heterotypic pairings of the parental Cx32*Cx43E1 junction. The asymmetry in the steady state conductancevoltage relation of the Cx32/Cx32N2E*Cx43E1 heterotypic junction qualitatively resembles that of the Cx32*Cx43E1/Cx26 junction, while the steady state conductancevoltage relation of Cx32N2E*Cx43E1/Cx26 qualitatively resembles that of the Cx32*Cx43E1/Cx32 heterotypic junction (compare Fig 1A, Fig B, Fig D, and Fig E). These results indicate that the negative charge substitution (N2E) reverses the gating polarity of Vj dependence in the Cx32*Cx43E1 hemichannel as it does in the wild-type Cx32 hemichannel. Closure of the Cx32N2E*Cx43E1 hemichannel occurs at smaller Vj and opposite polarity than the Cx32*Cx43E1 hemichannel. The N2E substitution also shifts the steady state conductancevoltage relation of the wild-type Cx32 channel (see
Voltage Dependence and Gating Polarity of Cx32*Cx43E1 and Cx32N2E*Cx43E1 Conductive Hemichannels
Macroscopic recordings.
A macroscopic recording of membrane currents in a Xenopus oocyte injected with Cx32*Cx43E1 RNA is presented in Fig 2 A. Depolarizations of the oocyte membrane to positive potentials from a holding potential of -90 mV result in a slowly developing outward current that does not plateau during the 30-s voltage step. Depolarization of oocyte membranes to voltages greater than +40 mV in Ca2+ containing bath solutions usually leads to the activation of substantial outward endogenous currents (probably Ca2+-activated Cl- currents). These endogenous currents can obscure those attributable to the activation of the Cx32*Cx43E1 conductive hemichannel (not shown).
|
Tail currents resulting from the repolarization of the oocyte membrane to the initial holding potential of -90 mV exhibit a complex time course. Typically, tail currents show an initial increase and then slowly return to steady state levels with a multiexponential time course (Fig 2 A). The time course of tail current relaxations is influenced by the concentration of Ca2+ in the bath solution. Reductions in Ca2+ concentration from 5 to 2 mM prolong the time required to reach steady state current levels (Fig 2 B). The replacement of Ca2+ with 2 mM Ba2+ alters the shape and time course of the tail currents; notably the initial increase in tail currents is absent (Fig 2 C). It is likely that the substitution of Ca2+ with Ba2+ reduces the contribution of Ca2+-activated Cl- currents that are endogenous to Xenopus oocytes (
A representative macroscopic recording of Cx32N2E*Cx43E1 conductive hemichannels in 5 mM Ca2+-containing bath solutions is presented in Fig 2 D. Unlike the hemichannels formed by the chimeric parental connexin, Cx32*Cx43E1, macroscopic currents plateau and decline at membrane potentials exceeding +30 mV. The decrease in macroscopic currents is consistent with the reversal of the gating polarity of Cx32N2E*Cx43E1 hemichannels in intercellular pairings (see Fig 1).
Single channel recordings of Cx32*Cx43E1 conductive hemichannels.
Cell-attached patch recordings of a single Cx32*Cx43E1 conductive hemichannel expressed in Xenopus oocyte membranes in divalent cation-free bath solutions are presented in Fig 3. At the negative membrane potentials shown (the resting membrane potential in these experiments was typically between 0 and -10 mV), the Cx32*Cx43E1 conductive hemichannel flickers between levels that correspond to fully open and subconductance levels. In these recordings, the current corresponding to full closures can be determined by loop gating closures (see Fig 5 B). The mean closed duration of the rapid "flickery" transitions is 1.5 ms at -70 mV (Fig 3 B). The entry of the channel into this "short-duration" substate does not substantially reduce the open probability of the channel. The open probability of the channel is 0.90 at -80 mV when channel closures occur by this mechanism. At larger negative potentials, channel closures to longer-lived subconductance levels become evident (for example, in the -80-mV trace in Fig 3 A and 4 A). The open probability of the channel shown in the longer current trace in Fig 4 A is
0.57 at -80 mV. At moderate positive membrane potentials, the open probability of the channel approaches 1.0 (Fig 3 A).
