Am J Physiol Cell Physiol 288: C1381–C1389, 2005.
First published January 26, 2005; doi:10.1152/ajpcell.00348.2004.
Inversion of both gating polarity and CO2 sensitivity of voltage gating
with D3N mutation of Cx50
Camillo Peracchia and Lillian L. Peracchia
Department of Pharmacology and Physiology, University of Rochester School of Medicine, Rochester, New York
Submitted 19 July 2004; accepted in final form 23 January 2005
cell communication; lens; gap junctions; chemical gating; channel
gating; Xenopus oocytes
GAP JUNCTIONS are regions of cell contact that mediate the
exchange of small cytosolic molecules via cell-cell channels. A
gap junction channel results from the extracellular interaction
of two hemichannels (connexons), each a hexamer of connexin
proteins. Connexins (Cx) contain four transmembrane domains, two extracellular loops, a cytoplasmic loop, a short NH2
terminus (NT), and a COOH terminus of variable length. The
sequences of cytoplasmic loop and COOH terminus vary
significantly among the members of the connexin family,
whereas those of the other domains are relatively well conserved (see Ref. 14 for review).
Gap junction channels are gated by transjunctional voltage
(Vj; 27) and increased intracellular [Ca2⫹] (10, 25) or [H⫹]i
(28, 33) via molecular mechanisms that are still poorly defined
(see Ref. 12 for review). At least two Vj-sensitive gates have
been identified: fast and slow. On the basis of their behavior at
the single channel level, fast Vj gate and chemical gate are
believed to be distinct: the former closes rapidly (⬍1 ms) but
incompletely, leaving a 20 –30% residual conductance,
whereas the latter closes slowly (8 –10 ms) but completely (5).
In contrast, slow Vj gate and chemical gate are likely to be the
Address for reprint requests and other correspondence: C. Peracchia,
Dept. of Pharmacology and Physiology, Univ. of Rochester School of
Medicine, 601 Elmwood Ave., Rochester, NY 14642-8711 (E-mail:
[email protected]).
http://www.ajpcell.org
same (5, 17, 20). Slow and fast Vj gates are in series, and each
hemichannel appears to have both gates. The slow gate closes
at the negative side of Vj in all connexin channels tested,
whereas the polarity of the fast Vj gating mechanism varies
among connexin channels (see Ref. 8 for review).
In the past, chemical and Vj gating have been studied almost
exclusively by testing chemicals and Vj gradients, respectively,
whereas minimal attention has been devoted to potential effects
of voltage on chemical gating or chemicals on voltage gating.
In the early 1990s, CO2 application was reported to increase
the Vj sensitivity of Cx32 channels (38). More recently chemical gating induced by CO2 was shown to be reversed by Vj
gradients positive at the mutant side of heterotypic channels
between Cx32 and various Cx32 mutants (17, 20), and chemical gating was reversed in insect cells by bilateral hyperpolarization (37). These observations suggest that chemical and
voltage gates may be sensitive to Vj and CO2, respectively.
We have recently reported that the speed and sensitivity of
Vj-dependent inactivation of junctional current (Ij) are increased by CO2 application in both Cx45 (21) and Cx32 (22,
41, 42) channels. Significantly, however, the effect of CO2induced acidification on Vj gating differs among connexin
channels, as CO2 decreases the Vj sensitivity of Cx40 (13),
Cx26 (22, 42), Cx50 (present study), Cx37 (C. Peracchia,
unpublished observations), and Cx38 (38) channels. This suggests that there are two distinct classes of connexin channels
whose Vj gating sensitivity reacts in opposite ways to CO2induced acidification.
As a hypothesis, we have proposed that the way in which the
Vj gating of connexin channels responds to CO2 may be related
to the polarity of Vj gating (13). Indeed, the fast Vj gates close
at the positive side of Vj (positive gaters) in Cx26, -37, -38, -40,
and -50 channels, and at the negative side in Cx32 and Cx45
channels (see Ref. 8 for review). To test this hypothesis, the
present study has monitored the CO2 sensitivity of Vj gating in
Cx50 channels (positive gaters; 4, 8, 35) and in channels made
of a Cx50 mutant (Cx50-D3N) in which the third residue,
aspartate (D), is replaced with asparagine (N). Cx50 is a
connexin expressed in eye lens fibers whose high gating
sensitivity to cytosolic acidification (9, 30, 40) has been proposed to play a role in the function of differentiating fibers (2).
The data show that CO2 decreases the Vj sensitivity of Cx50
channels, and that the point mutation D3N, which neutralizes
the negative charge in third position, reverses both the Vj
gating polarity of Cx50 channels (Cx50-D3N channels are
negative gaters) and their Vj sensitivity to CO2. A preliminary
account of this study has been published in abstract form (15).
The costs of publication of this article were defrayed in part by the payment
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0363-6143/05 $8.00 Copyright © 2005 the American Physiological Society
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Downloaded from http://ajpcell.physiology.org/ by 10.220.32.247 on June 16, 2017
Peracchia, Camillo, and Lillian L. Peracchia. Inversion of both
gating polarity and CO2 sensitivity of voltage gating with D3N
mutation of Cx50. Am J Physiol Cell Physiol 288: C1381–C1389, 2005.
First published January 26, 2005; doi:10.1152/ajpcell.00348.2004.—
The effect of CO2-induced acidification on transjunctional voltage
(Vj) gating was studied by dual voltage-clamp in oocytes expressing
mouse connexin 50 (Cx50) or a Cx50 mutant (Cx50-D3N), in which
the third residue, aspartate (D), was mutated to asparagine (N). This
mutation inverted the gating polarity of Cx50 from positive to negative. CO2 application greatly decreased the Vj sensitivity of Cx50
channels, and increased that of Cx50-D3N channels. CO2 also affected the kinetics of Vj dependent inactivation of junctional current
(Ij), decreasing the gating speed of Cx50 channels and increasing that
of Cx50-D3N channels. In addition, the D3N mutation increased the
CO2 sensitivity of chemical gating such that even CO2 concentrations
as low as 2.5% significantly lowered junctional conductance (Gj).
With Cx50 channels Gj dropped by 78% with a drop in intracellular
pH (pHi) to 6.83, whereas with Cx50-D3N channels Gj dropped by
95% with a drop in pHi to just 7.19. We have previously hypothesized
that the way in which Vj gating reacts to CO2 might be related to
connexin’s gating polarity. This hypothesis is confirmed here by
evidence that the D3N mutation inverts the gating polarity as well as
the effect of CO2 on Vj gating sensitivity and speed.
