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1 Department of Pharmacology, SUNY Upstate Medical University, Syracuse, NY 13210, USA2 Department of Pediatrics, University of Chicago, Chicago, IL 60637, USA
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Abstract |
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100 µM) whereas connexin43 (Cx43) is unaffected by identical concentrations of intracellular spermine. Replacement of two unique glutamate residues, E9 and E13, from the cytoplasmic amino terminal domain of Cx40 with the corresponding lysine residues from Cx43 eliminated the block by 2 mM spermine, reduced the transjunctional voltage (Vj) gating sensitivity, and reduced the unitary conductance of this Cx40E9,13K gap junction channel protein. The single point mutations, Cx40E9K and Cx40E13K, predominantly affected the residual conductance state (Gmin) and Vj gating properties, respectively. Heterotypic pairing of Cx40E9,13K with wild-type Cx40 in murine neuro2A (N2A) cells produced a strongly rectifying gap junction reminiscent of the inward rectification properties of the Kir (e.g. Kir2.x) family of potassium channels. The reciprocal Cx43K9,13E mutant protein exhibited reduced Vj sensitivity, but displayed much less rectification in heterotypic pairings with wtCx43, negligible changes in the unitary channel conductance, and remained insensitive to spermine block. These data indicate that the connexin40 amino terminus may form a critical cytoplasmic pore-forming domain that serves as the receptor for Vj-dependent closure and block by intracellular polyamines. Functional reciprocity between Cx40 and Cx43 gap junctions involves other amino acid residues in addition to the E or K 9 and 13 loci located on the amino terminal domain of these two connexins.
(Received 9 December 2003;
accepted after revision 23 April 2004;
first published online 23 April 2004)
Corresponding author R. D. Veenstra: Department of Pharmacology, SUNY Upstate Medical University, Syracuse, NY 13210, USA.Email: veenstrr{at}upstate.edu
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Introduction |
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Methods |
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Stable wild-type (wt) Cx40- and Cx43-transfected mouse neuro2A (N2A) cell cultures were prepared and maintained as previously described (Beblo et al. 1995). The mutant DNAs were generated using PCR site-directed mutagenesis according to established protocols (Vallette et al. 1989). The primary amino acid sequences for the cytoplasmic amino terminal (NT) domain of Cx40 and Cx43 are shown in Fig. 1A. The two glutamate (E) or lysine (K) residues present in Cx40 or Cx43, respectively, at positions 9 and 13, indicated by the arrowheads, were reciprocally mutated to generate the Cx40E9,13K and Cx43K9,13E double point mutations and the Cx40E9K, Cx40E13K, Cx43K9E, and Cx43K13E single point mutations. The mutant cDNAs were cloned into the pTracerTM-CMV2 vector (Invitrogen) using the Kpn1 and Xba1 sites. The plasmid construct was purified using a Plasmid Maxi Kit (Qiagen) and the sequence confirmed. Transient transfections were performed on N2A cells grown to 80–90% confluency in a 24-well culture dish (Falcon) using LipofectamineTM 2000 (Invitrogen) according to the manufacturer's instructions.
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Expression of mutant connexin proteins was confirmed by immunoblotting using Cx40- and Cx43-specific antibodies (Fig. 1B and C). N2A cells were harvested 72 h after transfection. Protein extracts from cells were prepared as described by Laing & Beyer (1995). Aliquots containing 10 µg of protein from parental or transfected HeLa and 100 µg of protein from parental or transfected N2A cells were separated by SDS-PAGE on 10% polyacrylamide gels and blotted onto Immobilon-P membranes (Millipore). Connexin-specific protein was detected using a rabbit antiserum directed against a bacterially expressed Cx40 carboxyl tail fusion protein (1 : 5000 dilution) or GST-Cx43 C-terminal fusion protein (1 : 20 000) (Kwong et al. 1998). Peroxidase conjugated goat anti-rabbit IgG (1 : 5000 dilution) was used as a secondary antibody. Immunoblots were developed with electrogenerated chemiluminiscence (ECL) reagents following the manufacturer's instructions (Amersham).