|
|
|
As illustrated in Fig 4 A, the channel adopts a "bursting" behavior at negative potentials. While the frequency of "short-duration" events (1.5 ms closed duration) increases as the membrane is hyperpolarized, much longer-lived transitions to at least one subconductance level appear (Fig 3 A and 4 A). The time that the channel resides at these longer-lived subconductance levels ranges from tens of milliseconds to tens of seconds. The events-list histogram of closures to substates of duration >50 ms at -80 mV, shown in Fig 4 B, is well fit by a sum of two exponentials with time constants, 1 = 56.8 ms and
2 = 593 ms. Events with durations longer than 3 s become more prevalent as the oocyte membrane is hyperpolarized beyond -80 mV, but because the closures are of such long duration, we were unable to record a sufficient number of events to estimate their time constant. As described above, the reduction in open probability caused by the entry of the channel into these "longer-lived" (>50 ms duration) substates largely accounts for the decrease in junctional currents observed in macroscopic recordings of intercellular channels and to the relaxation of tail currents in conductive hemichannels formed by the Cx32*Cx43E1 chimera (Fig 2). As discussed below, channel closures by loop gating have the same polarity and therefore are also likely to contribute to the tail currents observed at negative potentials.
Fig 5 A demonstrates that the Cx32*Cx43E1 conductive hemichannel can also enter into at least two other subconductance levels at negative membrane potentials (-100 mV in the case shown).
The Cx32*Cx43E1 conductive hemichannel also undergoes loop gating at sufficiently large membrane negative voltages (Fig 5 B, *). Loop gating transitions between the fully open and fully closed states are characterized by a series of stepwise reductions in conductance that give the overall appearance of "slow gating transitions." Loop gating closures become more prevalent at hyperpolarizing membrane potentials in both Cx46- and Cx32*Cx43E1 conductive hemichannels, although they are observed less frequently in cell-attached patches of Cx32*Cx43E1-expressing oocytes than those expressing Cx46 and tend to appear at more negative membrane potentials.
Similar complexities in kinetics and the existence of multiple subconductance levels (reflecting both Vj gating and loop gating) have been described in single-channel records of intercellular channels formed by Cx32 and Cx26 (see
The behavior of a single Cx32*Cx43E1 conductive hemichannel at positive membrane potentials is illustrated in Fig 5 C. Typically, the channel remains at the fully open conductance level when the membrane potential is less than +70 mV. However, at potentials greater than +70 mV, transitions to a single subconductance level are observed in long recordings. These transitions are infrequent and their polarity is opposite that of Vj gating and loop gating. It is likely that they are due to a third gating mechanism, and they may account for the slight relaxation of junctional currents observed in the macroscopic recordings of the heterotypic Cx32*Cx43E1/Cx26 junction when the Cx26 cell is made relatively negative (Fig 1 B). This third gating mechanism has the wrong polarity to account for the Vm dependence observed in Cx32*Cx43E1 homotypic junctions.
A comparison of the I-V relations of Cx32*Cx43E1 conductive hemichannels obtained with different voltage-ramp protocols is presented in Fig 6. In ±100-mV ramps of short duration (3 s) from an initial holding potential of approximately -10 mV (the resting potential of the oocyte when Cx32*Cx43E1 conductive hemichannels are expressed), channel closures are observed only at negative membrane potentials and primarily involve gating transitions to subconductance levels (i.e., Vj gating) (Fig 6A and Fig B). Transitions ascribable to loop gating are observed infrequently in current traces obtained with this voltage-ramp protocol. Channel closures to substates at positive membrane potentials are more likely to be observed in the I-V relations elicited by longer duration (8 s) voltage ramps (Fig 6C and Fig D).
|
The inward rectification of open-channel currents seen in cell-attached records is also evident when excised patches are placed in symmetric (ND98) salt solutions (not shown). This result indicates that the open-channel rectification is likely to arise from an asymmetry in the position of fixed charges residing in or near the channel pore (see
Single channel recordings of Cx32N2E*Cx43E1 conductive hemichannels.
