Correspondence to: Thaddeus A. Bargiello, Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Fax:Fax: 718-430-8821; E-mail:bargiell{at}aecom.yu.edu.
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Abstract |
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The fully open state of heterotypic gap junction channels formed by pairing cells expressing connexin 32 (Cx32) with those expressing connexin 26 (Cx26) rectifies in a way that cannot be predicted from the currentvoltage (IV) relation of either homotypic channel. Using a molecular genetic analysis, we demonstrate that charged amino acids positioned in the amino terminus (M1 and D2) and first extracellular loop (E42) are major determinants of the currentvoltage relation of the fully open state of homotypic and heterotypic channels formed by Cx26 and Cx32. The observed IV relations of wild-type and mutant channels were closely approximated by those obtained with the electrodiffusive model of Chen and Eisenberg (Chen, D., and R. Eisenberg. 1993. Biophys. J. 64:14051421), which solves the Poisson-Nernst-Plank equations in one dimension using charge distribution models inferred from the molecular analyses. The rectification of the Cx32/Cx26 heterotypic channel results from the asymmetry in the number and position of charged residues. The model required the incorporation of a partial charge located near the channel surface to approximate the linear IV relation observed for the Cx32*Cx26E1 homotypic channel. The best candidate amino acid providing this partial charge is the conserved tryptophan residue (W3). Incorporation of the partial charge of residue W3 and the negative charge of the Cx32E41 residue into the charge profile used in the Poisson-Nernst-Plank model of homotypic Cx32 and heterotypic Cx26/Cx32 channels resulted in IV relations that closely resembled the observed IV relations of these channels. We further demonstrate that some channel substates rectify. We suggest that the conformational changes associated with transjunctional voltage (Vj)-dependent gating to these substates involves a narrowing of the cytoplasmic entry of the channel that increases the electrostatic effect of charges in the amino terminus. The rectification that is observed in the Cx32/Cx26 heterotypic channel is similar although less steep than that reported for some rectifying electrical synapses. We propose that a similar electrostatic mechanism, which results in rectification through the open and substates of heterotypic channels, is sufficient to explain the properties of steeply rectifying electrical synapses.
Key Words: gap junctions, electrical rectification, rectifying electrical synapses, Poisson-Nernst-Plank, ion channel
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Introduction |
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Heterotypic gap junctions formed by two closely related connexins (Cx),1 Cx32 and Cx26, display marked but different asymmetries in the transjunctional voltage (Vj) dependence of initial and steady state conductances (
Although considerably less steep, the rectification of initial currents observed in the heterotypic Cx32/Cx26 junction qualitatively resembles that of a rectifying electrical synapse present in the abdominal nerve cord of the crayfish (
In their original report,
The connexins forming rectifying electrical synapses in vertebrates and invertebrates have not yet been identified and indeed the invertebrate gap junctions are formed by proteins encoded by another gene family with no primary sequence homology to the vertebrate connexin gene family (
The results of the molecular genetic studies described in this paper demonstrate that charged amino acid residues located in the amino terminus and first extracellular loop are major determinants of the rectification of the heterotypic Cx32/Cx26 channel. The rectifying IV relation of the heterotypic channel results from a structural asymmetry arising from differences in the number and position of fixed charges present in the Cx32 and Cx26 hemichannels. The different IV relations of homotypic channels can be explained by the different symmetrical charge distributions that result from homotypic pairings. Furthermore, we show that homotypic and heterotypic channels can enter substates that rectify. The nonlinearity of the IV relations of these substates is likely to result from the increased electrostatic effect of charged residues present in the amino terminus of one hemichannel that arises from a conformational change associated with Vj-dependent gating. This finding suggests that the change in conductance resulting from Vj gating may correspond to a narrowing of the entry of the connexin hemichannel near the cytoplasmic surface. We suggest that the rectification of single channel currents like that observed in the fully open state and substates of gap junction channels is responsible for the steep and rapid rectification observed in some electrical synapses.
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Materials and Methods |
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Construction of Chimeric Connexins, Site-directed Mutagenesis, RNA Synthesis, and Oocyte Injection
Chimeras of Cx26 and Cx32 were constructed by the procedure described by
Site-directed point mutations were constructed using oligonucleotide primers and the polymerase chain reaction. All chimeric constructs and point mutations were cloned into the plasmid vector, pGEM-7zf (+) (Promega Corp.) and sequenced in entirety. RNA was transcribed in vitro from linearized plasmid templates as described in l of 1
g/
l RNA was coinjected with 0.3
mol/
l 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 Recording of Xenopus Oocyte Pairs
Oocytes were devitellinized and paired 1224 h after RNA injection. Expression of junctional currents usually developed within 4 h of pairing. Only cells expressing <5 µS junctional conductance were employed to minimize the effects of access resistance on voltage dependence (. Coupled oocytes had equal resting membrane potentials that varied between -30 and -60 mV. Cells were voltage clamped to their resting potential (0 mV transjunctional voltage) and a family of junctional currents for each cell pair was generated by applying transjunctional voltages of ±5125 mV in an ascending series of 5-mV increments. Total pulse length was 20 s with a 90-s interpulse interval. Currents were digitized at two rates, first at 256 Hz for 2 s, and then at 28 Hz for the remainder of the trace. This allowed for greater accuracy in the measurement of initial currents while not requiring the collection of an excessive number of points during the extended time necessary for junctional currents to reach steady state. Each transjunctional voltage step was preceded by a 10-mV prepulse of short duration (100500 ms) that was used to normalize junctional currents within a family of current traces. In heterotypic junctions, current traces were scaled independently to prepulses of appropriate polarity of Vj since the prepulse amplitude elicited by a hyperpolarizing prepulse would not be the same as for a depolarizing prepulse administered to the same cell. Initial and steady state currents were determined by extrapolation of exponential fits to t = 0 and t =
. Initial (g0), steady state (g
), and residual (gmin) conductances were normalized to the value of initial conductance, g0 at Vj = 0 to obtain G0, G
, and Gmin. Additional details are provided in
Cell Transfections
The coding regions of wild-type and mutant connexins were cloned into pCI-neo (CMV promoter) vector (Promega Corp.) and transfected into the mouse Neuro-2a cell line and a second Neuro-2a cell line that expressed green fluorescent protein (GFP). Heterotypic channels were formed by mixing cell lines expressing different connexins, in which one member of the cell pair could be identified by the presence of GFP. In some cases, GFP expression was linked to that of a given connexin by the presence of an internal ribosomal entry site (IRES) sequence provided by H.-S. Shin (POSTECH, Pohang, South Korea). Transfected cell lines expressing exogenous connexins but not Cx45, which forms endogenous channels in Neuro-2a cell lines, were selected for single channel analysis. Cx45 channels are easily distinguished by their single channel conductance (~30 pS slope conductance), lack of substantial residual conductance states, and steep voltage dependence.
