Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642-8711
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ABSTRACT |
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Connexin channels are gated by transjunctional voltage (Vj) or CO2 via distinct mechanisms. The cytoplasmic loop (CL) and arginines of a COOH-terminal domain (CT1) of connexin32 (Cx32) were shown to determine CO2 sensitivity, and a gating mechanism involving CL-CT1 association-dissociation was proposed. This study reports that Cx32 mutants, tandem, 5R/E, and 5R/N, designed to weaken CL-CT1 interactions, display atypical Vj and CO2 sensitivities when tested heterotypically with Cx32 wild-type channels in Xenopus oocytes. In tandems, two Cx32 monomers are linked NH2-to-COOH terminus. In 5R/E and 5R/N mutants, glutamates or asparagines replace CT1 arginines. On the basis of the intriguing sensitivity of the mutant-32 channel to Vj polarity, the existence of a "slow gate" distinct from the conventional Vj gate is proposed. To a lesser extent the slow gate manifests itself also in homotypic Cx32 channels. Mutant-32 channels are more CO2 sensitive than homotypic Cx32 channels, and CO2-induced chemical gating is reversed with relative depolarization of the mutant oocyte, suggesting Vj sensitivity of chemical gating. A hypothetical pore-plugging model involving an acidic cytosolic protein (possibly calmodulin) is discussed.
cell communication; cell junctions; pH gating
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INTRODUCTION |
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GAP JUNCTIONS ARE MEMBRANE differentiations specialized for direct cell-to-cell exchange of metabolic and electrical signals. A gap junction channel is made of two hemichannels (connexons), each a hexamer of connexin proteins. Connexins have four transmembrane domains linked by two extracellular loops and a cytoplasmic loop (CL), a short NH2 terminus (NT), and a COOH terminus (CT) of variable length (for review see Ref. 13).
Gap junction channels close in response to changes in transjunctional voltage (Vj) or intracellular Ca2+ or H+ concentration (for review see Refs. 3, 9, and 14), but the molecular mechanisms of channel gating are still unclear. Vj and chemical gates are believed to be two distinct gates: the former closes the channel rapidly but incompletely, and the latter closes the channel slowly but completely (5).
Recently, we studied connexin mutants and chimeras expressed in Xenopus oocytes to identify domains of the rat connexin32 (Cx32) potentially involved in CO2-induced (low intracellular pH) channel gating. Cx32 is a connexin expressed in many organs, such as liver, pancreas, kidney, thyroid, and mammary gland, and in various cells of the nervous system, such as neurons, oligodendrocytes, and Schwann cells (for review see Ref. 3). Several Cx32 mutations have been shown to be relevant to the pathogenesis of a demyelinating disease known as the X-linked Charcot-Marie-Tooth disease (2, 3).
In Cx32, CL contains a domain relevant to CO2 gating sensitivity (23, 24), and NT (23) and 84% of CT (25, 28, 30) do not seem to play a role, whereas basic residues of the initial domain of CT (CT1) inhibit CO2 gating sensitivity (25, 28). Thus Cx32 appears to differ from Cx43, in which CT seems to play a major role in chemical gating (8) via a postulated ball-and-chain mechanism (6, 10). On the basis of our work on Cx32, we have proposed that CT1, a basic and partly hydrophobic domain, may interact electrostatically and hydrophobically with acidic and hydrophobic residues of CL1 and may inhibit gating by latching CL (26).
To further probe the chemical gating mechanism of Cx32, three new mutants were constructed. One mutant, the tandem, is a dimer in which two Cx32 monomers are concatenated NT-to-CT. With this mutant, each connexon (hemichannel) is made of three tandem connexins. In each connexon, three of the six NT chains are linked to three CT chains and the other three NT and CT chains are free. Another mutant, the 5R/E, is a Cx32 molecule in which five basic residues of CT1 (R215, R219, R220, R223, and R224) are replaced with an acidic residue (E). In the third mutant, the 5R/N, the five basic CT1 residues mentioned above are replaced with a neutral-polar residue (N). This mutant, when tested homotypically, generated channels more sensitive to CO2 than Cx32 (25, 28). All these mutations would be expected to interfere with the CT1-CL1 interaction. Homotypic pairing of tandem and 5R/E mutants did not produce functional junctions.
The data show that, in all these heterotypic channels, junctional conductance (Gj) increases with Vj gradients positive at the tandem, 5R/E, or 5R/N side and decreases with Vj of opposite polarity. In addition, positive Vj gradients partially reverse the Gj drop induced by CO2, suggesting that the chemical gate might be voltage sensitive.