Cell-attached recordings of single Cx32N2E*Cx43E1 conductive hemichannels are shown in Fig 7. In divalent cation-free solutions (Fig 7, AC), closures to subconductance levels are observed even at small positive membrane potentials (+20 mV). The voltage dependence of the open probability of homomeric Cx32N2E*Cx43E1 conductive hemichannels is presented in Fig 8 B for positive membrane potentials. No gating events to subconductance levels are observed at negative membrane potentials. However, loop-gating transitions are still observed at negative membrane potentials (Fig 7 C). Overall, these results are consistent with the macroscopic behavior of the Cx32N2E*Cx43E1 conductive hemichannel (Fig 2 D). The shift in the voltage dependence of the Cx32*Cx43E1 conductive hemichannel caused by the N2E substitution is similar to that observed for both Cx32N2E and Cx32N2E*Cx43E1 hemichannels when either is paired to form homotypic and heterotypic intercellular channels with Cx26 and Cx32 (Fig 1;
|
|
Interestingly, the N2E substitution causes a substantial change in the I-V relation of the fully open Cx32*Cx43E1 conductive hemichannel. As illustrated in Fig 7 D, there is a slight outward rectification of currents. The change in rectification most likely reflects the effect of the negative charge of the glutamic acid residue, which should be located near the cytoplasmic end of the channel. A comparable I-V relation to that observed can be obtained with the application of the 1-D Poisson-Nernst-Plank model of
Stoichiometry of Vj Gating Polarity Reversal by the N2E Substitution
The behavior of single heteromeric conductive hemichannels, formed by coinjecting RNA synthesized from mixtures of Cx32*Cx43E1 and Cx32N2E*Cx43E1 DNA templates is shown in Fig 8 A. Single heteromeric conductive hemichannels containing mixtures of Cx32N2E*Cx43E1 and Cx32*Cx43E1 are bipolar; that is, they can close to subconductance levels at both positive and negative membrane potentials, but remain open at membrane potentials around zero. This is most easily seen in the I-V relation shown in Fig 8 C. This contrasts the behavior of homomeric Cx32N2E*Cx43E1 conductive hemichannels, which close to subconductance levels only at positive membrane potentials. The observed bipolarity cannot be explained by a shift in voltage dependence resulting from a change in the chemical free energy of the channel, as closures are less frequent at membrane potentials around zero than at both larger positive and negative membrane potentials.
To define the subunit stoichiometry of Vj gating, we determined the distribution of gating polarities of single conductive hemichannels expressed in oocytes after injection with 20:1 and 1:20 RNA mixtures. In these experiments, the ratio of RNA is defined by the concentration of Cx32N2E*Cx43E1 compared with Cx32*Cx43E1. If both RNAs are expressed equally and the resulting protein subunits are assembled randomly into hexameric hemichannels, then the distribution of conductive hemichannel types will be predicted by the binomial distribution. In the 20:1 RNA mixture, 26.5% of channels should contain at least one Cx32*Cx43E1 subunit. In the 1:20 RNA mixture, 26.5% of channels should contain at least one Cx32N2E*Cx43E1 subunit. If the movement of a single appropriately charged subunit is sufficient to initiate Vj gating, then 26.5% of single conductive hemichannels are expected to display bipolar gating. If, however, at least two identical subunits are required, then only 3.28% of single conductive hemichannels are expected to display bipolar gating.
The symmetry of the binomial distribution provides a means of assessing the assumptions of equal efficacy of RNA translation and random assembly of mutant and wild-type subunits into hexameric conductive hemichannels. If all these assumptions are met, the ratio of bipolar to unipolar channels observed in 20:1 mixtures should be the same as that observed in 1:20 mixtures.
In 20:1 RNA mixtures of Cx32N2E*Cx43E1:Cx32*Cx43E1, 12 of 57 single conductive hemichannels observed (21.1%) displayed bipolar Vj gating similar to that illustrated in Fig 8 A, top. The remainder of conductive hemichannels (78.9%) were indistinguishable from channels obtained when only Cx32N2E*Cx43E1 RNA was injected. The distribution of channel types observed does not differ significantly from that predicted by binomial distribution if one Cx32*Cx43E1 subunit is sufficient to cause bipolar gating (2 = 0.015, P < 0.90). We conclude that the single Cx32*Cx43E1 subunit is sufficient to initiate Vj gating at negative membrane potentials.