Electrophysiological Recordings of Transfected Neuro-2a Cell Pairs
Single channel and macroscopic (1020 channels) records of gap junctions channels were obtained using double whole cell patch recordings (. All points histograms were constructed with a bin size of 0.05 pA and fit to a Gaussian using Origin 4.0 software (Microcal Software Inc.).
Poisson-Nernst-Plank Model
The Poisson-Nernst-Plank (PNP) model used is that of
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Results |
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voltage dependence of homotypic and heterotypic junctions formed by Cx32 and Cx26
Macroscopic Currents Obtained from Pairs of Xenopus Oocytes
Conductancevoltage relations and representative current traces of homotypic Cx32, Cx26, and heterotypic Cx32/Cx26 gap junctions expressed in pairs of Xenopus oocytes are shown in Figure 1. Both the initial and steady state conductancevoltage relations of homotypic gap junctions formed by Cx32 are symmetric about Vj = 0 (Figure 1 A). Initial conductance () is maximal at Vj = 0 and reduced by ~30% at large transjunctional voltages. At large transjunctional voltages, the steady state conductancevoltage relation decreases to a minimal conductance, Gmin, of ~0.3. In Cx26 homotypic junctions (Figure 1 B), initial conductance displays some dependence on the absolute voltage difference between the inside and outside of the cells (termed Vm or Vio), detected as asymmetry between the effects of depolarizing one cell and hyperpolarizing the other to the same extent (
) decreases only when the Cx26 side of the heterotypic junction is stepped to positive transjunctional potentials exceeding 40 mV. Gmin/G0 is ~0.2.
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Single-Channel Recordings of Cx26 and Cx32 in Transfected Neuro-2a Cell Lines
Homotypic Cx32 channels.
A record of a homotypic rat Cx32 single channel between Neuro-2a cells is shown in Figure 2. The single channel properties of rat Cx32 are similar to those that we have reported for human Cx32 (
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The boxed portion of the record shown in Figure 2 A (expanded in Figure 2 C) illustrates the presence of three different subconductances in rat Cx32 homotypic junctions: ~26 pS (S1), ~19 pS (S2), and ~10 pS (S3), determined from the all points histogram shown in Figure 2 D. We believe that these subconductances correspond to at least three distinct subconductance states of rat Cx32 homotypic channels (see also
Cx32 channels can also display gating that involves a complex series of transitions between fully open and fully closed states (Figure 2 B, positions 2 and 3; see also
The record also is suggestive of additional complexities in Vj-dependent gating. Homotypic rat Cx32 channels appear to be able to enter into either long- or short-lived subconductance states. For example, at the beginning of the trace shown in Figure 2 A, the channel flickered extensively, often entering a residual conductance state for <100 ms (exemplified by the transitions at position 2). In contrast, at position 3, the channel entered a 19-pS state that lasted 30 s and, later in the record, the channel entered a 10-pS state from which it had not exited when the Vj step was terminated 20 s later. In other records, we have observed that a Cx32 homotypic channel can remain in a residual conductance state for several minutes (data not shown). The presence of short- and long-lived subconductance states is also evident in the records shown in Figure 2 E and 3.
Figure 2 E shows that the IV relation of the fully open state of a single Cx32 channel is nearly linear, although currents do decrease slightly, by ~5%, for either polarity of large transjunctional voltages. A similar deviation from linearity is also observed in the conductancevoltage relations of initial currents of Cx32 recorded macroscopically in Neuro-2a cell lines that are expressing 1520 intercellular channels (Figure 2 F).
Figure 3 illustrates the gating and IV relations of Cx32 subconductance states as well as the fully open state. When the channel resided in the fully open state, the application of a 500-ms voltage ramp from -100 to + 100 mV (Figure 3 C) elicited a junctional current (Figure 3 A) that only slightly deviates from linearity. However, when the channel resided in a long-lived subconductance state, the application of the same voltage ramp resulted in a nonlinear change in junctional current (Figure 3 B). In this case, chord conductance decreased approximately twofold as the Vj ramp became more positive (from 2.4 pA at -100 mV to 1.2 pA at +100 mV). The return of junctional current to the same level (2.4 pA) as measured before the application of the voltage ramp makes it unlikely that the nonlinearity of junctional currents resulted from gating transitions between different subconductance states that could not be resolved in the record. Furthermore, reversing the time course of the voltage ramp (from +100 to -100 mV) while the channel resided in the same subconductance state reversed the time course of the rectification; junctional current now increased (from 1.2 to 2.0 pA) as the voltage ramp became more negative (not shown). Based on the negative Vj gating polarity of hemichannels formed by Cx32 (
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Figure 3 D illustrates the rectification observed in another cell pair using a similar test paradigm but starting from an initial voltage step of +100 mV and applying a voltage ramp of shorter duration (100 ms). Unlike the previous example, the application of a positive voltage to the stepped cell is expected to cause closure of the Vj gate in the hemichannel present in the unstepped member of the cell pair. In this case, junctional currents decreased nonlinearly as the voltage was ramped from +100 to -100 mV. The difference in the direction of rectification in these two cases is a consequence of the closure of opposite hemichannels. In the former case (Figure 3 B), it is likely that the Vj gate in the hemichannel in the stepped cell closed, whereas in the second case (Figure 3 D), it is likely that the Vj gate in the hemichannel in the unstepped cell closed.
In other instances, we have observed that the IV relations for other Cx32 substates do not rectify appreciably (data not shown). Thus, it appears that Cx32 hemichannels may adopt several different closed conformations. Although we have not examined the IV relations of all substates extensively, the available data suggest that the longer lasting substates rectify.
The linearity of the IV relation of the fully open Cx32 channel differs from expectation based on the 30% reduction in initial conductance observed for Cx32 homotypic junctions formed between pairs of Xenopus oocytes. Differential modification of the protein subunits or the presence of ancillary proteins in one of the two expression systems is unlikely to cause the different behavior of the initial currents, because initial currents in Cx26/Cx32 heterotypic junctions are almost identical in oocytes and transfected Neuro-2a cell lines (see Figure 1 C and 5 F). A possible explanation, described in the DISCUSSION, is that the rectification observed in oocytes arises from the difference in the intracellular ion composition of the two experimental systems. The concentration of permeable anions should be less in oocyte cell pairs than in Neuro-2a cells dialyzed with CsCl patch recording solutions.