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MATERIALS AND METHODS |
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Site-Directed Mutagenesis
Molecular biology protocols were generally as described by Sambrook et al. (20). Cx32 cDNA (12) was used in the construction of DNA mutants. Two wild-type Cx32 were linked to form a Cx32 tandem; a tandem is a Cx32 dimer composed of two Cx32 monomers concatenated NT-to-CT. Normal and modified Cx32 cDNAs were used in the construction of the tandem. The modification involved the deletion of a silent stop codon preceding the ATG initiation codon, which would be used in the second Cx32 monomer of the tandem. The strategy employed to create the 5R/E and 5R/N mutants of Cx32 has been previously described (25). All the mutations were verified by digestion of DNA with restriction enzymes and sequence analysis.Oligonucleotide Sequences
Oligonucleotides were synthesized by a DNA synthesizer (model 393, ABI, Foster City, CA). The sequences used to produce the 5R/E and 5R/N mutants are as follows (underscored letters represent mutated nucleotides): 5'-CCTTPreparation of cRNA
Wild-type and mutated forms of Cx32 cDNA were subcloned into pBluescript (Stratagene, Menasha, WI) or pGEM 3Z (Promega, Madison, WI) and used for in vitro synthesis of cRNA. cRNAs were transcribed from linearized plasmid with use of T7 or SP6 mMESSAGE mMACHINE (Ambion, Austin, TX) in the presence of the cap analog m7G(5')ppp(5')G (Ambion).Oocyte Preparation and Microinjection
Oocytes were prepared as previously described (15). Briefly, adult female frogs (Xenopus laevis) were anesthetized with 0.3% tricaine (MS-222), and the oocytes were surgically removed from the abdominal incision. The oocytes were placed in ND-96 medium. Oocytes at stage V or VI were subsequently defolliculated in 2 mg/ml collagenase (Sigma Chemical, St. Louis, MO) in Ca2+-free OR-2 medium for 80 min at room temperature. The defolliculated oocytes were injected with 46 nl (0.25 µg/µl) of antisense oligonucleotide complementary to endogenous Xenopus Cx38: 5'-GCTTTAGTAATTCCCATCCTGCCATGTTTC-3' (commencing at ntUncoupling Protocol
The oocyte chamber was continuously perfused at a flow rate of 0.6 ml/min by a peristaltic pump (Dyamax model RP-1, Rainin Instrument, Woburn, MA). The superfusion solution was ejected by a 22-gauge needle placed near the edge of the conical well containing the oocyte pair. The level of the solution in the chamber was maintained constant by continuous suction. Electrical uncoupling of oocyte pairs was induced by 3-15 min of superfusion (0.6 ml/min) of salines continuously gassed with 100% CO2. A ClMeasurement of Gj in Oocyte Pairs
All the experiments were performed using the standard double-voltage-clamp procedure for measuring Gj (21). After the insertion of a current and a voltage microelectrode in each oocyte, both oocytes were initially voltage clamped to the same holding potential (Vm), Vm 1 = Vm 2 (usuallyFor studying voltage dependence of
Gj, each oocyte
of the pair was first voltage clamped at 20 mV. Voltage steps of
20 mV (±120 mV maximum) and 20-s duration were then applied every
45 s to either oocyte of the pair while the other was maintained at
20 mV. To illustrate the relationship between steady-state Gj
(Gj ss)
and
Vj,
the normalized
Gj
(Gj ss/Gj max,
where
Gj max is maximum
Gj)
was plotted with respect to
Vj.
The curve was fitted to a two-state Boltzmann distribution of the
following form:
(Gj ss
Gj min)/(Gj max
Gj ss) = exp[
A(Vj
V0)],
where
V0
is the Vj
value at which the voltage-sensitive conductance is one-half the
maximal value,
Gj min
is the theoretical minimum normalized Gj,
and A = nq/kT is a constant expressing voltage
sensitivity in terms of number of equivalent gating charges
(n) moving through the entire
applied field (where q is the electron
charge, k is the Boltzmann constant,
and T is the temperature in Kelvin).
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RESULTS |
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Functional Tests of Mutants
Tandem and 5R/E mutants did not form functional channels when tested homotypically, even after 6-7 h of oocyte pairing. In heterotypic pairs between oocytes expressing tandem or 5R/E and oocytes expressing Cx32 wild type (tandem-32 and 5R/E-32, respectively), coupling developed slowly. Within 2-3 h after pairing, Gj was 0.56 ± 0.14 (SE) µS (n = 15) with tandem-32 and 0.34 ± 0.13 µS (n = 6) with 5R/E-32. In contrast, 5R/N mutants were functional homotypically (5R/N-5R/N) (26, 28) and heterotypically with Cx32 wild-type mutants (5R/N-32) (27). In 5R/N-32 pairs, Gj, measured with the conventional protocol (+20-mV voltage pulses of 2-s duration applied to 1 of the 2 oocytes every 10 s) within 2-3 h after pairing, was 2.03 ± 0.4 µS (n = 19). In homotypic 5R/N-5R/N and 32-32 pairs, Gj was 3.8 ± 1.3 (n = 8) and 4.2 ± 1.3 µS (n = 26), respectively.Sensitivity to Vj Pulses
Tandem-32 channels.