In oocytes injected with 1:20 mixtures of Cx32N2E*Cx43E1:Cx32*Cx43E1 RNA, 82 of 121 (67.8%) display behavior indistinguishable from conductive hemichannels formed by Cx32*Cx43E1. That is, these hemichannels close to subconductance levels at negative membrane potentials and transit to a subconductance level at positive membrane potentials exceeding 70 mV. The remaining conductive hemichannels (39 of 121, or 32.2%) display two distinct sets of transitions at depolarizing membrane potentials (Fig 8 A, middle and bottom). At positive membrane potentials, transitions are observed that are comparable with those observed in the parental Cx32*Cx43E1 homomeric conductive hemichannel described above. This gating is exemplified by the transition in Fig 8 A, *. The second population of gating transitions to subconductance levels have a substantially larger amplitude (Fig 8 A, middle and bottom) and occur more frequently at smaller depolarizing membrane potentials (not illustrated). The distribution of conductive hemichannel types observed in 1:20 RNA mixtures does not differ significantly from that predicted by the binomial distribution if one N2E subunit is sufficient to cause bipolar gating (2 = 0.017, P < 0.80). We conclude that a single Cx32N2E*Cx43E1 subunit is sufficient to initiate channel closure at positive membrane potentials.
The slight asymmetry observed in the number of bipolar conductive hemichannels observed after injection of 20:1 and 1:20 RNA ratios (21.1 and 32.2%, respectively) can be explained if the Cx32N2E*Cx43E1 subunit was present in a slight excess relative to Cx32*Cx43E1. This may have been due to systematic error in the determination of the concentration of DNA templates used to synthesize RNA (see MATERIALS AND METHODS). Alternatively, it may reflect a preferential rate of transcription or translation of the Cx32N2E*Cx43E1 construct, or a nonrandom assembly of the different subunits into hexameric hemichannels. However, the difference observed with the two RNA ratios does not alter the conclusion that a single appropriately charged subunit is sufficient to initiate Vj gating. If more than a single appropriately charged subunit were required to the reverse gating polarity of a hexameric hemichannel, then no more than 3.28% of channels would be expected to exhibit bipolar gating behavior in either 20:1 or 1:20 RNA mixtures. This is far less than the number observed.
The open probabilities of homomeric Cx32N2E*Cx43E1 conductive hemichannels and heteromeric conductive hemichannels containing at least one Cx32N2E*Cx43E1 subunit are plotted as a function of positive membrane potential in Fig 8 B. The observed open probability of the homomeric hemichannel is much less than that of the heteromeric hemichannel at comparable voltages. Also, the slope of the relation is steeper for the homomeric hemichannel. For simplicity, the changes in open probability at negative membrane potentials are not shown in Fig 8 B. Note, however, that in the case of the homomeric Cx32N2E*Cx43E1 hemichannel, a reduction in open probability at large negative voltages should result as a consequence of loop gating. While in the case of the heteromeric hemichannel shown, the reduction in open probability that would be evident at negative potentials should result from Vj gating as well as loop gating. Vj gating would be initiated by any of the five Cx32*Cx43E1 subunits.
The I-V relations shown in Fig 8 C illustrate two additional properties of the voltage gating of heteromeric conductive hemichannels that contain a single N2E subunit. First, it is apparent that a heteromeric hemichannel containing a single N2E subunit can make transitions to more than a single subconductance level at positive membrane potentials. This result indicates that the existence of multiple substates is not necessarily related to the number of subunits that are available to close for a given polarity of voltage. Second, the extrapolation of the I-V relation of the subconductance state that the hemichannel entered at negative membrane potentials (Fig 8 C, dotted line) does not correspond to the I-V relation of any of the subconductance levels entered into at positive potentials. This result suggests that the conformations of the substates of the heteromeric hemichannel differ when closure is initiated by the movement of the single N2E subunit than when initiated by one of the five Cx32*Cx43E1 subunits.