Electrophysiological recording
N2A cell cultures were washed 3–5 times with Hepes-buffered saline and placed on the stage of an inverted phase contrast microscope (Olympus IMT-2, Lake Success, NY, USA). The bath saline contained (mM): 142 NaCl, 1.3 KCl, 0.8 MgSO4, 0.9 NaH2PO4, 1.8 CaCl2, 4.0 CsCl, 2.0 TEACl, 5.5 dextrose, 10 Hepes (pH 7.4, titrated with 1 N NaOH), 310 mosmol kg–1. Junctional current recordings were obtained using conventional double whole-cell recording techniques with two Axopatch 1D (Axon Instruments, Union City, CA, USA) patch clamp amplifiers (Veenstra, 2001). Patch electrodes (PG52151-4, WPI, Inc., Sarasota, FL, USA) had tip resistances of 4–6 M
prior to gigaohm seal formation and patch break when filled with 140 mM KCl internal pipette solution (IPS). The standard KCl IPS contained (mM): 140 KCl, 4.0 CsCl, 2.0 TEACl, 3.0 CaCl2, 5.0 K4BAPTA, 1.0 MgCl2, 25 Hepes (pH 7.4, titrated with 1 N KOH), 310 mosmol kg–1. MgATP was added daily to achieve a final concentration of 3.0 mM. All experiments were performed at room temperature (20–22°C). All currents were digitized at 1–4 kHz after low pass filtering at 100–500 Hz (LPF 202 A, Warner Institute, Hamden, CT, USA). Analysis and curve fitting procedures were performed using Clampfit software (pCLAMP version 8.2, Axon Instruments, Inc.) All curve fitting was performed using the sum of squared errors minimization procedure and the standard error for each estimated parameter are provided. Final graphs were prepared using Origin version 6.1 or 7.0 software (OriginLab Corporation, Northampton, MA, USA).
Normalized steady-state junctional I–V and G–V relationships were obtained with continuous 200 ms mV–1 voltage ramps where V1 was varied from –40 to –140 or +60 mV in 1 mV increments (Veenstra, 2001). A rest interval of 15 s was included between each voltage ramp. Each V1 ramp was repeated four to five times per experiment and the I1 and I2 traces were ensemble averaged prior to calculation of Ij and gj. To ensure accuracy, quantitative methods were used to correct for junctional voltage errors resulting from the series resistance Rel of each patch electrode in the junctional conductance (gj) calculations according to the expression:
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Ionic blockade experiments
Spermine and tetrapentylammonium (TPeA) salts were added unilaterally as indicated for each experiment. Spermine HCl (Calbiochem, La Jolla, CA, USA) and TPeACl (Aldrich Chem Co., Milwaukee, WI, USA) were stored at –20°C as 500 mM stock solutions in 18 M cm water and diluted daily as required with KCl IPS. The final osmolarity of the TPeA + KCl IPS was not adjusted since the maximum dose of 5 mM TPeA altered the final IPS volume by 1% and the total osmolarity by 3% (Musa et al. 2001). For most spermine concentrations, the IPS osmolarity was altered by 1% (Musa & Veenstra, 2003). To determine the magnitude of polyamine (PA) block, a voltage protocol that sequentially altered the holding potential (
V1) of the PACl + KCl-containing cell (cell 1) from negative to positive and back to negative potentials relative to the KCl-containing cell (cell 2) in 30 s intervals was used. The common holding potential (V1 and V2) was –40 mV for both cells. Cell 1 was stepped to this common potential for 0.5 s during each Vj step and for 10 s between different –/+/–command voltages to assess any change in the non-junctional voltage clamp circuit. Vj= (V1+V1) –V2 and V1 was altered in 5 or 10 mV increments from 5 to 50 mV and Ij=–I2.