Homotypic Cx26 channels. A record of a single homotypic Cx26 channel between Neuro-2a cells is shown in Figure 4 A. Initially, the cells appeared to be uncoupled, as the application of a +60-mV voltage step to one member of the cell pair (Figure 4 A, position 1) did not change the current measured in the unstepped cell. The channel opened subsequently via a series of complex, poorly resolved events that resemble "loop gating" or "docking/formation currents." In the segment of the trace shown in Figure 4 B, (and expanded in Figure 4 C), the channel opened and closed by a similar series of complex gating events while a Vj of -60 mV was applied. In the record shown, the conductance of the fully open channel is 130 pS. In other records, we have observed the conductance of the fully open Cx26 channel to be ~150 pS at ±60 mV. There was a single Vj-dependent gating event (Figure 4 A, position 2) corresponding to a 104-pS transition that resulted in a 26-pS residual conductance state (substate). The residual conductance, ~20% of the fully open state, is in agreement with the Gmin/G0 of ~0.2 that is observed in macroscopic recordings. The relatively few gating transitions observed at ±60 mV is consistent with that expected, given a V1/2 of ~90 mV determined from the macroscopic conductancevoltage relation shown in Figure 1 B. The channel shows more gating at higher transjunctional voltages (±120 mV). It is likely that homotypic Cx26 channels may also enter into one or more substates with residual conductances lying between 10 and 30 pS, as shown in the current trace (Figure 4 E) and all points histogram (Figure 4 F). Cx26 junctional currents characteristically appear noisier when the channel resides in a high conductance state than when it resides in either a subconductance or the fully closed state. This suggests the existence of multiple open states whose average conductance is ~130150 pS.
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The IV relation of the open state of Cx26 homotypic channels is linear (Figure 4 G). In contrast to the expression of Cx26 in oocytes, there does not appear to be any dependence on Vm in Cx26-transfected cell lines, as the IV relation is symmetric at Vj = 0 over a ±120-mV ramp. The insensitivity of Cx26 hemichannels to Vj (V1/2 = ±90 mV) and the rapid opening of Cx26 hemichannels upon stepping to smaller transjunctional voltages (not shown) precluded the examination of the IV relation of Cx26 substates.
Heterotypic Cx32/Cx26 channels. A record of a heterotypic Cx32/Cx26 channel between Neuro-2a cells is shown in Figure 5. Both cells were initially held at 0 mV. At position 1 in the trace shown in Figure 5 A, the cell expressing Cx32 was stepped to -80 mV, eliciting a junctional current corresponding to a conductance of 120 pS (Figure 5 C). At position 2, the channel underwent an ~95-pS transition, which resulted in an ~25-pS subconductance state. The trace presented in Figure 5 B and the corresponding all points histogram (Figure 5 D) demonstrates the existence of at least three additional substates of ~20, 30, and 40 pS in the heterotypic channel. We ascribe these transitions to subconductance states resulting from Vj-dependent gating that could have occurred if either the Vj gate in the Cx32 or the Cx26 hemichannel or both gates had closed. Recall that the polarity of Vj-dependent gating is opposite in the two hemichannels with Cx32 closing for relatively negative Vj at its cytoplasmic face and with Cx26 closing for relatively positive Vj.
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The rectification of ionic currents through the fully open heterotypic channel is shown in Figure 5A and Figure F. In Figure 5 A, the voltage was stepped from -80 to +80 mV at position 3. The conductance of the fully open channel was ~2.5-fold greater at -80 (120 pS) than at +80 (50 pS) mV, (Figure 5 C). Figure 5 F shows the IV relation obtained by applying a ±120-mV ramp to the cell pair expressing Cx26. In this record, single channel currents rectify approximately threefold over the ±120 mV range.
Rectification of the same polarity was also observed in a substate of the heterotypic channels. In Figure 5 E, position 1, the voltage was stepped briefly from -100 to 0 mV, and then to +100 mV, but in this case the polarity of the Vj step was reversed while the channel resided in a 25-pS substate. The channel remained in this substate briefly before transiting to the fully open state. The conductance of the substate at +100 mV was 9.5 pS, compared with 25 pS at -100 mV, corresponding to a rectification of ~2.5-fold. The degree and polarity of rectification of this substate is similar to that observed for the rectification of the fully open state in this record (12545 pS or 2.8-fold at comparable voltages).
We observed discrete gating transitions (Vj dependent) from the fully open state when the Cx26-containing cell was depolarized or the Cx32-expressing cell was hyperpolarized. This result is consistent with the observed relaxation of steady state currents in macroscopic recordings of Cx32/Cx26 heterotypic junctions. On occasion, we observe complete channel closure via "loop gating" during the application of either polarity of Vj in this heterotypic junction (data not shown), which supports the hypothesis that this gating occurs by a mechanism distinct from Vj-dependent gating. If the "loop gate" is weakly voltage dependent, then this observation could explain the slight difference in the initial and steady state conductancevoltage relations that is observed in macroscopic records of some heterotypic junctions at polarities of transjunctional voltage that should not close the Vj gate. For example, there is a decrease in steady state junctional conductance at negative Vj in the conductancevoltage relations in Figure 6A and Figure B.
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molecular determinants of the voltage dependence of initial currents
As a means of establishing the mechanism underlying the rectification of junctional currents, we sought to localize the molecular determinants of the process. Our approach can be summarized by the following question: What amino acid residues in Cx26 and Cx32 must be exchanged to interconvert their IV relations in heterotypic as well as homotypic junctions?
The difference in the gating polarity of the steady state Vj dependence of Cx32 and Cx26 hemichannels is due to a single amino acid difference in the sequence of the NH2 terminus of these two connexins (
Role of the Amino Terminus of Cx32
Figure 6 A illustrates that the initial currents of the heterotypic Cx32/Cx32N2D junctions rectify similarly to those observed in wild-type Cx32/Cx26 heterotypic junctions when expressed in pairs of Xenopus oocytes. The effect appears to be due to the electrostatic contribution of the N2D mutation, as the voltage dependence of the initial conductance of Cx32N2E is similar to that of Cx32N2D (Figure 6 B), and substitutions with neutral amino acids Cx32N2Q and Cx32N2A have little or no effect on the voltage dependence of initial conductance (data not shown). These results suggest that charged amino acid residues in the amino terminus of Cx32 play a substantial role in the rectification of initial currents. However, studies of chimeras in which the first 11 amino acids of Cx26 were substituted for those of Cx32 suggest that residues in other domains may also play a role. For example, the chimera Cx32* Cx26(NT111), which results in the substitution of seven amino acid residues in Cx32, including N2D, did not express junctional currents when paired homo- or heterotypically with Cx26 or Cx32. This result was unexpected as the reciprocal domain substitution, Cx26* Cx32(NT111), forms functional channels [not shown, see also Verselis et al., 1994, where Cx26*Cx32(NT111) is designated as Cx26*32NT-V13]. Expression of junctional currents can be restored if the Cx32*Cx26(NT111) chimera also contains the cytoplasmic loop of Cx26; i.e., Cx32*Cx26(NT111+CL). This result suggests that the amino terminus and cytoplasmic loop of Cx26 may interact directly and that this interaction may be required for the expression of junctional currents.