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CO2-SENSITIVITY OF VOLTAGE GATING
MATERIALS AND METHODS
Oocyte Preparation and Microinjection
AJP-Cell Physiol • VOL
Measurement of Intracellular pH
For testing the effect of different CO2 concentrations on intracellular pH (pHi), oocytes were superfused for 40 min (0.6 ml/min) with
ND96 solutions gassed with 2.5%, 5.0%, 10%, or 30% CO2; these
CO2 concentrations reduce the pH of ND96 from 7.6 to 7.4, 6.85,
6.26, and 5.75, respectively. pHi measurements were performed with
the Dual-Wavelength Fluorescence Imaging and Photometry Systems
(InCyt; Intracellular Imaging, Cincinnati, OH), as previously described (16). The fluorescein derivative pH indicator BCECF (model
B-1151, Molecular Probes, Eugene, OR) was injected into oocytes in
amounts sufficient to reach an intra-oocyte concentration of ⬃100
␮M. After the injection, the oocytes were placed in the same conical
wells used for electrophysiology, modified by replacement of the
plastic floor with a glass coverslip. The conical wells were mounted
on the stage of a Nikon TMS microscope equipped for epifluorescence, and the oocytes were superfused with ND96 gassed with
different CO2 concentrations (see above). Specimen observation and
light measurements were performed with a Nikon Fluor ⫻10 objective. Light from a 300-W xenon arc illuminator passed through a
computer-controlled filter changer and shutter unit, containing 440and 490-nm band-pass filters and liquid light guide. Light emitted by
the oocytes (focusing on their bottom surface, closest to the glass
coverslip), was collected by a cooled charge-coupled device video
camera (model 4922-2010; COHU, San Diego, CA). Pairs of images
at the two wavelengths were collected in rapid succession, and [H⫹]i
was computer calculated online (model GP7-450, Gateway, N. Sioux
City, SD) by dividing the short wavelength by the long wavelength
image, after subtraction of the respective backgrounds. Calibration
curves were generated by rationing droplets of 0.1 M phosphate
buffers (pH 8.0, 7.5, 7.0, 6.5, 6.0) containing 13 ␮M BCECF.
RESULTS
Homotypic Cx50 Channels
Gj sensitivity to 30% CO2. The Gj sensitivity to CO2 of
channels made of Cx50 was tested by applying ⫺60 mV Vj
pulses (12-s duration) at 30-s intervals. With 10-min exposures
to 30% CO2, Gj,pk and Gj,ss dropped from 4.2 ⫾ 1.4 ␮s and
1.8 ⫾ 0.5 ␮s to 0.8 ⫾ 0.2 ␮s and 0.7 ⫾ 0.2 ␮s, respectively
(means ⫾ SE; n ⫽ 14; Fig. 1, A and B), and recovered to near
control values at a similar rate. Exposure to CO2 greatly
influenced both Vj sensitivity and Ij inactivation kinetics. Vj
sensitivity decreased dramatically, as Gj,ss/Gj,pk increased by
⬃78% (n ⫽ 14; Fig. 1B). Gj,pk, Gj,ss and Gj,ss/Gj,pk changed
with similar time course. The progressive change in Vj sensi-
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Oocytes were prepared as previously described (18). Briefly, adult
female Xenopus laevis frogs were anesthetized with 0.3% tricaine
(MS-222) and the oocytes were surgically removed from the abdominal incision. The oocytes were placed in ND96 medium containing
(in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES (pH 7.6
with NaOH). Oocytes at stage V or VI were subsequently defolliculated in 2 mg/ml collagenase (Sigma, St. Louis, MO) for 80 min at
room temperature in nominally Ca2⫹-free OR2 solution containing (in
mM) 82.5 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES (pH 7.6 with NaOH).
The defolliculated oocytes were injected with 46 nl (0.25 ␮g/␮l) of
antisense oligonucleotide complementary to endogenous Xenopus
Cx38: 5⬘-GCTTTAGTAATTCCCATCCTGCCATGTTTC-3⬘ (commencing at nt ⫺5 of Cx38 cDNA sequence; Ref. 3) with a Nanoject
apparatus (Drummond, Broomall, PA). The antisense oligonucleotide
blocks completely the endogenous junctional communication within
48 h. From 24 to 72 h later, 46 nl of mouse Cx50 wild-type cRNA
(⬃0.4 ␮g/␮l), or cRNA of a mouse Cx50 mutant in which the
aspartate residue in position 3 was replaced with asparagine (Cx50D3N), were injected into oocytes at the vegetal pole and the oocytes
were incubated overnight at 18°C. The oocytes were mechanically
stripped of their vitelline layer in hypertonic medium (18) and paired
at the vegetal poles in conical wells of culture dishes (Falcon Products, Becton Dickinson Labware, Franklin Lakes, NJ) filled with
ND96. All oocyte pairs were studied electrophysiologically 2–3 h
after being paired.
Measurement of junctional conductance and uncoupling protocols.
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. All of the experiments
were performed using the standard double voltage-clamp procedure
for measuring Gj (27). After the insertion of a current and a voltage
microelectrode in each oocyte, both oocytes were individually
clamped by two oocyte clamp amplifiers (model OC-725C, Warner
Instrument, Hamden, CT) to the same holding potential, Vm1 ⫽ Vm2
(⫺20 mV), so that no junctional current would flow at rest (Ij ⫽ 0).
For measuring junctional conductance (Gj) and CO2 sensitivity, a Vj
gradient was created by imposing a voltage step (V1) to oocyte 1 while
maintaining V2 at Vm; thus Vj ⫽ V1. The negative feedback current
(I2), injected by the clamp amplifier in oocyte 2 for maintaining V2
constant at Vm, was used for calculating Gj, as it is identical in
magnitude to Ij but of opposite sign (Ij ⫽ ⫺I2); Gj ⫽ Ij/Vj. Pulse
generation and data acquisition were performed with pCLAMP version 8.2.0.232 software (Axon Instruments, Foster City, CA) and a
DigiData 1322A interface (Axon). Ij and Vj were measured with
CLAMPfit (Axon), and the data were plotted with SigmaPlot (SPSS,
Chicago, IL).