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Results |
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Double whole-cell patch clamp recordings of gap junction currents (Ij) were obtained from homotypic pairs of connexin-transfected N2A cells after 24 h. The Vm of one cell (cell 1) was varied and the difference in whole-cell current of the second cell (cell 2) represented the negative of the net Ij (I2=–Ij, Veenstra, 2001). The unilateral addition of 2 mM spermine to the patch pipette of cell 1 achieved maximum block in wild-type (wt) Cx40 cell pairs (Fig. 2A–C). The same protocol failed to block Ij in Cx40E9,13K and Cx40E13K cell pairs, with intermediate amounts of block evident at Vj
+50 mV in Cx40E9K cell pairs (Fig. 2B and C). Further reductions in Cx40E9K Ij with 2 mM spermine at higher Vj values were complicated by the Vj gating present in these homotypic gap junctions above ±50 mV (see Fig. 3B and Table 1). We were unable to determine the voltage-dependent dissociation constants (Km(Vj)) for spermine blockade of this Cx40E9K gap junction since significantly less block was observed in the presence of unilateral 0.5 and 1.0 mM spermine. The decreased blockade near 0 mV in the mutant Cx40 gap junctions indicates that the tetravalent spermine molecule associates predominantly with the E13 locus, and to a lesser extent with the E9 locus, on the NT domain of Cx40.
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Homotypic mutant Cx40 gap junction Vj gating properties
To individually assess the effect of the E-to-K mutations of residues 9 and 13 on the Vj-dependent gating properties of Cx40 gap junctions, the steady-state Vj-dependent junctional conductance (gj) curves of homotypic wtCx40, Cx40E9K, Cx40E13K, and Cx40E9,13K, were determined by varying Vj between + and –140 mV in 1 mV, 200 ms increments (Fig. 3A–D). The Cx40E13K mutation nearly abolished all Vj gating inherent to wtCx40 whereas the Cx40E9K and Cx40E9,13K homotypic gap junctions exhibited a typical Boltzmann distribution for the normalized steady-state junctional conductance–voltage (Gj–Vj) curves (Fig. 3, Table 1). The half-inactivation voltage (V1/2) was larger in Cx40E9K than in Cx40E9,13K double point mutant gap junctions. The decrease in the V1/2 is indicative of a relative reduction in the open probability for the Cx40E9,13K gap junctions, despite the additional loss of equivalent gating charges (z) evident in the Boltzmann distributions for these two mutant connexin gap junctions. The homotypic Cx40E9K gap junctions also exhibited the complete loss of any residual Vj-insensitive Gj (Gmin). These data suggest that both the E9 and E13 loci contribute to the Vj gating properties of Cx40.
Heterotypic mutant/wild-type Cx40 gap junction Vj gating properties
Due to the inherent bilateral symmetry of all homotypic gap junctions, it becomes necessary to produce heterotypic gap junctions to assess the relative differences in the Vj gating properties of two related connexins. For this purpose, all three mutant Cx40 proteins were expressed in heterotypic combination with wtCx40 (Fig. 4). The steady-state junctional current–voltage (Ij–Vj) relationships for the Cx40E9K/wtCx40, Cx40E13K/wtCx40, and Cx40E9,13K/wtCx40 gap junctions all exhibit some degree of rectification, resulting in less current flow when Vj is positive in the wtCx40 cell. The corresponding Gj–Vj curves illustrate the asymmetry and the complex gating behaviour of these heterotypic gap junctions relative to homotypic wtCx40. In the Cx40E9K/wtCx40 and Cx40E9,13K/wtCx40 heterotypic cases, there was an increased V1/2 and a reduced gating charge valence (z) evident at negative Vj values. The Cx40E13K/wtCx40 heterotypic gap junction could not be fitted with a single Boltzmann curve owing to the biphasic Gj curve observed at negative Vj values (Fig. 4D). The heterotypic single point E9K and E13K Cx40 mutants resulted in a 50% reduction in slope with little or no increase in the V1/2 relative to wtCx40 at positive Vj values where the normal Cx40 Vj gating is expected to occur (Table 2). When both E-to-K point mutations were incorporated into the heterotypic Cx40 gap junction, the V1/2 was reduced to near zero with a further reduction in the apparent valence to approximately one equivalent charge (Fig. 4F). Gj was also observed to reduce to zero at positive Vj in the heterotypic Cx40E9K/wtCx40 and Cx40E9,13K/wtCx40 gap junctions, consistent with the zero Gmin properties of the Cx40E9K mutation. The reduced slope and increased V1/2 at negative Vj and the low V1/2 at positive Vj combine to produce a strongly rectifying Cx40E9,13K/wtCx40 gap junction reminiscent of the inwardly rectifying Kir2.1 potassium channel known to be blocked by spermine (Ficker et al. 1994; Lopatin et al. 1994). Hence, these results indicate that the E9K and E13K point mutations on the NT domain of Cx40 operate in concert to determine the Vj gating properties of Cx40.