The conductancevoltage relation of the heterotypic Cx32/Cx32*Cx26(NT111+CL) junction is shown in Figure 6 C. Notably, the initial conductance of this junction rectifies more steeply (five- vs. threefold over ±120 mV) than does the initial conductance of Cx32/Cx26 junctions (compare Figure 1 C and 6 C). Other Cx32 chimeric junctions, which contain the cytoplasmic loop of Cx26; Cx32*Cx26(CLCT)/Cx26 (Figure 6 D) and Cx32* Cx26CL/Cx26 (not shown) also display increased steepness in initial conductance.
The behavior of the heterotypic junction Cx26* Cx32(CLCT)/Cx26, in which the CL through CT domain of Cx26 is replaced by that of Cx32, is interesting as the initial conductance increases at larger transjunctional voltages of either polarity of Vj (Figure 6 E). This change in initial conductance corresponds to a super-linear IV relation. Also, the gating polarity of the Cx26*Cx32(CLCT) hemichannel appears to be reversed as steady state conductance only decreases when the Cx26 side of the junction is relatively positive or the Cx26*Cx32(CLCT) side of the junction is relatively negative. This result is surprising as both Cx26 and Cx26*Cx32(CLCT) hemichannels have a negatively charged residue at the second position (D2). We suggest that the chimera has a different conformation that effectively reduces the electrostatic contribution of the D2 residue to the voltage sensor of the Cx26* Cx32(CLCT) hemichannel. We did not observe the expression of junctional currents in homotypic pairings of this hemichannel.
The voltage dependence of initial currents in homotypic junctions formed by Cx32*Cx26(NT111+CL), and Cx32N2E (Figure 6F and Figure G) is substantially greater than that observed in Cx32 homotypic junctions. Initial conductance declines by ~50% over ±120 mVj in both Cx32N2E and Cx32*Cx26(NT111+CL) homotypic junctions. In addition, the shape of the initial conductancevoltage relations of these homotypic junctions differs markedly from that of Cx32 homotypic junctions, declining at small as well as large transjunctional voltages.
The initial conductance of heterotypic junctions Cx32N2D/Cx26, Cx32N2E/Cx26, and Cx32*Cx26(NT111 +CL)/Cx26 decreases for either polarity of applied Vj, but substantially more when the Cx26 side of the junction is relatively positive. The conductancevoltage relation of Cx32N2D/Cx26 (Figure 6 H) exemplifies the behavior of these junctions.
The single channel IV relations in Neuro-2a cells of the fully open Cx32*Cx26(NT111+CL) channel in homo- and heterotypic pairings with Cx32 and Cx26 are shown in Figure 7. In the case of the homotypic channel, Cx32*Cx26(NT111+CL), the IV relation of the fully open channel is sigmoidal. Currents decline by ~3540% at ±120 mV from that predicted by a linear fit of the current trace at ±20 mV (Figure 7 A). The IV relation of the fully open Cx32*Cx26(NT111+CL)/Cx26 channel is asymmetric, decreasing more when the Cx26 side of the channel is relatively positive (Figure 7 B) than when it is relatively negative (compare with Figure 6 H). Figure 7 C illustrates that junctional currents rectify when the Cx32/Cx32*Cx26(NT111+CL) heterotypic channel resides in the fully open state; increasing ~4.5-fold (±120 mV) when the Cx32 cell is made relatively negative (note that there are two channels in this record). This rectification is greater than that observed for wild-type Cx32/Cx26 heterotypic junctions (compare Figure 7 C with 5 F). In all cases, the degree and direction of rectification of Cx32*Cx26(NT111+CL) channels in Neuro-2a cells is reasonably well predicted by the macroscopic behavior of initial currents of this junction when they are expressed in pairs of Xenopus oocytes. There is similar correspondence between the single channel IV relation and the macroscopic conductancevoltage relation of the Cx32N2E homotypic junction (data not shown).
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Figure 7 D shows the IV relation of a Cx32*Cx26 (NT111+CL)/Cx26 channel substate that was obtained by applying a -120- to +120-mV ramp to the Cx26 side of the channel. The chord conductance of this substate decreased nonlinearly as the Cx26 side of the junction became more positive. Given the positive gating polarity of both Cx26 and Cx32*26(NT111+CL) hemichannels, this substate most likely resulted from the closure of the Cx32*Cx26(NT111+CL) hemichannel as the channel entered the substate when the Cx32*Cx26 (NT111+CL) cell was relatively positive.
In the case of the homotypic Cx32*Cx26(NT111+CL) channel illustrated in Figure 7 E, single channel currents rectified when the hemichannel in the unstepped cell entered a substate in response to the initial positive polarity of the voltage ramp that was applied to stepped cell (ramped from +120 to -120 mV). This would favor the closure of the Vj gate in the stepped cell as this cell would be relatively positive. As in Figure 7 D, chord conductance decreased nonlinearly through the substate when the voltage of the stepped cell became more negative.
As noted, the substitution of a single negative charge in the amino terminus of Cx32 is sufficient to make both the initial and steady state conductancevoltage relations of heterotypic mutant/Cx32 junctions resemble that of the Cx32/Cx26 heterotypic junction. However, none of the initial conductancevoltage relations of homotypic Cx32N2D, Cx32N2E, or Cx32* Cx26(NT111+CL) junctions resemble that of homotypic junctions formed by Cx26 (compare Figure 6F and Figure G, and Figure 7 A with Figure 1 B and 4 G). Also, the initial conductancevoltage relation of junctions formed by pairing these mutant hemichannels heterotypically with Cx26 is markedly asymmetric (illustrated by Figure 6 H and 7 B). These results indicate that the molecular determinants of initial conductance differ in Cx26 and Cx32 channels.
Role of the Amino Terminus of Cx26
When the Cx26 side of the heterotypic Cx26D2N/Cx26 junction is relatively positive, initial conductance increases as shown in Figure 8 A. Qualitatively, the voltage dependence of initial conductance is similar, but less steep than is observed in the Cx32/Cx26 junction. This result is consistent with the interpretation that the D2 residue in Cx26 plays a role in the process underlying the open channel rectification of Cx32/Cx26. However, the initial conductance of the Cx32/Cx26D2N junction (Figure 8 B) does not resemble that observed in Cx32 homotypic junctions as would be expected if the Cx26D2N mutation had converted Cx26 into Cx32. Nor does the initial conductance resemble that of junctions exemplified by Cx32N2D/Cx26 junctions (Figure 6 H), as would be expected if Cx32 and Cx26 made equal contributions to the rectification of initial conductance. In fact the initial conductance of the Cx32/Cx26D2N junction (Figure 8 B) rectifies more than that observed in the Cx26D2N/Cx26 junction (Figure 8 A) and, unlike the Cx32/Cx26 junction, conductance increases when the Cx32 side is relatively negative.