To test the effect of CO2 on Gj and Vj sensitivity, oocyte pairs were
superfused for 10 – 80 min (0.6 ml/min) with ND96 solutions gassed
with 2.5%, 5.0%, 10%, or 30% CO2, and Gj was measured by
applying voltage steps of ⫺60 mV (12-s duration) every 30 s to one
oocyte, while maintaining the other oocyte at Vm. Gj peak (Gj,pk), Gj
steady state (Gj,ss), and the ratio Gj,ss/Gj,pk were calculated in the
presence and absence of CO2. For comparing Cx50 and Cx50-D3N
channels in terms of Gj sensitivity to pHi, oocyte pairs were superfused for 30 – 40 min (0.6 ml/min) with ND96 solutions gassed with
2.5%, 5.0%, 10%, or 30% CO2 and Gj was measured by applying
voltage steps of ⫹20 mV (2 s duration) every 30 s to one oocyte while
maintaining the other oocyte at Vm.
For studying voltage dependence of Gj in the presence and absence
of CO2, a standard Vj protocol was used. Each oocyte was first voltage
clamped at ⫺20 mV. Voltage steps of ⫺10 mV (100 mV Vj maximum) and 25-s duration were applied every 45 s to either oocyte of
the pair, while maintaining the other at ⫺20 mV. The voltageinsensitive junctional conductance (Gj,max) was calculated using Ij
values elicited by Vj ⫽ ⫺10 mV because at this Vj there is no Ij decay.
To illustrate the relationship between Gj,ss and Vj, the ratio Gj,ss/Gj,max
was plotted with respect to Vj. The curve was fitted to a two-state
Boltzmann distribution of the form: (Gj,ss ⫺ Gj,min)/(Gjmax ⫺ Gj,ss) ⫽
exp[⫺A(Vj ⫺ V0)], where V0 is the Vj value at which Gj is one-half of
the value of Gj,max ⫺ Gj,min, Gj,max is Gj at Vj ⫽ 0 mV and Gj,min is
the theoretical minimum normalized Gj. A ⫽ ␩q/kT is a constant
expressing voltage sensitivity in terms of number of equivalent gating
charges, ␩, moving through the entire applied field, where q is the
electron charge, k is the Boltzmann constant, and T is the temperature
in Kelvin. The time constants (␶) of Ij decay at Vj ⫽ 100 mV, for Cx50
channels, and Vj ⫽ 50 mV, for Cx50-D3N channels, were calculated
by fitting each Ij curve to a two-term exponential function (␶1 and ␶2),
following baseline correction (CLAMPfit, Axon). Gj,ss was obtained
from the exponential fit (parameter “C” of CLAMPfit, Axon).
C1383
CO2-SENSITIVITY OF VOLTAGE GATING
tivity is clearly seen in the continuous Ij record displayed in
Fig. 1A. Exposure to either 5% or 10% CO2 did not significantly affect Gj or Vj sensitivity (data not shown).
Effect of CO2 on Vj sensitivity. To test the effect of CO2 on
Vj sensitivity, the Vj protocol was applied before (Fig. 2B) and
during (Fig. 2C) exposure to 30% CO2. Steady-state conditions
were reached after 15–20 min of CO2 superfusion (Fig. 2A).
After that, the CO2 superfusion was continued for as long as
45– 60 min, during which time the oocytes were retested with
the Vj protocol. At steady-state conditions, in 30% CO2, the
channels displayed a large decrease in Vj sensitivity (Fig. 2, C
and D) with respect to controls (absence of CO2; Fig. 2, B and
D). In plots of the relationship between Gj,ss/Gj,max and Vj (Fig.
2D), the Boltzmann values were the following: V0 ⫽ 24.53
mV, ␩ ⫽ 3.3 and Gj,min ⫽ 0.15, in the absence of CO2 (n ⫽
11), and V0 ⫽ 72.4 mV, ␩ ⫽ 1.4 and Gj,min ⫽ 0.32, in the
presence of CO2 (n ⫽ 7; see Table 1). Although the 30% CO2
data did not reach an asymptote, the degree of confidence was
⬎0.98 (␹2 ⫽ 0.00019 and 0.00054, for negative and positive
Vj, respectively). In Fig. 2D, the fitted function for control
conditions departs somewhat from the data points at Vj ⬎40
mV. This is also observed in other Vj records of Cx50 (4, 29,
40) and in those of Cx40 (1, 13) and Cx38 (36). The voltage
sensitivity under control condition is slightly lower than that
reported by an earlier study on Cx50 expressed in oocytes (39),
but higher than those reported for Cx50 expressed in N2A cells
(29, 40). Vj sensitivity returned to control values after prolonged CO2 washout (data not shown). The mean values of
AJP-Cell Physiol • VOL
Gj,pk and Gj,ss decreased slightly during the 30- to 60-min
period of exposure to 30% CO2 during which the Vj sensitivity
was tested by the Vj protocol, but the changes were not
statistically significant; Gj,pk decreased from 1.77 ⫾ 0.68 ␮s to
1.41 ⫾ 0.56 ␮s (means ⫾ SE; 20.3% drop; n ⫽ 9; P ⫽ 0.41)
and Gj,ss from 1.26 ⫾ 0.42 ␮s to 1.03 ⫾ 0.37 ␮s (means ⫾ SE;
18.3% drop; n ⫽ 9; P ⫽ 0.41); Gj,ss/Gj,pk remained virtually
unchanged (0.71 and 0.73, respectively).
Exposure to 30% CO2 also decreased the speed of Ij inactivation, as the time constants (␶1 and ␶2) of Ij decay at Vj ⫽ ⫾
100 mV increased from 6.7 ⫾ 0.4 and 0.68 ⫾ 0.03 s (means ⫾
SE; n ⫽ 20) to 12.1 ⫾ 1.9 and 1.1 ⫾ 0.1 s (means ⫾ SE; n ⫽
11; P ⫽ 0.0007 and 0.0009, respectively), corresponding to
81% and 63% increase, respectively (Fig. 2C, inset).