Finally, it is interesting to note the presence of a single N2E subunit reduces the inward rectification of currents passing through the open state of the conductive hemichannel relative to that of the parental Cx32*Cx43E1 conductive hemichannel (compare Fig 8 C with Fig 6). Typically, open hemichannel currents with a single N2E subunit rectify 1.5-fold over ±100 mV, compared with
2-fold for the homomeric Cx32*Cx43E1 hemichannel over this voltage range. This result supports the conclusion that charges in the amino terminus lie within the channel pore and play a substantial role in ionic permeation of channels formed by these connexins (see
Description of a Channel Endogenous to Xenopus Oocytes
Single-channel records of the channels described above are often "contaminated" by an endogenous channel that is commonly found in Xenopus oocytes and may be mistaken for exogenously expressed conductive hemichannels formed by connexins. The endogenous Xenopus oocyte channel has a conductance of 2025 pS at 30 mV and 30 pS at -50mV (Fig 9 A). The channel becomes active at depolarizing membrane potentials and displays "use dependence," in that its activity at a given hyperpolarizing membrane potential is greater when the hyperpolarizing step is preceded by a membrane depolarization (Fig 9 B). The open probability of the channel appears to decrease as a function of time at hyperpolarizing membrane potentials (Fig 9 B), suggestive of a "rundown" mechanism. Another interesting feature of the channel is the long delay in channel reopening that is observed when the membrane potential is reversed from -90 to +90 mV (Fig 9 B). The I-V relation of the open state of this channel is essentially linear (Fig 9 C). Overall, the channel displays voltage-gating properties that are similar to mechanosensitive channels in Xenopus oocytes described by
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The major aim of this paper was to further elucidate the mechanism of transjunctional voltage gating by determining the stoichiometry of polarity reversal by a negative charge substitution (N2E) in the amino terminus of a Cx32 chimera (Cx32*Cx43E1). This chimera forms both intercellular channels and conductive hemichannels in Xenopus oocytes. This property has allowed us to compare the gating polarity of hemichannels paired to form intercellular channels with that of unapposed hemichannels (conductive hemichannels) and to determine the stoichiometry of polarity reversal.
In our previous studies, we demonstrated that the polarities of Vj dependence of homomeric hemichannels formed by two Group I connexins, Cx26 and Cx32, are opposite when they are paired to form intercellular channels in Xenopus oocytes (
The conclusions of our earlier study (
In this paper, we demonstrate that Cx32*Cx43E1 homomeric conductive hemichannels close at negative Vj, as do wild-type Cx32 hemichannels in heterotypic pairings with Cx32 and Cx26 hemichannels and that the polarity of Vj dependence is reversed by the N2E substitution. We could not obtain stably transfected cell lines to examine the Vj-dependent gating of single homotypic and heterotypic intercellular channels formed by the chimera, but we infer that the mechanism is similar based on single-channel recordings of conductive hemichannels formed by Cx32*Cx43E1 and Cx32N2E*Cx43E1.
The results presented in this paper indicate that the mechanisms of voltage-dependent gating of Cx32*Cx43E1 conductive hemichannels are for the most part comparable with those of intercellular Cx32 channels described by
At negative potentials, the conductive hemichannel makes rapid transitions between the fully open state and closed substates (Vj gating) and "slow" (complex) transitions to the fully closed state (loop gating). Our results indicate that Vj gating of Cx32*Cx43E1 conductive hemichannels is complex; characterized by the presence of at least three distinct substates and at least four kinetically distinguishable substates with mean closed durations of 1.5, 55, 600, and >1,000 ms. The negative charge substitution, N2E, reverses the gating polarity of all these substates. The simplest interpretation of this result is that all of these gating events are initiated by, or result from, a conformational change in the NH2 terminus.
The polarity of loop gating in Cx32*Cx43E1 conductive hemichannels is the same as that reported for Cx46 by
The closure of Cx32*Cx43E1 conductive hemichannels at positive membrane potentials exceeding +70 mV was unexpected as comparable closures are not observed in single channel records of heterotypic intercellular channels formed by Cx32 and Cx26, nor in Cx46 conductive hemichannels (see
Stoichiometry of the Polarity Reversal of Vj Gating
The results presented in this study demonstrate that a single connexin subunit can initiate Vj gating. The bipolarity of heteromeric hemichannels containing oppositely charged NH2 termini can be explained if the closures to substates that are observed at positive membrane potentials are initiated by the movement of an N2E subunit, while the closures to substates that are observed at negative potentials are initiated by the movement of a wild-type subunit. Thus, in effect, a hemichannel contains six voltage sensors, one in each subunit, that function individually but not necessarily independently to initiate Vj gating. This view is supported by all the available data.