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The results obtained with the Cx26D2N mutation must be interpreted cautiously, as the mutation causes a substantial shift in the steady state conductancevoltage relation. A simple implication of this result is that there is a higher probability that the mutant hemichannel resides in a substate at Vj = 0. Therefore, it is likely that currents passing through one or more substates make a substantial contribution to the initial currents measured macroscopically in Xenopus oocytes when the experimental paradigm described in MATERIALS AND METHODS is employed. A large shift in the steady state conductancevoltage relation of the Cx26D2N hemichannel towards positivity explains the failure to observe junctional currents in the homotypic Cx26D2N junctions. The conductancevoltage relation inferred for Cx26D2N hemichannels from Cx26D2N/Cx26 junctions predicts that junctional conductance of homotypic Cx26D2N junctions would be substantially reduced at all transjunctional voltages. We have not yet examined the single channel currents of Cx26D2N mutations.
The conductancevoltage relation of Cx26 chimeras containing the NH2 terminus and first transmembrane domains of Cx32, Cx26*Cx32(NT+TM1), do not display large shifts in their steady state conductancevoltage relations. Consequently, they provide a means of examining the effect of neutralization of the negative charge of the D2 residue on the voltage dependence of initial conductance. Figure 8 C illustrates that the initial conductance of the heterotypic junction, Cx26*Cx32(NT+TM1)/Cx26, is similar to that of Cx32/Cx26. This result suggests that the Cx26 chimera has adopted Cx32 properties. However, the initial conductance of Cx26*Cx32(NT+TM1)/Cx32 junction is markedly asymmetric (Figure 8 D), decreasing more when the Cx32 hemichannel is stepped to positive transjunctional voltages. The substitution of either N2D or the first eight amino acids of Cx26 into the amino terminus of the Cx26*32(NT+TM1) chimera results in hemichannels, Cx26*Cx32(N2D+NT+TM1), and Cx26* Cx32(NT922+TM1), whose properties are indistinguishable from those of wild-type Cx26. The conductancevoltage relations of Cx26*Cx32(NT922+TM1)/Cx26 and Cx26*Cx32(NT922+TM1)/Cx32 are shown in Figure 8E and Figure F). These results indicate that amino acids downstream of TM1 in addition to the charged aspartate residue (D2) are responsible for the linear initial conductancevoltage relation of Cx26 homotypic junctions.
Role of the First Extracellular Loop
Our previous work indicated that the E1 domain of Cx26 was likely to be involved in the voltage dependence of initial currents, because a chimera in which the first extracellular loop of Cx32 was replaced with the E1 domain of Cx26 to form Cx32*Cx26E1 demonstrated an appreciable asymmetry in the conductancevoltage relation of initial currents when paired with Cx32 (
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The changes in voltage dependence of the Cx26E1 substitution into Cx32 are likely to involve amino acid residues located at the TM1/E1 border as the double mutation, Cx26(K41E,E42S) (previously termed Cx26* ES) displays a steeper initial conductancevoltage relation than does Cx32/Cx26 when it is paired with Cx32 (
The reciprocal chimera, Cx26*Cx32E1, displays a large shift in the steady state conductancevoltage relation in heterotypic pairings with Cx32, as illustrated in Figure 9 F. Although initial conductance increases in this heterotypic junction with positivity on the Cx26*Cx32E1 side, the large shift in the steady state conductancevoltage relation makes it likely that substates of the Cx26*Cx32E1 hemichannel make a substantial contribution to the initial conductance determined macroscopically and this possibility complicates any quantitative interpretation. We did not observe the expression of junctional currents in Cx26*Cx32E1 homotypic pairings, an observation that may be explained by a negative shift in the V1/2 of both hemichannels conductancevoltage relations.
Substitution of Other Domains Does Not Alter the Voltage Dependence of Initial Currents
Reciprocal substitutions of the second extracellular loop of Cx26 and Cx32 have no effect on the voltage dependence of steady state or initial conductance in homo- and heterotypic pairings (
These data indicate that substitutions involving the second extracellular loop, the four transmembrane domains and the carboxyl terminus have little or no effect on the voltage dependence of initial conductance observed in junctions formed by Cx26 and Cx32. However, as described above, changing amino acids positioned in the amino terminus and first extracellular loop appear to have pronounced effects. Although the cytoplasmic loop alters the voltage dependence of initial conductance, it is not clear if this is the result of a direct electrostatic effect or an indirect effect caused by changes in the conformation of the amino terminus.
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Discussion |
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Molecular Determinants of Electrical Rectification
Based on the single channel data presented in this paper, it is apparent that the voltage dependence of initial conductance observed in the heterotypic Cx32/Cx26 channel arises as a consequence of the rectification of ionic currents through open channels rather than by conformational changes associated with voltage-dependent gating. It is remarkable that the IV relations of both Cx32 and Cx26 homotypic channels expressed in Neuro-2a cell lines are essentially linear. Evidently, the union of Cx32 and Cx26 hemichannels results in an asymmetry in the structure of the heterotypic channel that produces the rectifying currentvoltage relation. The union of like hemichannels to form a complete intercellular channel always results in symmetric, although not necessarily linear, IV relations. For example, the IV relation of homotypic Cx32*Cx26(NT111+CL) channels is sigmoidal (Figure 7A and Figure E). Asymmetric conductancevoltage relations may also result if the channels display dependence on the absolute voltage difference between the inside and outside of the cells (termed Vi-o or Vm). However, this form of voltage dependence does not underlie the open channel IV relations described in this study.
Our previous results have indicated that charged amino acid residues, positive (M1) and negative (D2) in Cx26 and positive only (M1) in Cx32, are responsible for the difference in the Vj -dependent gating polarity of Cx26 and Cx32 hemichannels. We proposed that these residues are located near the cytoplasmic entrance of the channel pore based on their ability to sense transjunctional voltage and the accepted membrane topology of gap junctions. These charges may account for the slight anion selectivity of homotypic Cx32 channels (
The observations that the substitutions of the neutral second amino acid residue of Cx32 with negatively charged amino acid residues alter the initial conductancevoltage relation and that neutral amino acid substitutions do not change initial conductance are consistent with an electrostatic effect. The increased steepness of the rectification observed in the Cx32/Cx32* Cx26(NT111+CL) junction (Figure 6 C and 7 C) may arise from an interaction between the CL and NT domains that alters the position of the negatively charged residue located in the amino terminus of this chimera. Alternatively, it may reflect an electrostatic effect of charged residues that differ in the CL domain in Cx32 and Cx26. Of the 38 amino acids present in the cytoplasmic loop of Cx26, 50% are charged, 12 positive and 7 negative. Of the 37 amino acid residues in the CL domain of Cx32, 6 are positively and 5 are negatively charged. Nine charges (five positive and four negative) are conserved between the two connexins. Consequently, it is difficult to assess whether the effect of the cytoplasmic loop on open channel rectification is a direct effect of specific charged amino acid residues, or if this domain exerts its effect indirectly by changing the conformation of the NH2 terminus.