Heterotypic Cx50/Cx50-D3N Channels
Heterotypic channels were generated by pairing oocytes
expressing Cx50 with oocytes expressing Cx50-D3N. These
channels displayed an asymmetrical Vj behavior consistent
with a reversal of Vj polarity resulting from the D3N mutation
(neutralization of the negative charge in position 3). With Cx50
hemichannels at the positive side of Vj, the magnitude of Ij
inactivation increased progressively with increasing Vj gradients (Fig. 3A, bottom traces), whereas with Cx50 at the negative side of Vj, Ij inactivation was negligible (Fig. 3A, top
traces). This indicates that the D3N mutation has switched the
Vj gating polarity from positive (Cx50) to negative (Cx50-
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Fig. 1. Sensitivity of junctional conductance
(Gj) and transjunctional voltage (Vj) gating to
30% CO2 in homotypic connexin 50 (Cx50)
channels. Vj pulses of ⫺60 mV, 12-s duration, were applied at 30-s intervals. A: the
progressive changes in Gj and Vj sensitivity
are obvious in the continuous junctional current (Ij) record. Inset: note the reversible
changes in Ij inactivation. B: with 10-min
exposures to 30% CO2, peak Gj (Gj,pk) and
steady-state Gj (Gj,ss) decrease reversibly by
82.4% and 62.7%, respectively (n ⫽ 14). The
rates of uncoupling and recovery are similar.
Gj,ss/Gj,pk increases by 62% (n ⫽ 14), indicating that Vj sensitivity is greatly decreased
by CO2 application.
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CO2-SENSITIVITY OF VOLTAGE GATING
D3N), such that with Cx50 at the positive side of Vj the gates
of both Cx50 and Cx50-D3N hemichannels are activated,
whereas with Cx50 at the negative side of Vj neither hemichannel’s gate is activated. In plots of the relationship between
Gj,ss/Gj,max and Vj, with Cx50-D3N at the negative side of Vj
(Fig. 3B, right), the Boltzmann values were: V0 ⫽ 43.2 mV,
␩ ⫽ 2.82, and Gj,min ⫽ 0.0035 (n ⫽ 11) (see Table 1). Note
that Gj,min is nearly zero, which contrasts with the behavior of
homotypic Cx50 channels (Fig. 2D).
Homotypic Cx50-D3N Channels
Gj sensitivity to 5% CO2. The D3N mutation rendered
Cx50-D3N channels considerably more sensitive to CO2 than
wild-type Cx50 channels, such that even CO2 concentrations as
Table 1. Boltzmann parameters of voltage gating in
presence and absence of CO2
Cx50/Cx50
Cx50/Cx50 (30% CO2)
Cx50/Cx50-D3N (Cx50-D3N negative)
Cx50-D3N/Cx50-D3N
Cx50-D3N/Cx50-D3N (2.5% CO2)
n
V0, mV
␩
Gj, min
11
7
11
14
12
24.53
72.4
43.2
46.09
36.15
3.3
1.4
2.82
1.83
2.04
0.15
0.32
0.0035
0.0085
0.02
Values are means ⫾ SE; n, no. of experiments. Cx50, connexin 50; D3N,
residue 3: aspartate (D), mutates to asparagine (N); V0, Vj at which Gj is one
half of the value of Gj,max ⫺ Gj ,min; ␩, number of equivalent gating charges;
Gj,min, theoretical minimum normalized junctional conductance.
AJP-Cell Physiol • VOL
low as 2.5% were sufficient to significantly affect Gj and Vj
sensitivity. With 10 min, exposures to 5% CO2, Gj,pk and Gj,ss
dropped from 1.7 ⫾ 0.6 and 0.7 ⫾ 0.2 ␮s to 0.45 ⫾ 0.15 and
0.09 ⫾ 0.02 ␮s, respectively (means ⫾ SE; n ⫽ 6; Fig. 4, A
and B), and recovered to near control values at a slower rate.
The CO2 exposure increased significantly the Vj sensitivity
because Gj,ss/Gj,pk decreased by ⬃48% (n ⫽ 6; Fig. 4B). The
time courses of Gj,pk and Gj,ss/Gj,pk are well matched (Fig. 4B),
as seen with homotypic Cx50 channels as well (Figs. 1B and
2A). The progressive change in Vj sensitivity is clearly visible
in the continuous Ij record shown in Fig. 4A.
Effect of CO2 on Vj sensitivity. The effect of CO2 on the Vj
sensitivity of Cx50-D3N channels was tested by applying the
Vj protocol before (Fig. 5B) and during (Fig. 5C) exposure to
2.5% CO2. Steady-state conditions were reached after 15–20
min of CO2 superfusion (Fig. 5A). After that time, the CO2
superfusion was continued for as long as 45– 60 min and the
oocytes were retested with the Vj protocol while they were
maintained in 2.5% CO2. At steady-state conditions, in 2.5%
CO2, the channels displayed a significant increase in Vj sensitivity (Fig. 5, C and D) with respect to controls (absence of
CO2; Fig. 5, B and D). In plots of the relationship between
Gj,ss/Gj,max and Vj (Fig. 5D), the Boltzmann values were the
following: V0 ⫽ 46.09 mV, ␩ ⫽ 1.83 and Gj,min ⫽ 0.0085, in
the absence of CO2 (n ⫽ 14), and V0 ⫽ 36.15 mV, ␩ ⫽ 2.04
and Gj,min ⫽ 0.02, in the presence of CO2 (n ⫽ 12) (see Table
1); the ⬃10 mV shift in V0 (Fig. 5D) is statistically significant
(P ⫽ 0.0002 and 0.003, for negative and positive Vj, respec-
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Fig. 2. Effect of 30% CO2 on the Vj sensitivity of homotypic Cx50 channels. The standard Vj protocol (Vj steps of ⫺10 mV (100
mV max, and 25-s duration) applied every
45 s) was tested before (B) and during (C)
exposure to 30% CO2. Steady-state conditions of partial uncoupling are reached within
15–20 min of CO2 superfusion (A). Exposure
to 30% CO2 drastically decreases the Vj sensitivity (C and D) with respect to controls (B
and D). In plots relating Gj,ss/Gj,max to Vj (D),
the Boltzmann values are the following: Vj at
which Gj is one half of the value of Gj ,max ⫺
Gj ,min (V0) ⫽ 24.53 mV, number of equivalent gating charges (␩) ⫽ 3.3, and minimum
Gj (Gj,min) ⫽ 0.15, in the absence of CO2
(n ⫽ 11), and V0 ⫽ 72.4 mV, ␩ ⫽ 1.4 and
Gj,min ⫽ 0.32, in the presence of CO2 (n ⫽ 7;
see Table 1). Exposure to 30% CO2 also
decreases the speed of Ij inactivation. The
decrease in both Vj sensitivity and gating
speed can be seen in C (inset), which shows
the time course of Ij at Vj ⫽ 100 mV both
before (control) and during CO2 exposure
(30% CO2). The time constants (␶1 and ␶2) of
Ij decay at Vj ⫽ ⫾ 100 mV increased by 81%
and 63%, respectively, with CO2 application.