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± 100 mV, only to close completely within milliseconds at positive Vj (Fig. 4H). At the same large negative Vj values, Ij increased slowly and then decreased slightly to less than instantaneous values. These data indicate that closure of the Vj gate, rather than open channel current rectification, is the predominant mechanism for the current rectification at positive Vj values. Conductance properties of mutant NT Cx40 single channels
It is of interest to know if the functional alterations in spermine blockade and Vj gating of Cx40 gap junctions associated with these acidic-to-basic charge substitutions at positions 9 and 13 have any effect on their single channel conductances. Figure 5A illustrates the typical response of a Cx40 gap junction to a Vj pulse of +80 mV, a rapid reduction in Ij to minimum steady-state values. Superimposed on this steady-state Ij baseline are discrete current fluctuations of about 12 pA in amplitude that correspond to a unitary conductance (
j) of 150 pS. The difference in the peak current amplitudes of the all points histogram from this and other Vj pulses were taken as the mean single channel current (Ij) values for the wtCx40 gap junction channel. The composite Ij–Vj relationship for the wtCx40 gap junction channel from three such experiments had a slope conductance of 157.9 ± 3.3 pS, consistent with earlier findings (Musa et al. 2001; Musa & Veenstra, 2003).
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j relative to wtCx40 (Fig. 5D). The heterotypic combination of these two connexin channels revealed a modest rectification of the channel Ij–Vj relationship that distinctly correlated with the two j values of the homotypic Cx40E9K and wtCx40 gap junction channels (Fig. 5E and F). This heterotypic gap junction acquired the channel conductance properties of the wtCx40 channel at positive Vj and of the Cx40E9K channel at negative Vj values. Single channel current fluctuations in homotypic Cx40E13K or heterotypic Cx40E9,13K/wtCx40 gap junctions were not resolved due to the lack of Vj gating and the reduced amplitude of the observed current fluctuations. However, unitary current fluctuations were observed on one occasion in a homotypic Cx40E9,13K cell pair (Fig. 5G and H). At a Vj of –80 mV, the current amplitude was less than 3 pA in this example. The slope conductance was 31.6 ± 1.9 pS, a mere 20% of the wtCx40 j. The low gj recordings shown in Fig. 6 exemplify the channel conductance and gating properties of the mutant and wt Cx40 channels. The homotypic wtCx40 and Cx40E9K gap junctions exhibit obvious gating behaviour with increasing Vj characterized by large quantal current fluctuations associated with the closure of multiple channels (Fig. 6A and B). By comparison, quantal current fluctuations were not readily resolved in the lower noise recordings from similar low gj Cx40E13K and Cx40E9,13K gap junctions (Fig. 6C and D). Again, inclusion of the E9K point mutation restores Vj gating properties to the mutant Cx40E13K gap junction (Fig. 6D), confirming previous observations about the Vj gating properties of these three homotypic mutant Cx40 gap junctions.
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The effects of the K-to-E mutations of residues 9 and 13 on the Vj-dependent gating properties of Cx43, wtCx43, Cx43K9E, and Cx43K9,13E gap junctions were also determined. The Cx43K9E mutation only moderately reduced the equivalent gating charge of this homotypic gap junction relative to wtCx43 (Fig. 7, Table 3). In contrast to Cx40, the K9E mutation in Cx43 produced a dramatic increase in the Gmin to more than double the wtCx43 value. Albeit the opposite effect to that observed at this exact locus in Cx40, the data again indicate a role for the NT domain in the formation of the Gmin state. We were not able to obtain similar data with the Cx43K13E mutation since its expression levels were too high to permit reliable quantitative assessment of the Vj gating properties of this homotypic mutant gap junction (see Table 4). The Vj gating properties of Cx43 were apparently restored by the inclusion of the K13E substitution into the Cx43K9E mutant protein as indicated by the return to essentially wtCx43 Vj gating parameters. The reciprocal double point Cx43K9,13E mutant gap junctions remained insensitive to block by 2 mM spermine (data not shown).