Cx32 junctions displayed a characteristic sensitivity to
Vj.
As shown in Fig.
1A,
Ij
decayed exponentially with time for
Vj > ±40
mV. In contrast, tandem-32 channels displayed a unique
Ij-Vj behavior (Fig. 1A). With the
tandem side negative, as
Vj
was increased in 20-mV steps from 20 to 120 mV, the initial and final
Ij
progressively decreased to very low conductance values, and the
channels appeared to be
Vj
sensitive even at the lowest
Vj.
With the tandem side positive, as
Vj
increased in steps from 20 to 120 mV,
Ij
progressively increased to high values, and
Ij
recorded at the end of the pulse was greater than the initial
Ij,
as if
Vj
caused an increase rather than a decrease in
Ij.
Only at the largest
Vj
gradients (100-120 mV), two fairly conventional
Ij
levels started to appear (Fig. 1A). The asymmetric
Ij-Vj
behavior of tandem-32 and other heterotypic mutant-32 junctions is
clearly demonstrated by plotting normalized Gj
(Gj ss/Gj max)
vs.
Vj
(Fig. 1, C and
D).
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5R/E-32 channels. 5R/E-32 channels subjected to the conventional Vj protocol behaved as tandem-32 channels (Fig. 1A). With the 5R/E side negative, initial and final Ij progressively decreased to very low values, and the channels appeared to be Vj sensitive even at the lowest Vj. With the 5R/E side positive, Ij progressively increased to very high values, and with Vj gradients as high as 80 mV Ij measured at the end of the pulse was greater than the initial Ij. Only with the largest Vj gradients (100-120 mV) did two fairly conventional Ij levels start to appear (Fig. 1A). As for tandem-32 channels, the relationship between normalized Gj (Gj ss/Gj max) and Vj clearly demonstrates the asymmetric Ij-Vj behavior of 5R/E-32 junctions (Fig. 1D).
5R/N-32 channels. The behavior of 5R/N-32 channels exposed to the conventional Vj protocol was not as asymmetric as that of tandem-32 or 5R/E-32 channels (Fig. 1A). With Vj > 20 mV, initial and final Ij were higher with the 5R/N side positive than negative at each comparable Vj value (Fig. 1A). This moderate asymmetry is displayed in Fig. 1C, which plots the normalized Gj (Gj ss/Gj max) vs. Vj and the two-state Boltzmann fit for 5R/N-32 and 32-32 channels. The Boltzmann values of 5R/N-32 channels are as follows: V0 = 52.6 mV, n = 2.4, and Gj min = 0.11 (n = 3) for the 5R/N side negative and V0 = 77.2 mV, n = 2.4, and Gj min = 0.22 (n = 3) for the 5R/N side positive. Those of homotypic 32-32 and 5R/N-5R/N channels are similar: V0 = 59.5 mV, n = 2.1, and Gj min = 0.29 (n = 7) and V0 = 59.8 mV, n = 2.3, and Gj min = 0.21 (n = 5), respectively. On the basis of data described below, the asymmetric voltage behavior of mutant-32 channels may indicate that progressive exposure to prolonged Vj gradients that make the mutant side negative or positive progressively closes or opens, respectively, an increasing number of channels.
Effect on Gj of Trains of Long Vj Pulses
The asymmetric Ij-Vj behavior of mutant-32 channels (Fig. 1A) suggested that Vj-dependent channel opening and closing processes might have a long time constant (
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Tandem-32 channels. With Vj pulses positive at the tandem side, Ij behaved as previously described (cf. Fig. 2A, left trace, with Fig. 1B). With the tandem side negative, initial and final Ij levels progressively decreased to very low values (Fig. 2A, right trace). Gj, measured with the conventional protocol (+20-mV pulses of 2-s duration applied to 1 oocyte every 10 s) immediately after the train of Vj pulses positive at the tandem side (pulse train 1), was 130% greater than that measured before the train of Vj pulses (Fig. 2B) and recovered in 3-4 min (Fig. 2B). Gj measured immediately after the train of 60-mV Vj pulses (pulse train 2) negative at the tandem side decreased to 23% of control values (Fig. 2B). This behavior suggested Vj-dependent channel opening and closing processes, with long time constants, that could reflect the function of a slow Vj-sensitive gate.