It is apparent that the substitution of a negative charge into NH2 terminus of Cx32 does not recreate the properties of Cx26 homotypic junctions, since the IV relations of Cx32*Cx26(NT111+CL) and Cx32N2E homotypic channels are sigmoidal (e.g., Figure 7 A). Therefore, other charged residues must also influence ionic flux in Cx26 channels. The asymmetry in the voltage dependence of initial currents observed in the Cx32*Cx26E1/Cx32 junction (Figure 9 A) indicates that the E1 domain of Cx26 is also involved. The negatively charged E42 residue in Cx26 is the best candidate, as the initial conductancevoltage relation of the point mutation Cx32S42E paired heterotypically with Cx32 is almost identical to that of Cx32*Cx26E1/Cx32 (Figure 9 E). The view that E42 plays a role in the expression of the voltage dependence of initial conductance is further supported by the increased steepness of the voltage dependence of initial conductance observed in the Cx32/Cx26(K41E,E42S) junction (
Electrodiffusive Model of the Observed Electrical Rectification
The results presented in this paper indicate that charged amino acid residues located in the amino terminus and first extracellular loop play a major role in shaping the IV relations of channels formed by Cx26 and Cx32. In the following sections, we examine whether these charges can account for the IV relations of wild-type and mutant channels presented in this paper by using the electrodiffusive model of
The PNP model of
In this study, we adopt a different strategy since we have very limited ionic data for any given channel (at best two ionic conditions for Cx32 channels), but we have information describing how the IV relations are changed by substitutions of charged amino acid residues in different regions of Cx32 and Cx26 in several different pairing combinations. We seek to determine whether we can use our molecular studies to derive a set of charge distributions that when incorporated into the PNP model will lead to IV relations comparable with those observed in homotypic and heterotypic pairings of wild-type and mutant hemichannels. In our use of PNP theory, we vary only the magnitude, the position, and the width of fixed charges that we infer from our molecular studies. We use a fixed pore geometry of 7 Å in radius and 120 Å in length. The cation and anion mobilities are set to values of the aqueous bulk solution mobilities of Cs and Cl (2.06 and 2.03 · 10-5 cm2 s-1), the solutions used in single channel experiments that were performed. The ion concentrations were 150 mM in the symmetric salt case, and 15 and 150 mM in the case of 1:10 salt gradients. The dielectric constant for the channel pore and the membrane were fixed at 80 and 4, respectively (the default values of the PNP computer program). The IV relations obtained were not very sensitive to changes in the value of the dielectric constant assigned to the channel pore in the 150-mM salt case using fixed charges > ±1 e. The PNP model appears to be quite sensitive to reductions in the dielectric constant of the pore when ionic concentrations and the values assigned to fixed charges are both reduced (for example, to 15 mM and 0.1 e, respectively, for the charge distribution shown in Figure 10 C). This observation most likely reflects the "charge screening" property of the PNP model. The input file for the pore surface charge was generated using the PNP Windows computer program of Stephen Traynelis' group (Emory University, Atlanta, GA) using the program's defaults (sigmoid, smooth charge points = 7). In the case where more than two charges are modeled, the charge input file was assembled in overlapping pairwise combinations using Microcal Origin software and saved as PAS or TXT files in Programmers Notepad. Output files (IV relations, anion and cation fluxes, and chord conductances) were generated using the PNP computer program of Dr. Duan P. Chen (Rush Medical College, Chicago, IL) using the unmodified Poisson Equation. The Fortran version of the computer program was compiled in C++ by Brady Trexler (Albert Einstein College of Medicine).
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We restrict our use of the PNP model to channels for which we have single channel records. We did not attempt to use any fitting algorithms, as we feel that this approach is premature in the absence of a larger data set that includes a variety of ionic conditions. The availability of additional ionic data would be helpful in distinguishing among alternative charge distribution models, but such data are lacking at present, and additional ionic data are not easily obtained for intercellular channels expressed in cells.
Models of Wild-Type Channels
Initially, we explored a PNP model of Cx32 with a charge step of +6 e located at either end of the open channel, with the rationale that each of the six connexin subunits that form a hemichannel would contribute one positive charge. The IV relation of a channel with this charge distribution that resulted with the PNP model was quite similar to the IV relation observed for Cx32 homotypic channels in symmetric 150-mM CsCl solutions (deviating from linearity by ~5% at ±120 mV, not illustrated). However, although not unexpectedly, the PNP-modeled channel was almost ideally anion selective in 15 mM:150 mM CsCl solutions. Virtually all the current was carried by the anion over the entire voltage range explored in the model (±120 mV) and the predicted reversal potential was approximately -58 mV in the 1:10 salt gradient of this ionic strength. A reversal potential approximating that obtained for Cx32 homotypic channels (approximately -10 mV; see Figure 10 A) could be obtained by incorporating a baseline charge of -1.5 e. The baseline charge in the computer model could be interpreted as a smearing of a negative charge along the length of the pore surface, perhaps reflecting the negative charge contribution of backbone carbonyls or the negative charge of side chains of pore-lining amino acids. Amino acids such as serine, which are believed to carry partial negative charges (see note 4 in
Although the shapes of the IV relations observed correspond closely to those obtained with the PNP model with the charge distribution and parameters used, the predicted single channel conductance is substantially greater than observed (227 vs. 70 pS at ±60 mV). This problem may be solved by reducing the diffusion coefficients of the ions in the pore, as done by
The results of the molecular analysis indicate that the IV relation of Cx26 channels is likely to be determined by external positive and negative charges and an internal negative charge. Following these considerations, we modeled the charge distribution of Cx26 as illustrated in Figure 10 E. The charge associated with the D2 residue was set to -2 e, the positive charge in the amino terminus set to +1 e, and the internal charge (E42) was set to -1 e as this residue may be located at some distance from the channel lining. We justify the increased magnitude of the D2 charge by considering that negative charge substitutions into the amino terminus of Cx32 appear to dominate the effect of the fixed positive charge in polarity reversal of Vj dependence. We also incorporated a baseline charge of +0.25 e to make the Cx26 channel slightly cation selective in 150 mM:15 mM CsCl gradients (Figure 10 F) in consideration of the results reported by
An alternative charge distribution model (Figure 10 H) also results in a linear IV relation and a slightly cation selective channel (not shown), but in this case the predicted single channel conductance is identical to that observed (150 pS, not shown).
We modeled the charge distribution of the heterotypic Cx26/Cx32 channel (Figure 10 I) by merging the charge distribution of half the Cx26 channel shown in Figure 10 E (i.e., covering the electrical distance from 0.0 to 0.5) with that of Cx32, shown in Figure 10 C (covering the electrical distance from 0.5 to 1.0). The charge distribution at the point of contact was recalculated by using the Windows PNP program of Traynelis to remove the sharp discontinuity that would otherwise result. The IV relation resulting from the PNP model (Figure 10 J) is almost identical to that observed for single Cx26/Cx32 channels, with current increasing approximately threefold when the Cx26 side of the channel is made relatively positive (-12.4 pA at -120 mV to 36.2 pA at 120 mV). The single channel conductance of the modeled channel can be made identical to that observed by reducing ion mobilities to 0.85 · 10-5 cm2 s-1.