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CO2-SENSITIVITY OF VOLTAGE GATING
tively). Vj sensitivity returned to control values after prolonged
CO2 washout (data not shown). The mean values of Gj,pk and
Gj,ss decreased slightly during the 30 – 60 min period of exposure to 2.5% CO2 during which the Vj sensitivity was tested by
the Vj protocol, but the changes were not statistically significant; Gj,pk decreased from 2.28 ⫾ 1.02 to 2.02 ⫾ 0.91 ␮s
(means ⫾ SE; 11.4% drop; n ⫽ 6; P ⫽ 0.188) and Gj,ss from
0.86 ⫾ 0.37 to 0.73 ⫾ 0.33 ␮s (means ⫾ SE; 15% drop; n ⫽
6; P ⫽ 0.26); Gj,ss/Gj,pk remained virtually unchanged (0.38
and 0.36, respectively).
Exposure to 2.5% CO2 increased the speed of Ij inactivation.
The slow time constant (␶1), measured at Vj ⫽ ⫾ 50 mV,
decreased from 9.5 ⫾ 1.0 s (n ⫽ 8) to 5.6 ⫾ 0.9 s (means ⫾
SE; n ⫽ 11; P ⫽ 0.01), corresponding to a 41% drop (Fig. 5C,
inset). In contrast, ␶2 did not change significantly, being 1.2 ⫾
0.2 s (means ⫾ SE; n ⫽ 8) under control conditions and 1.4 ⫾
0.1 s (means ⫾ SE; n ⫽ 11) in 2.5 CO2. Note that Gj,min is
nearly zero both in the presence and absence of CO2. In this,
homotypic Cx50-D3N channels behave like heterotypic Cx50/
Cx50-D3N channels (Fig. 3B), but differ from homotypic Cx50
channels (Fig. 2D).
Gj Sensitivity of Cx50 and Cx50-D3N Channels to pHi
To test the effect of different CO2 concentrations on pHi,
oocytes were superfused for 40 min with ND96 solutions
Fig. 4. Sensitivity of Gj and Vj gating to 5%
CO2 in homotypic Cx50-D3N channels. Vj
pulses of ⫺60 mV, 12-s duration, were applied
at 30-s intervals. A: changes in Gj and Vj
sensitivity are clearly seen in the continuous Ij
record. Inset: note the reversible changes in Ij
inactivation. B: with 10-min exposures to 5%
CO2, Gj,pk and Gj,ss decrease reversibly by
73.4% and 86.9%, respectively (n ⫽ 6). The
rate of recovery is slower than that of uncoupling. Gj,ss/Gj decreases by 46.5% (n ⫽ 6; B),
indicating that Vj sensitivity is significantly
increased by CO2 application.
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Fig. 3. Vj sensitivity of heterotypic Cx50/Cx50-residue 3 aspartate (D) mutated to asparagine (N) (D3N). With Cx50 at the positive side of Vj, the Ij inactivation
increases progressively with Vj (A, bottom traces), whereas with Cx50 at the negative side of Vj, Ij inactivation is negligible (A, top traces). This asymmetrical
Vj behavior, consistent with the inversion of Vj polarity of Cx50-D3N channels from positive to negative, is clearly apparent in the relationship between Vj and
normalized Gj (Gj,ss/Gj,max), plotted with the Vj sign relative to the Cx50 hemichannel (B). The Boltzmann values for Cx50-D3N at the negative side of Vj are
the following: V0 ⫽ 43.2 mV, ␩ ⫽ 2.82, and Gj,min ⫽ 0.0035 (n ⫽ 11) (see Table 1). Note that Gj,min is nearly zero, indicating that at high Vj all of the channels
are closed.
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CO2-SENSITIVITY OF VOLTAGE GATING
gassed with 2.5%, 5.0%, 10%, or 30% CO2 (Fig. 6A). Steady
state was reached after 15–20 min of CO2 superfusion (Fig.
6A). pHi reversibly dropped from control values of 7.73 ⫾ 0.05
(means ⫾ SE, n ⫽ 14) to 7.37 ⫾ 0.02 (means ⫾ SE; n ⫽ 4),
7.19 ⫾ 0.06 (means ⫾ SE; n ⫽ 2), 6.98 ⫾ 0.07 (means ⫾ SE;
n ⫽ 3), and 6.83 ⫾ 0.03 (mean ⫾ SE; n ⫽ 5), respectively
(Fig. 6A). The mean pHi of 7.73 measured under control
conditions in these experiments is close to that previously
reported by us (pHi ⫽ 7.63; Ref. 17) and Sasaki et al. (pHi ⫽
7.69; Ref. 26). At control conditions, the pHi of oocytes
perfused with ND96 solutions (pH 7.6) varied among batches
taken from different Xenopus females, as reported by Cicirelli
et al. (7).
The Gj of Cx50-D3N channels was significantly more sensitive to lowered pHi than that of Cx50 channels (Fig. 6B).
After 30- to 40-min superfusion of ND96 gassed with different
Fig. 6. Effect of different CO2 concentrations on intracellular pH (pHi) (A) and Gj (B). With 40-min superfusion of ND96 solutions gassed with 2.5%, 5.0%,
10% or 30% CO2, pHi reversibly dropped from 7.73 ⫾ 0.05 (means ⫾ SE; n ⫽ 14) to 7.37 ⫾ 0.02 (means ⫾ SE; n ⫽ 4), 7.19 ⫾ 0.06 (means ⫾ SE; n ⫽ 2),
6.98 ⫾ 0.07 (means ⫾ SE; n ⫽ 3), and 6.83 ⫾ 0.03 (means ⫾ SE; n ⫽ 5), respectively (A). Steady state was reached after 15–20 min of CO2 superfusion (A).