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The wtCx43 channel has a lower conductance of approximately 97 pS (Wang & Veenstra, 1997). The channel current recordings and composite Ij–Vj relationship from three Cx43 cell pairs yielded similar results: mean j= 92.2 ± 2.7 pS (data not shown). In contrast to the double point mutations in Cx40, the Cx43K9,13E double point mutant protein still formed a homotypic gap junction channel with no discernable change in j: mean j= 101.1 ± 1.3 pS (data not shown).
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Discussion |
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Despite the virtual elimination of spermine block by the E9K and E9,13K mutations in Cx40, approximately 50% of the block by 5 mM TPeA remained with this mutation (Fig. 2D, Musa et al. 2001). TPeA block was postulated to occur by steric hindrance, i.e. the approximate pore and ionic diameters are effectively equivalent at a narrow internal restriction site. Both TPeA and spermine inhibit Cx40 Ij by reducing channel open probability to near zero with little or no effect on the main state conductance of the single channel conductance. This type of block resembles altered channel gating more than ‘flickery’ permeation block and is associated with prolonged association times with the channel, consistent with bimolecular binding. The polyamine block of the Kir2.1 channel also exhibits some degree of a ‘gating’ type of block atypical of the flickery ‘open’ channel block associated with truly impermeant ions (Lopatin et al. 1995; Lee et al. 1999; Guo & Lu, 2000b). The elimination of spermine block accompanied by the partial retention of TPeA block could be explained by reduced monovalent cation partitioning into the Cx40 pore with no change in the internal pore diameter. We have yet to perform the necessary ionic substitution experiments to estimate the relative cation/anion conductances and pore diameters of the mutant Cx40 and Cx43 channels (Veenstra, 1996; Beblo & Veenstra, 1997; Wang & Veenstra, 1997).
Interestingly, the E9K point mutation reduced the gating charge of Cx40 by 25% while the E13K effaced the typical Boltzmann distribution of the Gj–Vj curve (Fig. 3, Table 1). Each homologous Cx40 K-for-E or Cx43 E-for-K substitution results in a net valence change of ±2 per locus. Yet the homotypic E9K and E9,13K mutations in Cx40 reduced the apparent gating charge valence by only 1 and 2 equivalent charges, respectively. The homotypic E13K mutation effectively eliminated the Vj gating of Cx40 altogether, as it did to the blockade by spermine. The E9K mutation similarly produced only a 32% diminution of Cx40 j but, when combined with the E9K mutation, it resulted in an 80% reduction in j (Figs 5 and 6). The E13K mutation j could not be assessed individually.
It is possible that the E13K substitution, which results in a neutral E12K13 locus instead of the rare occurrence of a highly acidic double glutamate locus at the midpoint of the connexin amino terminal domain sequences, is vital to the structural conformation of this putative pore-forming domain. The E9K locus, by virtue of its more internal location according to the domain-hinge-domain motif proposed by Purnick et al. (2000a), may produce a lesser effect since the presence or absence of the E12,13 locus closer to the cytoplasm plays a greater role in determining the partitioning of permeable cations into the mouth of the channel. For this to be true, the charge density must also be greater at the E12,13 site, suggesting that the pore diameters are not dramatically 
different at these two sequential sites on the Cx40 NT domain. Whether these effects are the result of point charge reversal or disruption of secondary structure can be further assessed by conservative (D) or polar neutral (Q, N) amino acid substitutions at positions 9 and 13 of Cx40.