5R/E-32 channels. 5R/E-32 channels displayed a similar Ij behavior (Fig. 2C) but, with the 5R/E side positive, only the first pulse resulted in a monophasic Ij increase, whereas subsequent pulses progressively assumed a more conventional Ij behavior: an initial Ij peak followed by exponential Ij decay (Fig. 2C, left trace). However, initial and final Ij levels increased exponentially over the first six to seven pulses (Fig. 2C, left trace). With the 5R/E side negative, initial and final Ij levels progressively decreased to very low conductance values (Fig. 2C, right trace). Gj increased by 110% during the train of Vj pulses (pulse train 1) positive at the 5R/E side (Fig. 2D) and recovered exponentially in 4-5 min (Fig. 2D). In contrast, Gj reversibly dropped to 31% of control values with pulses (pulse train 2) negative at the 5R/E side (Fig. 2D).
5R/N-32 channels. 5R/N-32 channels displayed an Ij behavior (Fig. 2E) qualitatively similar to that of tandem-32 (Fig. 2A) and 5R/E-32 (Fig. 2C) channels. With the 5R/N side positive, the first pulse resulted in a monophasic Ij increase from initial to final Ij, whereas subsequent pulses generated a more conventional Ij behavior: an initial Ij peak followed by exponential Ij decay (Fig. 2E, left trace). Initial and final Ij levels increased sizably between the first and the second pulse but only slightly with subsequent pulses (Fig. 2E, left trace). In contrast, with the 5R/N side negative, initial and final Ij levels progressively decreased to lower conductance values (Fig. 2E, right trace). Gj increased by 11% during the application of the train of Vj pulses (pulse train 1) positive at the 5R/N side (Fig. 2F) and recovered to control values in 4-5 min (Fig. 2F). In contrast, Gj reversibly dropped to 63% of control values with pulses (pulse train 2) negative at the 5R/N side (Fig. 2F).
32-32 channels. The application of similar trains of Vj pulses of either polarity to homotypic 32-32 channels (Fig. 2G) caused a small and reversible Gj drop to ~90% of control values (Fig. 2H). This indicates that, albeit to a lesser extent, repeated Vj pulses affect a slow Vj-sensitive gate in homotypic Cx32 wild-type channels as well.
Effect on Gj of Steady-State Vj
The ability of trains of long Vj pulses of opposite polarity to progressively increase or decrease Gj in tandem-32, 5R/E-32, and 5R/N-32 junctions suggested that these channels might also be sensitive to prolonged, steady-state Vj gradients. If so, steady-state Vj protocols could enable one to better define the magnitude and kinetics of this process.Tandem-32 channels.
In oocyte pairs in which each oocyte was initially clamped at
Vm = 20 mV
(Vj = 0), the establishment of steady-state
Vj = 40 mV (tandem side positive) increased
Gj
exponentially by 262 ± 64% (mean ± SD,
n = 4), with
= 0.88 ± 0.2 (SD)
min (n = 4; Fig.
3A and
B);
Gj
was measured by the conventional protocol: +20-mV pulses of 2-s
duration applied to one oocyte every 10 s. Similar results were
obtained by simultaneously depolarizing the tandem side and
hyperpolarizing the Cx32 side or by just hyperpolarizing the Cx32 side
in oocytes initially clamped at
20 mV (Fig.
3A and B) or
40 mV.
Vj
gradients of 10 and 20 mV increased
Gj
by 10-20% and 80-130%, respectively (data not shown).
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5R/E-32 channels.
Similar results were obtained with 5R/E-32 channels. Exposure to
Vj = 40 mV (5R/E side positive) increased
Gj
by 182 ± 50% (mean ± SD,
n = 5), with = 1.28 ± 0.33 (SD) min (n = 5; Fig. 3,
C and
D).
Gj
increased by as much as 400% with
Vj = 60 mV. On return to
Vj = 0 mV,
Gj
recovered slowly (Fig. 3C), with
= 2.04 ± 0.35 min (n = 4). When
Vj
was reversed from positive to negative at the 5R/E side,
Gj
drastically decreased (Fig. 3D) to
14.8 ± 3.3% (n = 4) of control
values (measured at
Vj = 0), with
= 0.39 ± 0.07 min
(n = 4). As for tandem-32
channels, on return to
Vj = 0 mV from
Vj = 40 mV (5R/E side positive),
Gj
increased abruptly before dropping (Fig.
3C) because of the reopening of conventional
Vj-sensitive
gates. Indeed, as expected, this abrupt increase in
Gj
was not observed when the
Vj
polarity was reversed from positive to negative (Fig.