Cx50-D3N channels were much more sensitive to lowered pHi than Cx50 channels (B). Gj was measured by applying voltage steps of ⫹20 mV (2-s duration,
30-s intervals) to one oocyte, while maintaining the other oocyte at Vm. After 30 – 40 min superfusions of ND96 gassed with different CO2 concentrations, the
Gj of Cx50 channels dropped to 22.2 ⫾ 4.4% (means ⫾ SE; n ⫽ 12) and 81.8 ⫾ 2.1% (means ⫾ SE; n ⫽ 5) of control values with 30% and 10% CO2,
respectively, whereas CO2 concentrations of 5% (n ⫽ 3) and 2.5% (n ⫽ 2) had no effect of Gj (B). In contrast, in Cx50-D3N channels Gj dropped to 4.8 ⫾ 1.2%
(means ⫾ SE; n ⫽ 5) and 44.9 ⫾ 5.2% (means ⫾ SE; n ⫽ 6) of control values with 5% and 2.5% CO2, respectively, whereas CO2 concentrations of 10% (n ⫽
3) and 30% (n ⫽ 2) caused complete electrical uncoupling (B).
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Fig. 5. Effect of 2.5% CO2 on the Vj sensitivity of homotypic Cx50-D3N channels.
The standard Vj protocol [Vj steps of ⫺10
mV (100 mV max, and 25-s duration) applied every 45 s] was tested before (B) and
during (C) exposure to 2.5% CO2. Steadystate conditions of partial uncoupling are
reached within 15–20 min of CO2 superfusion (A). Exposure to 2.5% CO2 significantly
increases the Vj sensitivity (C and D), with
respect to controls (B and D). In plots relating Gj,ss/Gj,max to Vj (D), the Boltzmann
values are the following: V0 ⫽ 46.09 mV,
␩ ⫽ 1.83 and Gj,min ⫽ 0.0085, in the absence
of CO2 (n ⫽ 14), and V0 ⫽ 36.15 mV, ␩ ⫽
2.04 and Gj,min ⫽ 0.02, in the presence of
CO2 (n ⫽ 12) (see Table 1); the ⬃10 mV
shift in V0 (D) is statistically significant (P ⫽
0.0002 and 0.003, for negative and positive
Vj, respectively). Exposure to 2.5% CO2 also
increases the speed of Ij inactivation. C,
inset: the increase in both Vj sensitivity and
gating speed is exhibited, which shows the
time course of Ij at Vj ⫽ 50 mV both before
(control) and during CO2 exposure (2.5%
CO2).
CO2-SENSITIVITY OF VOLTAGE GATING
CO2 concentrations, Gj of Cx50 channels dropped to 22.2 ⫾
4.4% (means ⫾ SE; n ⫽ 12) and 81.8 ⫾ 2.1% (means ⫾ SE;
n ⫽ 5) of control values with 30% and 10% CO2, respectively,
whereas CO2 concentrations of 5% (n ⫽ 3) and 2.5% (n ⫽ 2)
had no effect of Gj (Fig. 6B). In contrast, in Cx50-D3N
channels Gj dropped to 4.8 ⫾ 1.2% (means ⫾ SE; n ⫽ 5) and
44.9 ⫾ 5.2% (means ⫾ SE; n ⫽ 6) of control values with 5%
and 2.5% CO2, respectively, whereas CO2 concentrations of
10% (n ⫽ 3) and 30% (n ⫽ 2) caused complete cell-cell
uncoupling (Fig. 6B).
DISCUSSION
AJP-Cell Physiol • VOL
as low as 5%, corresponding to a drop in pHi to just 7.19.
Because longer exposures to 5% CO2 would have closed all of
the Cx50-D3N channels, a concentration as low as 2.5% CO2
was needed to enable us to reach steady-state conditions and
yet have a sufficient number of operational channels for measuring the effect of CO2 on Vj sensitivity. This is the reason
why the effect of CO2 appears less striking with Cx50-D3N
channels than with Cx50 channels. Very likely, a higher CO2
concentration might have had greater effects on both Vj sensitivity and gating speed, if it had been possible to test it.
The high CO2 sensitivity of Cx50-D3N channels may make
them the most sensitive connexin channels yet known. Indeed,
at sea level (PB ⫽ 760 torr, gas saturated with water vapor at
37°C) 5% and 2.5% CO2 correspond to PCO2 values of 36 and
18 torr, respectively. Because cells of average human tissue
operate at PCO2 values of ⬃48 torr (PCO2 of mixed venous
blood), if this connexin mutant were present in a living organism, cells expressing Cx50-D3N would be expected to be
constantly in uncoupled state. The reason for this high CO2
sensitivity is unclear, but it might relate to the loss of the
negative charge in position 3 resulting from D/N mutation.
This also points to the extreme relevance of NH2 terminus
domain to chemical gating. Significantly, this domain is likely
to be a calmodulin (CaM) binding site, and a model of
chemical gating that envisions a direct CaM-Cx interaction has
been proposed (for reviews, see Refs. 12 and 19). In Cx32, this
NH2 terminus domain has been found to bind CaM in Ca2⫹dependent way with a Kd of 27 nM (32). Because CaM
interacts best with basic amphiphilic helical domains, perhaps
the removal of a negative charge from this site enhances its
CaM binding efficiency.
Evidence that the D3N mutation inverts the gating polarity
of Cx50 from positive to negative confirms similar data on
Cx26 (D2N mutation; 11, 42). Significantly, in Cx32 the
reverse mutation (Cx32-N2D) inverts the polarity of Cx32
from negative to positive (11, 23, 34, 42). The data on Cx50
and Cx50-D3N are consistent with the hypothesis that the
manner in which Vj gating responds to CO2 may be related to
the gating polarity of the connexin (13). Recently, this hypothesis has also been strengthened by evidence that Cx32-N2D
mutant channels (positive gaters) decrease in Vj gating sensitivity during CO2 application (42).
The D3N mutation in Cx50 enables the channels to close
completely at high voltages: with Vj ⫽ 100 mV, Gj,min is
virtually zero in both heterotypic Cx50/Cx50-D3N and homotypic Cx50-D3N channels. This is interesting, because gating
to full channel closure is believed to reflect the behavior of the
slow Vj gate (5, 17, 20). The function of the slow Vj gate is
usually hidden, in the absence of chemical uncouplers, in all
connexin channels except in Cx45 channels (6), but it manifests itself in various disparate channels made of connexin
mutants (17, 20, 23, 31). This suggests that the D3N mutation
of Cx50 may unmask or unlatch the slow Vj gate. Of course,
the potential effect of the D3N mutations on slow gating could
only be properly evaluated at the single channel level.