The charge distribution model formed by combining the right half of the charge distribution shown in Figure 10 C with the left side of the charge distribution shown in Figure 10 H also resulted in an IV relation that resembled that of Cx32/Cx26 heterotypic channels, rectifying 2.7-fold at ±120 mV (increasing when the Cx26 side of the channel is positive).
Models of Cx32*Cx26(NT111+CL) Channels
In the model of the Cx32*Cx26(NT111+CL) channel, the charge profile of Cx26 shown in Figure 11 A was modified by removing the central charge (-1 e) associated with E42 in Cx26 and moving the external charges outward (the charge step was initiated at an electrical distance of 0.025 rather than 0.1), but the magnitude of the external charges was not altered. The change in position of these charges was required to produce a charge distribution model that could replicate the experimentally observed IV relations in all pairing combinations involving the Cx32*Cx26(NT111+CL) hemichannel. The baseline charge was increased by 0.25 e to 0.5 e to maintain the expected moderate cation selectivity of this channel (Erev = -10 mV in 1:10 salt gradient, not shown). The charge profiles for Cx26/Cx32*Cx26(NT111+CL) and Cx32*Cx26(NT111+ CL)/Cx32 are shown in Figure 11B and Figure C, respectively. The IV relations given by the PNP model for the homotypic Cx32*Cx26(NT111+CL) channel, and heterotypic Cx26/Cx32*Cx26(NT111+CL) and Cx32* Cx26(NT111+CL)/Cx32 channels closely resemble the observed IV relations (compare Figure 11, DF with Figure 7, AC). The small difference in the modeled and observed single channel conductances could be further reduced by decreasing the mobilities of ions in the PNP model (1.4 · 10-5 cm2 s-1 for the homotypic channel, 1.2 · 10-5 cm2 s-1 for the heterotypic Cx26 channel, and 0.9 · 10-5 cm2 s-1 for the heterotypic Cx32 channel).
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The IV relations of homotypic junctions formed by Cx32N2E and Cx32N2D and their heterotypic junctions with Cx32 and Cx26 can be explained using charge distribution models similar to those employed to explain the behavior of the Cx32*Cx26(NT111+CL) channels (not shown). However, using half of the charge distribution model shown in Figure 10 H for Cx26 to form a model of Cx26/Cx32*Cx26(NT111+CL) channel produced a nearly symmetric sigmoidal IV relation (not shown) that did not correspond to the IV relation observed experimentally. As we were not able to find an alternate charge distribution model for Cx32*Cx26(NT111+CL) that would provide the observed IV relation, we believe that the Cx26 charge distribution model shown in Figure 10 H is unlikely to be correct.
Models of Cx32*Cx26E1
As detailed above, there is good qualitative agreement between the IV relations obtained with the PNP model using the charge distributions inferred from the molecular studies and those observed in this study. However, the linearity of the IV relation of the Cx32*Cx26E1 homotypic channel cannot be explained by charge distribution models using only the identified charges analyzed so far. We were surprised to be unable to obtain a linear IV relation for Cx32*Cx26E1 using the PNP model with the identified external and internal charges (M1 and E42). The IV relation of a channel containing only the central charges (E42) is linear (not shown), while the IV relation of a channel containing an external charge at either end can be linearized by moving the charges centrally (not shown) in the PNP analysis. Yet all models that considered both charges together were characterized by substantially sigmoidal IV relations (not shown). A nearly linear IV relation could be obtained by the PNP model if one incorporated a partial negative charge near the ends of the charge distribution model for Cx32*Cx26E1. The conserved tryptophan residue at the third amino acid position (W3) is a reasonable candidate for the formation of this negative charge if one proposes that the tryptophan residue adopts a conformation such that the negative component of the electrostatic surface potential of the aromatic ring of the tryptophan residue is oriented parallel to the channel pore. We did not consider the positive component of the electrostatic surface potential. The aromatic ring of the tryptophan residue has a permanent quadrupole moment and it is believed that its electrostatic effect is important in cation interactions (
interactions at conserved aromatic amino acids may be responsible for the ion selectivity of potassium channels.
The charge distribution used and the resulting IV relation of this class of model after the application of the PNP model are illustrated in Figure 12A and Figure D. However, the incorporation of a negative charge at the W3 position alters the predicted IV relation of Cx32 homotypic channels and Cx26/Cx32 heterotypic channels. The IV relation of Cx32 becomes linear and notably the predicted reversal potential in a 1:10 salt gradient is close to that observed without the incorporation of a baseline charge (not shown). Also, the predicted IV relation of Cx26/Cx32 rectifies much less, approximately twofold over ±120 mV (not shown). Interestingly, a channel with the charge distribution shown in Figure 12 B has a predicted IV relation that closely resembles wild-type homotypic Cx32 channels (Figure 12 E). In this case, the magnitude of the charge at W3 was reduced to -0.25 e, and a central charge of -0.5 e was introduced. It is possible that this charge corresponds to residue E41 in Cx32 and that the charge profile shown in Figure 12 B more closely approximates that of Cx32 than the one shown in Figure 10 C. Consequently, we explored the effect of incorporating the negative charges associated with residues W3 and E41 in a model of the heterotypic Cx26/Cx32 channel. The modeled charge distribution is shown in Figure 12 C. The resulting IV relation closely resembles that of Cx26/Cx32, rectifying 2.8-fold (-14.3 pA at -120 mV/40.7 pA at +120 mV). The single channel conductance of the modeled channel is three times greater than that actually observed, but can be adjusted by reducing the intrapore ionic mobilities of both the anion and cation to 0.7 · 10-5 cm2 s-1 from the bulk solution mobilities of Cs and Cl. The IV relation of the resulting channel is shown in Figure 12 F. The incorporation of a negative charge (-0.5 e) associated with E41 into the charge distribution models of Cx32*Cx26(NT111+CL) channels (Figure 11) does not substantially alter the resulting IV relations obtained with the PNP model. The effect of this charge on the IV relation obtained for the Cx26/Cx32*Cx26(NT111+CL) channel is illustrated in Figure 13.