3D).
5R/N-32 channels.
5R/N-32 channels behaved qualitatively as tandem-32 and 5R/E-32
channels. The establishment of
Vj = 40 mV (5R/N side positive) increased
Gj
reversibly and exponentially by 23 ± 19.5% (mean ± SD,
n = 18; Fig. 3,
E and
F), with = 0.78 ± 0.32 (SD)
min (n = 11). With reestablishment of
Vj = 0 mV,
Gj
recovered, with
= 2.4 ± 0.9 min
(n = 4). When the channels were
subjected to Vj
negative at the 5R/N side from
Vj = 0 mV (Fig. 3E) or
Vj = 40 mV (5R/N side positive; Fig.
3F),
Gj
dropped to 35.2 ± 17.3% (n = 10)
of control values (measured at
Vj = 0), with
= 1.05 ± 0.61 min
(n = 9). On return to
Vj = 0 mV from
Vj = 40 mV, 5R/N side positive,
Gj
increased abruptly, then dropped (Fig.
3E); this was not observed with
Vj
reversal from positive to negative (Fig.
3F), as described for tandem-32
(Fig. 3B) and 5R/E-32 (Fig. 3D) channels.
32-32 channels. In homotypic 32-32 junctions the application of 40-mV Vj gradients to either oocyte decreased Gj to 67.7 ± 8.2% (mean ± SD, n = 7; Fig. 3G). On Vj application, Gj dropped rapidly in the first 10-20 s (Fig. 3G, more obvious with 1st Vj application) and then slowly over the following 4-5 min, eventually reaching a steady-state value. The rapid drop and the slow decay may result from the sequential closures of the conventional (fast) Vj gate and the slow Vj gate, respectively.
Effect of Simultaneous Depolarization or Hyperpolarization of Both Oocytes
The heterotypic channels were tested for sensitivity to Vm by depolarizing or hyperpolarizing both oocytes in 20-mV steps starting from Vm =CO2 Sensitivity
Homotypic 32-32 channels were weakly sensitive to CO2, as previously reported (22). Gj decreased to 85 ± 5% (mean ± SE, n = 7) with a 3-min exposure to CO2 (Fig. 4A) and to 47 ± 4.8% with a 15-min exposure (n = 16; Fig. 4B) at a maximum rate of ~9%/min. Tandem-32 channels were more sensitive to CO2 than 32-32 channels. Gj dropped to 43 ± 8% (n = 5) with 3-min exposures to CO2 (Fig. 4) and to 6.4 ± 5% (n = 4) with 15-min exposures (Fig. 4B) at a maximum rate of ~13%/min. 5R/E-32 channels were much more sensitive to CO2 than 32-32 channels (Fig. 4). Gj dropped to zero (n = 3) with 3-min exposures to CO2 at a maximum rate of ~37%/min (Fig. 4).
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As previously reported (26), the sensitivity of 5R/N-32 channels was also greater than that of 32-32 channels. Gj decreased to 37.5 ± 6.4% (mean ± SE, n = 3) with 3-min exposures to CO2 (Fig. 4B) and to ~0% (n = 4) with 15-min exposures (Fig. 4B) at a maximum rate of ~21%/min. Thus the CO2 sensitivity of all these channels ranked as follows: 5R/E-32 > 5R/N-32 > tandem-32 > 32-32.
Effect on Gj of Steady-State Vj During CO2-Induced Uncoupling
Oocyte pairs expressing mutant-32 or 32-32 channels were subjected to Vj gradients of different polarity during exposure to 100% CO2. In mutant-32 channels, Gj, reduced to very low values by exposure to CO2 at Vj = 0 mV, increased dramatically and reversibly with Vj gradients that made the tandem (Fig. 5A), 5R/E (Fig. 5C), or 5R/N (Fig. 5D) side positive by 40 mV. Similar results were obtained by simultaneously depolarizing the mutant side and hyperpolarizing the Cx32 side or by just hyperpolarizing the Cx32 side (Fig. 5, A, C, and D). With the same Vj gradient, Gj increased more during uncoupling or recovery from uncoupling than during maximal uncoupling. This was most obvious with 5R/E-32 channels (Fig. 5C), inasmuch as their CO2 sensitivity is so high that Gj remains 0 µS for several minutes even with CO2 exposures as short as 3-5 min (Figs. 4A and 5C). With tandem-32, 5R/E-32, and 5R/N-32 channels, the application of Vj = 40 mV (mutant side positive) during CO2 uncoupling (Fig. 5, A, C, and D) increased Gj by ~1,000, ~2,600, and ~600%, respectively. This is noteworthy, because in the absence of CO2, similar Vj applications increased Gj by only ~260, ~180, and ~23%, respectively (see above). These data indicated that Vj is able to open tandem-32, 5R/E-32, and 5R/N-32 channels that had been closed by the CO2 treatment. In contrast, Vj gradients that made the mutant side negative dramatically and reversibly reduced Gj to very low values (Fig. 5, A, C, and D). When a similar protocol was tested on 32-32 channels, the application of 40-mV Vj gradients of either polarity always resulted in a significant drop of Gj (Fig. 5B). The magnitude of the Gj drop indicates that also in the case of 32-32 channels Vj affects the conventional Vj gate and the slow Vj gate.