The present model for Vj-dependent gating assumes that there are two identical gates in series but with the opposite orientation on each side of a homotypic gap junction channel (Harris et al. 1981; Verselis et al. 1994). The ‘voltage sensor’ for the ‘fast’Vj-dependent gating was localized within the NT domain of the connexins. This voltage-sensing domain was postulated to reside within the Vj field owing to a highly conserved glycine hinge at position 12 originally identified in Cx26 (Purnick et al. 2000a). Recent evidence has indicated that only one Vj gate needs to close to occlude the pore (Oh et al. 2000). Furthermore, charged amino acid substitutions up to position 10 of the NT domain of Cx26 can alter the voltage gating polarity and sensitivity of this connexin (Purnick et al. 2000b). Pairing two connexins with opposite voltage polarities for Vj-dependent inactivation in a heterotypic configuration (each half of the gap junction being composed of a different connexin) results in a rectifying Gj–Vj curve due to the asymmetric gating and channel conductance properties (Verselis et al. 1994; Bukauskas et al. 1995). Cx40 and Cx43 are similarly proposed to close with positive and negative Vj polarities, respectively, which are also of opposite sign to the charged amino acid residues on the NT domain (Valiunas et al. 2000).
Our data from the homotypic and heterotypic pairings of the Cx40E9 (and/or)13K mutants with themselves or wild-type Cx40 confirm that there are alterations in the Vj gating properties of these gap junctions (Figs 3 and 4, Tables 1 and 2). The heterotypic combinations of the Cx40 E9K and E13K mutations with wtCx40 produced asymmetric Gj–Vj curves with reduced slopes at both polarities and shifted V1/2 values. The rectifying Ij–Vj curve of the heterotypic wtCx40/Cx40E9,13K gap junction, obtained in the absence of spermine, strongly resembled the I–V curve for wtCx40, obtained in the presence of 2 mM unilateral spermine, and the inward rectifier Kir2.1 channel in the presence of 10–100 µM intracellular spermine (Ficker et al. 1994; Lopatin et al. 1994), further supporting the hypothesis that these E9,13 loci are critical to the polyamine block of the wtCx40 gap junction channel (Fig. 4E). All of the heterotypic results were reported with Vj relative to the wtCx40 cell. If it is assumed that the positive Vj gating parameters reflect the Vj gating properties of the wtCx40 hemichannel relative to the mutant hemichannel, then both the E9K and E13K substitutions reduce the valence by 1 while only the E13K mutation affects the open–closed probability equilibrium at positive Vj values. Combining both mutations in Cx40 shifts the V1/2 by negative 40 mV with only a slight additional reduction (0.5) in valence.
One hypothesis that could account for the differential functional effects of these two charge reversal mutations is that the combination of both point mutations reverses the Vj gating polarity of Cx40 from positive to negative. Each individual mutation reduces the positive gating relative to wtCx40 and the Cx40E9,13K double mutation effectively reverses it. One cannot definitively distinguish between a simple electrostatic voltage shift towards more negative Vj values and reversal of the Vj gating polarity in the Cx40E9,13K mutant gap junctions, but the lack of any shift in the Gmax in the heterotypic steady-state Gj–Vj curves by both single point mutations does not favour an electrostatic mechanism. The proposed positive Vj gating polarity of wtCx40 hemichannels is further supported by the observation of the wtCx40 j at positive Vj values (Fig. 5; Valiunas et al. 2000). The observation of the mutant Cx40E9K j at negative Vj values (relative to wtCx40) is indicative of an operative positive Vj gate in the mutant Cx40E9K hemichannel. At all positive Vj values, the ohmic instantaneous Ij decayed essentially to zero within 1 s (Fig. 4G and H). The brief appearance of a subconductance state in these records is consistent with the gating of the wtCx40 channels at positive Vj while the complete closure of the Cx40E9,13K/wtCx40 gap junction is consistent with the zero Gmin state attributed to the Cx40E9K mutation. The zero Gmin state was also accomplished by the complete closure of several homotypic Cx40E9K gap junction channels (Fig. 5C and E) without the occurrence of a subconductance state as observed in the presence of wtCx40 in the heterotypic gap junctions (Fig. 4G and H).