The reason why CO2 decreases the Vj sensitivity of positive
gaters, such as Cx26 (42), Cx40 (13), Cx37 (C. Peracchia,
unpublished observations), Cx38 (38), Cx50 (this study) and
Cx32-N2D (42), and increases that of negative gaters, such as
Cx32 (42), Cx45 (21), and Cx50-D3N (this study), is unclear.
The molecular basis of gating polarity is also only partly
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This study describes the effect of CO2 on chemical and Vj
gating sensitivities of channels made of Cx50 wild-type or a
Cx50 mutant (Cx50-D3N) in which the aspartate (D) in position 3 was mutated to asparagine (N). The data show that this
mutation inverts the Vj gating polarity of Cx50 from positive
(4, 8, 35) to negative and that CO2 decreases both Vj gating
sensitivity and inactivation speed of Cx50 channels but increases those of Cx50-D3N channels. Evidence from this study
strengthen the hypothesis that the response of Vj gating sensitivity to CO2-induced cytosolic acidification may be related to
the Vj gating polarity of the connexin (13).
During CO2 superfusion, the time course of the Gj,ss/Gj,pk
ratio matched closely that of Gj,pk in both Cx50 wild-type and
Cx50-D3N channels. Because the drop in Gj,pk reflects the
number of channels closed by CO2 (chemical gating), a possible interpretation is that the changes in Vj sensitivity are
mechanistically related to chemical gating. Perhaps there is an
interplay between the chemical (slow) gate and the Vj sensor of
the fast Vj gate.
In both Cx50 and Cx50-D3N channels, the Vj-dependent Ij
decay is best fit by a two-term exponential function, suggesting
the presence of two gating components. In Cx50 channels both
time constants (␶1 and ␶2) are CO2 sensitive as they increase
significantly with CO2, indicating that CO2 reduces the speed
as well as the sensitivity of Cx50 Vj gating. In contrast, in
Cx50-D3N channels ␶1 is significantly decreased by CO2,
indicating that both the speed and sensitivity of Vj gating are
increased by CO2.
The Boltzmann fits correspond well to the data points in Vj
records of Cx50-D3N channels (Figs. 3B and 5D), but diverge
somewhat at Vj gradients higher than ⫾ 40 mV in control
records of Cx50 channels (Fig. 2D). This divergence is also
seen in other records of Cx50 channels (4, 29, 40) and in those
of Cx40 (1, 13) and Cx38 (36) channels, but not in records of
Cx32 (36) and Cx45 (21) channels. Because Cx50, Cx40, and
Cx38 channels are positive gaters, whereas Cx32, Cx45, and
Cx50-D3N channels are negative gaters, this phenomenon may
also be related to gating polarity. Perhaps it involves competition vs. cooperation between fast and slow Vj gates, as in
positive gaters; fast and slow Vj gates are believed to operate at
opposite ends of the channel, whereas in negative gaters they
are likely to operate at the same end of the channel.
As previously reported (9, 30, 40), Cx50 channels are among
the most CO2-sensitive connexin channels tested. Indeed, Gj
drops by as much as 78% with exposures to just 30% CO2,
corresponding to a drop in pHi to 6.83. However, even more
striking is the CO2 sensitivity of the Cx50-D3N mutant channels, as Gj drops by as much as 95% with CO2 concentrations
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CO2-SENSITIVITY OF VOLTAGE GATING
ACKNOWLEDGMENTS
The authors thank Joey T. Chen for excellent technical assistance.
GRANTS
This study was supported by National Institute of General Medical Sciences
Grant GM-20113.
REFERENCES
1. Anumonwo JMB, Taffet SM, Gu H, Chanson M, Moreno AP, and
Delmar M. The carboxyl terminal domain regulates the unitary conductance and voltage dependence of connexin40 gap junction channels. Circ
Res 88: 666 – 673, 2001.
AJP-Cell Physiol • VOL
2. Baldo GJ, Gong X, Martinez-Wittinghan FJ, Kumar NM, Gilula NB,
and Mathias RT. Gap junctional coupling in the lens from ␣8 connexin
knockout mice. J Gen Physiol 118: 447– 456, 2001.
3. Barrio LC, Suchyna T, Bargiello T, Xu LX, Roginski RS, Bennett
MVL, and Nicholson BJ. Gap junctions formed by connexins 26 and 32
alone and in combination are differently affected by applied voltage. Proc
Natl Acad Sci USA 88: 8410 – 8414, 1991.
4. Beahm DL and Hall JE. Hemichannel and junctional properties of
connexin 50. Biophys J 82: 2016 –2031, 2004.
5. Bukauskas FF and Peracchia C. Two distinct gating mechanisms in gap
junction channels: CO2-sensitive and voltage-sensitive. Biophys J 72:
2137–2142, 1997.
6. Bukauskas FF, Bukauskiene A, Verselis VK, and Bennett MVL.
Coupling asymmetry of heterotypic connexin 45/connexin 43-EGFP gap
junctions: Properties of fast and slow gating mechanisms. Proc Natl Acad
Sci USA 99: 7113–7118, 2002.
7. Cicirelli MF, Robinson KR, and Smith LD. Internal pH of oocytes: a
study of the mechanism and role of pH changes during maturation. Dev
Biol 100: 133–146, 1983.
8. Harris AL. Emerging issues of connexin channels: biophysics fills the
gap. Q Rev Biophys 34: 325– 472, 2001.
9. Lin JS, Eckert R, Kistler J, and Donaldson P. Spatial differences in gap
junction gating in the lens are a consequence of connexin cleavage. Eur
J Cell Biol 76: 246 –250, 1998.
10. Loewenstein WR. Permeability of membrane junctions. Ann NY Acad Sci
137: 441– 472, 1966.
11. Oh S, Abrams CK, Verselis VK, and Bargiello TA. Stoichiometry of
transjunctional voltage-gating polarity reversal by a negative charge substitution in the amino terminus of a connexin32 chimera. J Gen Physiol
116: 13–31, 2000.
12. Peracchia C. Chemical gating of gap junction channels. Roles of calcium,
pH and calmodulin. Biochim Biophys Acta 1662: 61– 80, 2004.
13. Peracchia C, Chen JT, and Peracchia LL. CO2 sensitivity of voltage
gating and gating polarity of gap junction channels– connexin40 and its
COOH-terminus truncated mutant. J Membr Biol 200: 105–113, 2004.