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DISCUSSION |
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This study shows that oocytes expressing tandem, 5R/E, or 5R/N mutants have altered sensitivity to CO2 and Vj when paired with oocytes expressing Cx32 wild-type mutants. When Vj is depolarized with respect to the mutant-expressing oocyte, Gj increases rather than decreases. Positive Vj at the mutant side is also effective in reopening channels closed by 100% CO2.
Tandem and 5R/E mutants did not display functional channels in homotypic configuration. A possibility is that tandem and 5R/E mutants assume conformations unsuitable for homotypic interaction. In the tandem mutant the NH2-to-COOH terminus linkage may render opposite dimers too rigid for interaction but able to interact heterotypically with more flexible Cx32 monomers. However, the architecture of tandems in connexons is difficult to predict, and in the absence of structural information the possibility that only one of the two monomers of a tandem is normally inserted in the membrane cannot be discounted.
In tandem-32 and 5R/E-32 pairs,
Gj
is low when measured at
Vj = 0 mV but increases dramatically with depolarization of the mutant-expressing oocyte. This large increase in
Gj
with positive Vj
at the mutant side could be explained in a number of ways: new channel
formation, increase in single-channel open probability (Po), increase
in single-channel conductance
(j), or a combination of the
above. Although it is possible that positive
Vj
favors channel formation, this is considered unlikely, because it would mean that channels formed by positive
Vj
at the mutant side are removed on return to
Vj = 0 mV and that negative
Vj
at the mutant side removes newly formed and preexisting channels. One
would have to assume that channel formation and removal are based on the same mechanism, which is unlikely. Because
Gj
measured at Vj = 0 mV did not significantly increase after the first 2-3 h of
pairing, we believed that at that time the channel population had
reached some stability, at least in terms of new channel formation.
Whether the observed phenomenon is due to changes in
Po
and/or j can only be determined
at the single-channel level. Interestingly,
j was found to be sensitive to
Vj
in heterotypic Cx32/Cx26 channels (4). However, in this case, even
Vj
as high as ±100 mV did not change
j by >50% in either
direction, whereas in our case Vj
as low as 40 mV (tandem or 5R/E side positive) increased
Gj by as much as two- to threefold and
Vj
as low as 60 mV (tandem or 5R/E side negative) reduced
Gj
to nearly zero. Therefore, this is likely to be a gating phenomenon
involving changes in
Po.
Tandem, 5R/E, and 5R/N mutants may display the function of a slow,
Vj-sensitive gate.
If it is assumed that this is a channel gating phenomenon, does it involve the conventional Vj-sensitive gate or another gate, a slow gate? Several elements suggest that we might be dealing with a slow gate distinct from the conventional Vj gate. The presumed slow gates appear to open and close with time constants on the order of minutes, whereas Vj gates close with time constants of a few seconds. Vj gradients of 40-60 mV (mutant side negative) close most of the channels, whereas even with Vj gradients of ±120 mV a 20-25% residual conductance is usually observed (5). Even a Vj gradient as low as 10 mV (tandem side positive) significantly increases Gj, whereas the conventional Vj-sensitive gate is insensitive to Vj < 20-40 mV. Furthermore, the distinct behavior of the two gates is clearly manifested by the abrupt increase in Gj observed with return to Vj = 0 mV from positive Vj at the mutant side (Fig. 3, A, C, and E). Because the conventional Vj gate of Cx32 is believed to close with negative potentials (22), the abrupt increase in Gj is likely to mark the reopening of some of the Cx32 Vj gates. This interpretation is further confirmed by the observation that this abrupt increase in Gj is absent when Vj is reversed from positive to negative at the mutant side; in this case, the behavior of the conventional Vj gate is masked, because, with Vj reversal, whereas the Vj gate of the Cx32 hemichannel opens that of the mutant hemichannel closes, or vice versa. Recently, additional evidence for a distinction between a slow gate and a conventional Vj gate has come from our preliminary data (unpublished observations) on the behavior of heterotypic channels between Cx26 wild-type and Cx26 mutant (4basic/E) channels, in which, as in 5R/E, all four basic residues of CT were mutated to E (R215E, K220E, K222E, and R223E). Interestingly, these channels behaved qualitatively as 5R/E-32 channels when exposed to Vj in the presence and absence of CO2, despite the fact that the conventional (fast) Vj gates of Cx26 and Cx32 are sensitive to opposite voltage polarities (22).