Our results show that the voltage sensitivity of a hemichannel correlates with the number of appropriately charged subunits that are available to gate. In the case illustrated in Fig 8 B, the probability that a Cx32N2E*Cx43E1 homomeric hemichannel (six identical subunits) closes at positive membrane potentials is substantially greater than that of a heteromeric hemichannel containing a single Cx32N2E*Cx43E1 subunit. This result can be explained if the open probabilities of heteromeric and homomeric hemichannels reflect the number of subunits that are available to initiate Vj gating. For example, in an independent gating model (no subunit interactions), the open probability of a homomeric hemichannel can be predicted from the binomial distribution using estimates of the probability that a single subunit of a heteromeric hemichannel will initiate Vj gating. If, for example, the open probability of a heteromeric hemichannel containing a single negatively charged subunit at a given positive voltage were 0.95, then the probability that all six subunits of a homomeric hemichannel formed by six negatively charged subunits would be open at the same voltage would be 0.74. However, the reduction in open probability that is observed at positive membrane potentials is much greater than predicted by a simple independent model (Fig 8 B). For example, at voltages where the open probability of a heteromeric hemichannel containing a single N2E subunit is 0.95, the corresponding open probability of the homomeric N2E hemichannel is <0.2. The deviation from independence can be explained by postulating the existence of electrostatic interactions between subunits, which are likely to alter the free energy (stability) of the open and closed channel states. It is reasonable to expect that electrostatic attractions among oppositely charged subunits would stabilize the open state of a heteromeric hemichannel, while electrostatic repulsion among identically charged subunits would destabilize the open state of a homomeric hemichannel.
The difference in slope of the two open probabilityvoltage relations illustrated in Fig 8 B is likely to reflect the movement of a greater amount of charge during voltage gating of the homomeric hemichannel than of the heteromeric hemichannel at positive potentials. A simple explanation for this observation is that, while the closure of the homomeric hemichannel in response to voltage may be initiated by the movement of the voltage sensor in a single subunit, ultimately all six subunits could move and contribute to the calculated gating charge. Whereas, in the heteromeric hemichannel, the slope of the relation may only reflect the movement of the charge contributed by the single N2E subunit. The possibility that multiple subunits respond to changes in membrane voltage may explain the multiexponential time course of current relaxations that is usually observed in macroscopic recordings of homomeric hemichannels at higher membrane potentials.
Allosteric Models of Vj Gating
While previous models have assumed that gating of intercellular channels (gap junctions) is likely to involve a concerted movement of all six connexin subunits (see, for example,
|
The I-V relation of the heteromeric conductive hemichannel presented in Fig 8 C is also consistent with an individual gating model like that illustrated in Fig 10 B. If gating resulted from a simple concerted mechanism, the set of substate conformations at negative potentials are likely to be the same as the set of substate conformations at positive potentials. This does not appear to be the case. Rather, the conformations of the "substates" of the hemichannel differ when closure is initiated by an N2E-containing subunit than when closure is initiated by a Cx32*Cx43E1 subunit. The multiplicity of substates that arise from the movement of the single N2E-containing subunit, which is apparent in Fig 8 C, suggests that a single subunit can adopt more than one "closed" conformation (see below).
Models of Vj Gating and Structural Implications
The ability of a single connexin subunit to initiate Vj gating suggests several refinements and additions to the models of Vj-dependent gating that we have previously proposed (
Previously, we suggested (
As discussed above, it is possible that both short- and long-range electrostatic interactions (such as among charges at the TM1/E1 border and NH2 terminus) exist. However, these interactions appear limited to changing the likelihood that an individual subunit will respond to a given change in applied voltage, rather than determining the net valence of the voltage sensor and the likelihood that all six subunits will make a concerted transition. The ability of a charge substitution at the TM1/E1 border to suppress the reversal of Cx32 gating polarity by N2D can be explained if the TM1/E1 substitution caused a shift in the voltage dependence of loop gating that obscured the polarity of Vj gating. Thus, it is possible that the reversal of polarity by Cx32N2D was not suppressed by the charge substitution at the TM1/E1 border and consequently that charges at this position are not part of the Vj sensor.