The complex biphasic gating behaviour observed at –120 mV in the Cx40E9,13K/wtCx40 gap junctions suggests that the mutant Cx40E9,13K hemichannel possess bipolar Vj gating properties since characteristics of the mutant Cx40 hemichannel are observed at both negative and positive Vj values. The sequence of positive and negative amino acids in the Cx40E9,13K mutant protein is DKEK, similar to the DKDK sequence found in wtCx43 at positions 3, 9, 12, and 13. These results suggest that all of the point charges do not contribute equally to the gating charge valence of Cx40. Since all of the Boltzmann distribution measurements were obtained relative to the homologous mutant Cx40 or wtCx40 protein, they reflect relative differences in the transjunctional voltage distribution across a symmetric or asymmetric Cx40 gap junction. Hence, each charge reversal mutation in Cx40 may account for an apparent unitary reduction in gating charge valence, but the double E-to-K mutation may at least partially reverse the gating polarity of Cx40. Vj polarity reversal would be expected to increase the cooperativity of channel closure at positive Vj in a contingent gating scheme as originally proposed by Harris et al. (1981).
We postulate that the Vj gradient can drop entirely across the E9,13K loci of Cx40 as indicated by the complete inhibition of gj by spermine, which certainly interacts with these sites, and the zero Gmin observed with the inclusive Cx40E9K homotypic and heterotypic gap junctions. These alterations in Vj gating occur despite the conservation of an aspartate residue at position 3 (D3) in Cx40 and Cx43 that is analogous to the D or N substitutions found at position 2 owing to the addition of a glycine at position 2 in both Cx40 and Cx43. Furthermore, the glycine hinge at position 12 in Cx26 and Cx32 is absent in Cx40 and Cx43 where the analogous position 13 is occupied by the respective E or K residue (Verselis et al. 1994; Purnick et al. 2000a; Harris, 2002). This possible interpretation is called to question by the observations in Cx43 where the reciprocal K9,13E double point mutation did not confer spermine block to the Cx43 gap junction. The results were also not as obvious in regard to the homotypic and heterotypic Vj gating properties of the Cx43 mutants (Figs 7 and 8, Tables 3 and 5). The V1/2 was negligibly altered while the valence was, respectively, decreased or increased by approximately 0.5 or 1.0 in the homotypic or heterotypic Cx43K9E and Cx43K9,13E combinations with wtCx43. This is in contrast to the sublinear additive decreases in valence observed with the reciprocal Cx40 mutations. Similarly, significant changes in Cx43 j were not observed with the K9,13E mutations. If there was any effect at all, the Cx43K9,13E mutation resulted in at most a 10% increase in Cx43 j. However, as the sequence information in Fig. 1 indicates, there are other amino acid differences, albeit not involving precise reciprocal charge substitutions, between the amino terminal domains of Cx40 and Cx43 that may also contribute to the overall structure of the putative ‘polyamine receptor’ of Cx40. It is also possible that interdomain interactions that have yet to be defined are important in this inhibitory bimolecular interaction.
It was proposed, at least for Cx43, that the fast Vj gate operates by a ball-and-chain mechanism where the CT domain forms the subcondutance state by interacting with a receptor associated with the channel pore (Moreno et al. 2002). The Cx40E9K gap junction channel closed completely with rapid kinetics and with both fast (< 10 ms) and slow (> 10 ms) transition times whereas the Cx43K9E gap junction had an increased Gmin. These data suggest that the position 9 locus is somehow integrally involved in determining the channel's ability to close. The ability of spermine to completely inhibit Cx40 gj is consistent with the hypothesis that spermine occludes the Cx40 pore by serving as an exogenous inactivation particle in its interactions with the NT domain of Cx40. The CT domain of Cx43, however, does not bind to the NT domain of Cx43. Instead it interacts with the second half of the cytoplasmic loop domain of Cx43 nearest to the third transmembrane domain and is involved in the pH gating of this gap junction (Duffy et al. 2002). We have alternatively proposed that the actions of the CT domain on fast Vj gating occur through intermediate interactions and that some other portion of the connexin molecule serves as the endogenous inactivation particle (Veenstra, 2003). The structure and function of the NT domains of Cx40 and Cx43 should continue to be investigated using both site-directed mutagenic and chimeric domain-swapping approaches. It is not clear how each locus of point charge contributes to the total gating charge of Cx40 and Cx43 except that the results are obviously not linear. The conserved D3 and D/E12 point charges on the NT domains of Cx40 and Cx43 should also be examined in this context to determine what role they play in the Vj-dependent closure and how pore occlusion is accomplished in these gap junctions.
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References |
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