Is the slow gate active in wild-type connexins as well? In homotypic 32-32 channels, exposure to steady-state Vj (40 mV) of either polarity decreased Gj to ~68%. The time course of Gj decay had a fast and a slow component, suggesting that also in these channels fast and slow Vj gates are activated. However, with 32-32 channels the Gj drop was much smaller than with mutant-32 channels, indicating that the effect of negative Vj on the slow gate is less pronounced in wild-type Cx32 than in mutant hemichannels.
Most intriguing is the observation that Vj gradients (mutant side positive) significantly increase Gj in oocytes partially or totally uncoupled by CO2. This raises the possibility that the chemical gate is voltage sensitive and that the chemical gate and the slow gate are the same. Interestingly, the effect of voltage on chemical gating has been recently reported in insect cells (29). In this case, however, the chemical gate appears to be sensitive to Vm rather than to Vj. The idea that the slow gate and the CO2-sensitive gate are the same may be corroborated by the observation that with mutant-32 channels a Vj application (mutant side positive) during CO2-induced uncoupling increases Gj by a much larger fraction than in the absence of CO2. We believe that positive Vj has a greater effect on Gj in the presence than in the absence of CO2, because with CO2 a larger fraction of slow gates are in the closed state. This further indicates that Vj and CO2 compete for driving the same gating mechanism. An additional piece of evidence favoring the idea that the chemical gate and the slow gate may be the same comes from preliminary data on oocytes in which calmodulin (CaM) expression was inhibited with antisense oligonucleotides to CaM mRNA (15, 17). Within 24-48 h after the injection of CaM antisense oligonucleotides, the slow gating behavior of tandem-32, 5R/E-32, and other mutant-32 channels completely disappeared (17) and tandem-32 channels manifested a symmetrical Vj sensitivity virtually identical to that of 32-32 channels. This observation, in conjunction with our previous data showing complete loss of CO2 gating sensitivity after the same treatment with CaM antisense oligonucleotides (15), suggests that CaM may be involved in chemical gating and slow gating mechanisms. On this basis, a gating mechanism viewing CaM (or another small acidic protein) as a negatively charged channel-plugging molecule ("cork" gating model) is being considered (see below).
Data from this and previous studies (25, 27, 28) show that homotypic 5R/N-5R/N and heterotypic tandem-32, 5R/E-32, and 5R/N-32 channels are more sensitive to CO2 than 32-32 channels. If we assume that the hemichannels of heterotypic channels gate independently from each other (27), this would indicate that, in tandem-32, 5R/E-32, and 5R/N-32 pairs, most of the channels close at the mutant hemichannel side. If this were the case, the observed increase in Gj at positive Vj would be expected to reflect primarily the behavior of the slow gates of tandem, 5R/E, and 5R/N hemichannels rather than that of Cx32 hemichannels. Because Vj gradients activate the conventional Vj gate as well, the increase in Gj observed at positive Vj on the mutant side indicates that the consequence of slow gates reopening significantly outweighs the effect of Vj gates closing. Thus the increase in Gj in the presence and absence of CO2 with positive Vj at the mutant side would be even greater if the conventional Vj gate were inactive, and the opposite would be expected with negative Vj. However, the effect of 40-mV Vj gradients on conventional Vj gating is relatively small.
With 32-32 channels, Vj gradients of either polarity always caused further drops in Gj during exposure to CO2. Because in this case the CO2-sensitive gates are expected to close symmetrically, one would predict Vj gradients to complement the effect of CO2 at the negative side and oppose it at the positive side; thus the Gj drop may result from the activation of conventional Vj-sensitive gates only. However, the magnitude of the Gj drop is greater than expected on the basis of the weak sensitivity of 32-32 channels to Vj gradients of ±40 mV. Therefore, it is likely that the Gj drop reflects the effect of Vj on conventional Vj gates and slow gates.