The results presented in this paper demonstrate that Vj gating is complex and includes gating events of markedly different duration and the existence of several substates. The short duration closures (1.5 ms, Fig 3), which are too brief to account for the current relaxations observed in macroscopic recordings of intercellular channels and conductive hemichannels formed by Cx32*Cx43E1 are likely to differ mechanistically from the longer duration closures (Fig 4 B). Yet, as the gating polarities of both short- and long-duration events are reversed by the N2E substitution, they all must be related to movements of the NH2 terminus. We propose that the NH2 terminus is flexible and that the brief closures might reflect "local" changes in the conformation of the NH2 terminus. We propose that the longer duration closures, which contribute more to the changes in conductance that are observed macroscopically, are initiated by another movement of the NH2 terminus and are ultimately mediated by the dynamic properties of a proline kink motif found in the second transmembrane helix (P87 in Cx32) of all connexins (
Based on the molecular and computational studies described in
Finally, all individual gating models, such as the one depicted in Fig 10 B, require that the interactions among the six subunits that form a hemichannel be sufficiently "elastic" to allow a single subunit to undergo conformational changes without forcing the adjacent subunits to adopt the same conformation. An explanation for how this may occur is suggested by the 3-D structure of a gap junction channel reported by
The model that emerges from our studies is that the first and second transmembrane helices of each of the six connexin subunits interact to form a functional domain, which contributes to the formation of the channel pore and plays a central role in the mechanism of Vj gating. Our results indicate that the connexin Vj sensor resides in the NH2 terminus and that the movement of the voltage sensor in a single connexin subunit is sufficient to initiate the set of conformational changes that result in channel closure to subconductance levels. We propose that hemichannel "closure" does not arise by a concerted mechanism but rather results from the changes in the conformation of individual subunits. Substates resulting from Vj gating may arise in two ways: a given subunit may adopt one of several different "closed conformations" and more than one subunit may adopt a closed conformation.
![]() |
Footnotes |
---|
1 Abbreviations used in this paper: E1, first extracellular loop; TM1, first transmembrane segment; Vj, transjunctional voltage.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We thank Angele Bukauskiene and Joe Zavilowitz for technical assistance and Brady Trexler for his comments and permission to cite some of his unpublished work.
This work was supported by the National Institutes of Health grant GM 46889.
Submitted: 16 December 1999
Revised: 7 April 2000
Accepted: 11 April 2000
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bennett, M.V.L., Barrio, L.C., Bargiello, T.A., Spray, D.C., Hertzberg, E.L., and Saez, J.C. 1991. Gap junctions: new tools, new answers, new questions. Neuron. 6:305-320[Medline].
Bukauskas, F.F., and Weingart, R. 1994. Voltage-dependent gating of single gap junction channels in an insect cell line. Biophys. J. 67:613-625[Abstract].
Bukauskas, F.F., Elfgang, C., Willecke, K., and Weingart, R. 1995. Biophysical properties of gap junction channels formed by mouse connexin40 in induced pairs of transfected HeLa cells. Biophys. J. 68:2289-2298[Abstract].
Bukauskas, F.F., and Peracchia, C. 1997. Two distinct gating mechanisms in gap junction channels: CO2 sensitive and voltage sensitive. Biophys. J. 72:2137-2142[Abstract].
Castro, C., Gomez-Hernandez, J.M., Silander, K., and Barrio, L.C. 1999. Altered formation of hemichannels and gap junction channels caused by C-terminal connexin-32 mutations. J. Neurosci. 19:3752-3760
Chen, D., and Eisenberg, R. 1993. Charges, currents and potentials in ionic channels of one conformation. Biophys. J. 64:1405-1421[Abstract].
Ebihara, L., Berthoud, V.M., and Beyer, E.C. 1995. Distinct behavior of connexin56 and connexin46 gap junctional channels can be predicted from the behavior of their hemi-gap-junctional channels. Biophys. J. 68:1796-1803[Abstract].
Ebihara, L. 1996. Xenopus connexin38 forms hemi-gap-junctional channels in the nonjunctional plasma membrane of Xenopus oocytes. Biophys. J. 71:742-748[Abstract].
Gil, Z., Magelby, K.L., and Silberberg, S.D. 1999. Membrane-pipette interactions underlie delayed voltage activation of mechanosensitive channels in Xenopus oocytes. Biophys. J. 76:3118-3127
Harris, A.L., Spray, D.C., and Bennett, M.V. 1981. Kinetic properties of a voltage-dependent junctional conductance. J. Gen. Physiol. 77:95-117[Abstract].