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Extension of the Model to Other Channels
We did not attempt to apply PNP theory to model the behavior of the remaining junctions for which we have no single-channel data. We expect that the IV relations that can be inferred from the macroscopic conductancevoltage relations could be predicted by the charge distribution models we present for Cx32, Cx26, Cx32*Cx26(NT1-11+CL) and Cx32*Cx26E1 with only minor modifications. The only notable exception is the superlinear IV relation that would correspond to the macroscopic conductancevoltage relation of the Cx26* Cx32(CLCT)/Cx26 heterotypic junction (Figure 6 E). The apparent reversal of the Vj gating polarity of the Cx26*Cx32(CLCT) hemichannel suggests that a conformational change has taken place that effectively removes the D2 residue from the transjunctional field. In barrier models, superlinear IV relations are easily explained by the presence of a dominant central barrier. We did not find a reasonable way to obtain a superlinear IV relation with the PNP model, although superlinear IV relations have been obtained with the PNP2 model (
Summary of the Application of the PNP Model
The preceding sections illustrate how the results of the molecular analyses could be used in conjunction with the PNP model of
It is important to stress that the set of charge distribution models provided above are probably not unique, in that there are likely to be other sets of charge distribution models that may also consistently describe the available set of experimentally observed IV relations. This statement is not meant to imply that all charge distribution models that were examined in the current study could satisfactorily account for the entire experimental data set. For example, the class of charge distribution model for Cx26 illustrated in Figure 10 H did not give rise to the experimentally observed IV relation for the Cx26/Cx32*Cx26(NT111+CL) channel, although it worked reasonably well in reproducing the IV relations of Cx26 homotypic and Cx32/Cx26 heterotypic junctions. We were unable to find an alternate charge distribution of Cx32*Cx26(NT111+CL) that could remedy this discrepancy and continue to adequately describe the behavior of other channels. Similarly, the model appeared to be quite sensitive to changes in the position of charges located near the entry of the Cx32*Cx26 (NT111+CL) hemichannel. As described above, the charges in this region had to be moved outward relative to those used in the charge distribution model of Cx26 to satisfactorily reproduce the IV relations of homotypic and heterotypic channels observed for this chimera. The PNP model appears to be sensitive to changes in the position of charges in the class of charge distribution model exemplified by the Cx32* Cx26(NT111+CL) chimera, but, as described below, less sensitive to other changes in charge distribution.
We did not attempt to rigorously explore the sensitivity of the PNP model to alterations in the input parameters. As stated previously, the PNP model does not appear to be sensitive to changes in the assigned values for dielectric constant of the membrane and pore, but this insensitivity may be related to the ionic conditions and charge distributions used in our study. In some cases, the model did not appear to be very sensitive to rather substantial changes in charge distribution. This is illustrated in Figure 13AC, where two different charge distribution models of the Cx26/Cx32*Cx26(NT111+CL) channel result in very similar IV relations with the PNP model in the symmetric 150-mM salt case. The two charge distribution models differ only by the presence of an internal negative charge (-0.5 e), which we propose to correspond to residue E41 in Cx32 (see above). However, the two charge distribution models can in principle be discriminated in other pairing configurations or by varying the ionic concentration parameter used in the PNP model. For example, the IV relations obtained for the two different models of Cx32*Cx26 (NT111+CL) differ somewhat when they are used to form homotypic channels (Figure 13 D). Also, the IV relations (and predicted reversal potential) of the two different heterotypic Cx26/Cx32*Cx26(NT111+CL) channels differ slightly when asymmetric salts (15 mM inside:150 mM outside) are used (Figure 13 E). The incorporation of the negative charge shifts the reversal potential from -10 to 0 mV. Greater differences in the IV relations are obtained with the PNP model in the symmetric salt case when the concentration of ions is reduced to 15 mM from 150 mM (Figure 13 F). Although it is not clear whether the differences in the IV relations shown in Figure 13 can be resolved experimentally, it seems at least theoretically possible that the PNP model is able to discriminate among different charge distribution models when the model's input parameters are varied.
The results of our molecular studies in conjunction with PNP theory have enabled us to identify at least one internally consistent set of charge distributions that can explain the observed rectification of several hetero- and homotypic channels. This finding supports the view that charged amino acid residues in the NT and E1 domain of Cx32 and Cx26 are important molecular determinants of electrical rectification. The questions, if the charge distributions models derived here are unique and if the PNP model can provide a "unique" solution in all cases needs to be addressed further, but this aim is computationally and experimentally intensive and beyond the scope of the current paper.
Substate Rectification
We cannot legitimately apply the PNP model to explain the rectification observed in substates of Cx32 and Cx32*Cx26(NT111+CL) hemichannels as the available model considers the surface charge distribution of channels of right circular cylindrical geometry. While the changes in conformation associated with Vj gating to substates may not substantially change the magnitude of surface charge, it is likely that reductions in pore radius would increase the electrostatic effect of charges located in constricted regions. We illustrate this by considering the charge distribution models shown in Figure 14. If one increases the magnitude of the external charges in the simple charge distribution models of Cx32 and Cx32*Cx26(NT111+CL) to mimic an increased electrostatic effect of these charges, then the IV relations obtained rectify in the same direction as observed (compare Figure 14C and Figure D, with Figure 3 B and 7 E). When the Cx32 homotypic channel enters a substate in response to negative Vj, currents decrease as Vj becomes more positive (Figure 14 C). In homotypic Cx32*Cx26(NT111+CL) channels, current increases as the cell containing the hemichannel rendering in a substate becomes positive (Figure 14 D). Note that Cx32 and Cx32*Cx26(NT111+CL) hemichannels close for opposite polarities of transjunctional voltage.
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Rectification of Electrical Synapses
The results obtained from the application of the PNP model can provide a mechanistic explanation for the generation of steeply rectifying electrical synapses originally reported by
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Voltage-dependent gating is unlikely to be the sole determinant of the electrical rectification observed in electrical synapses because steady state conductance at these synapses is reached very rapidly within 800 µs (
The rectification of gap junction open states and substates described in this study provides a mechanism that would allow for a nearly instantaneous increase in conductance in response to presynaptic depolarization. Electrodiffusive models (PNP theory and Nernst-Plank with electroneutrality assumption) predict steeply rectifying IV relations for channels in which positive and negative charges are located at opposite ends of an intracellular channel (p-n junction). Thus, the steepness of the IV relations attainable by p-n junctions can adequately explain the observed behavior of electrical synapses and obviates the need to invoke voltage gating. Recently, several fish connexins (
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Footnotes |
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Dr. Rubin's current address is Joshua B. Rubin, Dana Farber Cancer Institute, Boston, MA 02115.
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Acknowledgements |
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We thank Drs. A. Finkelstein, S. Slatin, P. Kienker, M. Colombini, D. Chen, and Dr. O. Andersen for helpful discussions and insights into the use of electrodiffusive models, Brady Trexler for compiling the PNP computer program, Dr. H.-S. Shin for providing the IRES vector, and Dr. P. Brink for providing the Neuro-2a cell line expressing Cx32*Cx26E1.
This work was supported by the National Institutes of Health grant GM46889. Michael Bennett is the Sylvia and Robert S. Olnick Professor of Neuroscience.
Submitted: February 18, 1999; Revised: May 26, 1999; Accepted: June 18, 1999.
1used in this paper: CL, cytoplasmic loop; CT, COOH terminus; Cx, connexin; E, extracellular loop; IV, currentvoltage; LS channel, large diameter synthetic ion channel; NT, NH2 terminus; PNP, Poisson-Nernst-Plank; TM, transmembrane domain
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References |
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