If it is assumed that the observed phenomena reflect the behavior of a slow gate, why do they manifest themselves preferentially in these mutants? One can only speculate at this stage. In previous studies we have proposed that CL is relevant for CO2-induced gating of Cx32 channels (23, 24) and that CT1 may inhibit gating by limiting CL mobility via electrostatic and hydrophobic interactions with CL1 (26). If present, these interactions could maintain the channel open by latching CT1 to CL1. CO2 would close the channel by initiating a mechanism that releases CL from CT. If this were true, one would expect any weakening of the CL1-CT1 interaction to result in a mixture of open and closed channels. In tandem, 5R/E, and 5R/N mutants, CL1-CT1 interactions might be weaker for the following reasons. In the tandem mutants, three of the six CT and NT chains are linked, which may prevent three of the six CT1 domains from reaching CL1 domains, if indeed both monomers are inserted in the membrane. In 5R/E mutants the replacement of five basic residues of CT1 (R215, R219, R220, R223, and R224) with acidic residues (E) would cause CT1 and CL1 to repel each other, inasmuch as electrostatic CL1-CT1 interactions are postulated to occur between basic CT1 residues (particularly R215 and R219) (25, 28) and acidic CL1 residues (El02, E109, and D113). In 5R/N mutants the removal of positive charges from CT1 would eliminate electrostatic interactions yet leave unaffected hydrophobic interactions. This would be expected to weaken but not prevent CT1-CL1 interactions, which could be the reason why 5R/N-32 channels were not affected by steady-state Vj gradients as strongly as tandem-32 or 5R/E-32 channels. Consistent with this idea may also be recent data for increased CO2 sensitivity in mutants in which E102 was replaced with R (26) or G (19) residues, mutations that would be expected to weaken potential CL1-CT1 charge interactions; interestingly, the latter is a mutation reported to occur in some patients with Charcot-Marie-Tooth disease (7, 19).
Of course, the CT1-CL1 association-dissociation hypothesis is based on many assumptions, the foremost of which is the CL1-CT1 interaction, which at this stage relies primarily on circumstantial evidence (26). Therefore, the phenomena described here could very well be related to different molecular domains. Whatever conformational changes connexins undergo with uncouplers, a likely possibility is that these changes render the channel mouth more accessible to a negatively charged plugging molecule (cork gating model).
The cork gating hypothesis envisions slightly different scenarios for 32-32 and mutant-32 channels. In the former, CaM would have limited accessibility to the channel mouth under normal cytosolic conditions (absence of uncouplers) and in the absence of Vj gradients. Uncouplers would change CaM and/or connexin conformation, making the channel mouth accessible to a CaM lobe, which would bind electrostatically and hydrophobically to the channel's mouth, closing the channel completely. The latter would be accessible to a CaM lobe even without uncouplers, but CaM would bind loosely, only electrostatically, such that positive Vj would dislodge it slowly and negative Vj would lodge it in a greater number of channels. With uncouplers, CaM would plug the channel more efficiently, by interacting electrostatically and hydrophobically. This would imply two closed states: closed state 1 would involve both types of interaction and would not be reversed by Vj, and closed state 2 would involve only electrostatic interactions, and so it would be sensitive to Vj. The presence of both types of interaction in closed state 1 could explain why at maximum uncoupling positive Vj is less effective (Fig. 5C). Because the channel's mouth (18) and the CaM lobes are ~2.5 nm in diameter, a CaM lobe could fit well in the channel's mouth.
In conclusion, data on chemical and voltage gating characteristics of three Cx32 mutants (tandem, 5R/E, and 5R/N) expressed heterotypically with Cx32 wild type indicate that Vj gradients activate a slow gating mechanism that appears to be distinct from the conventional Vj gating mechanism. The presumed slow gate opens at relatively positive Vj and closes at negative Vj, following exponential courses with long time constants. In addition, a positive Vj at the mutant side appears to reopen channels closed by CO2, raising the possibility that the chemical gate and the Vj-sensitive slow gate are the same. The slow gate appears to be present in homotypic Cx32 channels as well, but since these channels are all open in the absence of Vj, the slow gate of Cx32 wild-type channels manifests itself only with relatively negative Vj gradients. This gate could be a pluglike cytosolic molecule (possibly CaM).
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ACKNOWLEDGEMENTS |
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The authors thank Dr. Eric C. Beyer (Washington University) for providing the cDNA clone for the rat liver connexin32, Dr. Basilio A. Kotsias (Instituto Investigaciónes Médicas, Buenos Aires, Argentina) for useful criticism, and Drs. Peter G. Shrager and Ted B. Begenisich (University of Rochester) for helpful comments on the manuscript.
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FOOTNOTES |
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This study was supported by National Institute of General Medical Sciences Grant GM-20113.
A preliminary account of this study has been published in abstract form (16).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: C. Peracchia, Dept. of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester, 601 Elmwood Ave., Rochester, NY 14642-8711 (E-mail: cpera{at}pharmacol.rochester.edu).
Received 6 November 1998; accepted in final form 15 March 1999.
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