Hertzberg, E.L., Disher, R.M., Tiller, A.A., Zhou, Y., and Cook, R.G. 1988. Topology of the Mr 27,000 liver gap junction protein. Cytoplasmic localization of amino- and carboxyl termini and a hydrophilic domain which is protease-hypersensitive. J. Biol. Chem 263:19105-19111
Kado, R.T., and Baud, C. 1981. The rise and fall of electrical excitability in the oocyte of Xenopus laevis. J. Physiol. 77:1113-1117.
Lazark, A., and Peracchia, C. 1993. Gap junction gating sensitivity to physiological internal calcium regardless of pH in Novikoff hepatoma cells. Biophys. J. 65:2002-2012[Abstract].
Milks, L.C., Kumar, N.M., Houghten, R., Unwin, N., and Gilula, N.B. 1988. Topology of the 32-kd liver gap junction protein determined by site-directed antibody localizations. EMBO (Eur. Mol. Biol. Organ.) J. 7:2967-2975[Abstract].
Neyton, J., and Trautmann, A. 1985. Single channel currents of an intercellular junction. Nature. 317:331-335[Medline].
Oh, S., Ri, Y., Bennett, M.V.L., Trexler, E.B., Verselis, V.K., and Bargiello, T.A. 1997. Changes in permeability caused by connexin 32 mutations underlie X-linked Charcot-Marie-Tooth disease. Neuron. 19:927-938[Medline].
Oh, S., Rubin, J.B, Bennett, M.V.L., Verselis, V.K., and Bargiello, T.A. 1999. Molecular determinants of electrical rectification of single channel conductance in gap junctions formed by connexins 26 and 32. J. Gen. Physiol. 114:339-364
Paul, D.L., Ebihara, L., Takemoto, L.J., Swenson, K.I., and Goodenough, D.A. 1991. Connexin46, a novel lens gap junction protein, induces voltage-gated currents in nonjunctional plasma membrane of Xenopus oocytes. J. Cell Biol. 115:1077-1089[Abstract].
Rettinger, J. 1999. Novel properties of the depolarization-induced endogenous sodium conductance in the Xenopus laevis oocyte. Pflügers Arch. 437:917-924.
Ri, Y., Ballesteros, J.A., Abrams, C.K., Oh, S., Verselis, V.K., Weinstein, H., and Bargiello, T.A. 1999. The role of a conserved proline residue in mediating conformational changes associated with voltage gating of Cx32 gap junctions. Biophys. J. 76:2887-2898
Rubin, J.B., Verselis, V.K., Bennett, M.V.L., and Bargiello, T.A. 1992. Molecular analysis of voltage dependence of heterotypic gap junctions formed by connexins 26 and 32. Biophys. J. 62:183-195[Abstract].
Trexler, E.B., Bennett, M.V.L., Bargiello, T.A., and Verselis, V.K. 1996. Voltage gating and permeation in a gap junction hemichannel. Proc. Natl. Acad. Sci. USA. 93:5836-5841
Trexler, E.B., Bukauskas, F.F., Bennett, M.V.L., Bargiello, T.A., and Verselis, V.K. 1999. Rapid and direct effects of pH on connexins revealed by the Cx46 hemichannel preparation. J. Gen. Physiol. 113:721-742
Unger, V.M., Kumar, N.M., Gilula, N.B., and Yeager, M. 1999. Three dimensional structure of a recombinant gap junction membrane channel. Science. 283:1176-1180
Unwin, P.N. 1987. Gap junction structure and the control of cell-to-cell communication. Ciba Found. Symp. 125:78-91[Medline].
Veenstra, R.D. 1996. Size and selectivity of gap junction channels formed from different connexins. J. Bioenerg. Biomembr. 28:327-337[Medline].
Verselis, V.K., Bennett, M.V.L., and Bargiello, T.A. 1991. A voltage-dependent gap junction in Drosophila melanogaster. Biophys. J. 59:114-126[Abstract].
Verselis, V.K., Ginter, C.S., and Bargiello, T.A. 1994. Opposite voltage gating polarities of two closely related connexins. Nature. 368:348-351[Medline].
Weber, W.M. 1999. Endogenous ion channels in oocytes of Xenopus laevis: recent developments. J. Membr. Biol. 170:1-12[Medline].
Zimmer, D.B., Green, C.R., Evans, W.H., and Gilula, N.B. 1987. Topological analysis of the major protein in isolated intact rat liver gap junctions and gap junction-derived single membrane structures. J. Biol. Chem. 262:7751-7763