14. Peracchia C, Lazrak A, and Peracchia LL. Molecular models of
channel interaction and gating in gap junctions. In: Handbook of Membrane Channels–Molecular and Cellular Physiology, edited by Peracchia
C. San Diego, CA: Academic, 1994, p. 361–377.
15. Peracchia C and Peracchia LL. Gating polarity determines the response
of voltage gating to CO2 in gap junction channels made of Cx50 or its
D3N mutant (Abstract). Mol Biol Cell 15: 267a, 2004.
16. Peracchia C, Wang XG, Li LQ, and Peracchia LL. Inhibition of
calmodulin expression prevents low-pH-induced gap junction uncoupling
in Xenopus oocytes. Pflügers Arch 431: 379 –387, 1996.
17. Peracchia C, Wang XG, and Peracchia LL. Is the chemical gate of
connexins voltage sensitive? Behavior of Cx32 wild-type and mutant
channels. Am J Physiol Cell Physiol 276: C1361–C1373, 1999.
18. Peracchia C, Wang XG, and Peracchia LL. Chemical gating of gap
junction channels. Methods 20:188 –195, 2000.
19. Peracchia C, Wang XG, and Peracchia LL. Behavior of chemical- and
slow voltage-sensitive gating of connexin channels: the “cork” gating
hypothesis. In: Gap Junctions–Molecular Basis of Cell Communication in
Health and Disease, edited by Peracchia C. San Diego, CA: Academic,
2000, p. 271–295.
20. Peracchia C, Wang XG, and Peracchia LL. Slow gating of gap junction
channels and calmodulin. J Membr Biol 78: 55–70, 2000.
21. Peracchia C, Young KC, Wang XG, and Peracchia LL. Is the voltage
gate of connexins CO2-sensitive? Cx45 channels and inhibition of calmodulin expression. J Membr Biol 195: 53– 62, 2003.
22. Peracchia Young C, KC, Wang XG, Chen JT, and Peracchia LL. The
voltage gates of connexin channels are sensitive to CO2. Cell Comm Adhes
10: 233–237, 2003.
23. Purnick PEM, Oh SH, Abrams CK, Verselis VK, and Bargiello TA.
Reversal of the gating polarity of gap junctions by negative charge
substitutions in the N-terminus of connexin 32. Biophys J 79: 2403–2415,
2000.
24. Purnick PEM, Benjamin DC, Verselis VK, Bargiello TA, and Dowd
TL. Structure of the amino terminus of a gap junction protein. Arch
Biochem Biophys 381: 181–190, 1975.
25. Rose B and Loewenstein WR. Permeability of cell junction depends on
local cytoplasmic calcium activity. Nature 254: 250 –252, 2000.
288 • JUNE 2005 •
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Downloaded from http://ajpcell.physiology.org/ by 10.220.32.247 on June 16, 2017
understood, but there is good evidence that charged residues at
the initial segment of NT play a role (34). A standing hypothesis envisions the NT domain as the voltage sensor, located
within the channel’s mouth (11, 23, 24, 34). Positive and
negative gaters would have an acidic or a basic residue at the
initial sequence of NT, respectively. With the establishment of
a Vj gradient, the NT of positive gaters would move away from
the channel’s mouth (toward the cytoplasm) at the positive side
of Vj, enabling channel gating by another connexin domain or
an accessory molecule. Conversely, in negative gaters NT
would move away from the channel’s mouth at the negative
side of Vj. Positive gaters would be so by virtue of the acidic
residue in the third position (D3), which is absent in negative
gaters, except in Cx43.
With CO2-induced cytosolic acidification, histidine residues
are likely to be protonated, resulting in the addition of positive
charges to relevant domains of connexins and/or accessory
proteins. A possibility is that protonated histidines affect the
function of the Vj sensor in opposite ways depending on the
gating polarity of the connexin. However, preliminary experiments with the histidine reagent diethylpyrocarbonate (DEPC)
do not support this hypothesis, as 20-min applications of 0.5–1
mM DEPC (pH 6.0) to oocytes expressing Cx50 channels
altered neither the CO2 sensitivity of Vj gating nor the effect of
CO2 on Gj. If indeed histidine protonation does not play a role,
an alternative mechanism could involve an interplay between
the Vj sensor and chemical gate. Because the chemical gate is
likely to be negatively charged (17, 20), if this gate were to
come in proximity of the Vj sensor, before closing the channel
completely, it could potentially hinder or facilitate the Vjinduced displacement of the sensor from the channel lumen in
positive or negative gaters, respectively. Significantly, the
absence of a DEPC effect on CO2 induced uncoupling efficiency seriously questions the potential role of histidine protonation in the uncoupling mechanism as well.
In conclusion, this study shows that CO2 induced cytosolic
acidification decreases both sensitivity and speed of Vj gating
in channels made of mouse Cx50 and increases those of
channels made of a Cx50 mutant (Cx50-D3N), in which the
aspartate in position 3 is mutated to asparagine. In addition, the
data indicate that the D3N mutation greatly increases chemical
gating sensitivity to CO2 and may unmask the activity of the
slow Vj gate. Because the D3N mutation inverts the gating
polarity of Cx50 from positive (4, 8, 35) to negative, the
opposite effect of CO2 on Cx50 and Cx50-D3N Vj gating
sensitivity confirms the hypothesis that the way, in which Vj
gating responds to CO2 may be related to the polarity of Vj
gating.
CO2-SENSITIVITY OF VOLTAGE GATING
AJP-Cell Physiol • VOL
35. Verselis VK and Veenstra RD. Gap junction channels: permeability and
voltage gating. In: Advances in Molecular and Cell Biology. Stamford,
CT: JAI, 2000, vol. 30, p. 129 –192.
36. Wang XG and Peracchia C. connexin 32/38 chimeras suggest a role for
the second half of inner loop in gap junction gating by low pH. Am J
Physiol Cell Physiol 271: C1743–C1749, 1996.
37. Weingart R and Bukauskas FF. Long-chain-alkanols and arachidonic
acid interfere with the Vm-sensitive gating mechanism of gap junction
channels. Pflügers Arch 435: 310 –319, 1998.
38. Werner R, Levine E, Rabadan-Diehl C, and Dahl G. Gating properties
of connexin32 cell-cell channels and their mutants expressed in Xenopus
oocytes. Proc R Soc Lond B Biol Sci 243: 5–11, 1991.
39. White TW, Bruzzone R, Wolfram S, Paul DL, and Goodenough DA.
Selective interactions among the multiple connexin proteins expressed in
the vertebrate lens: the second extracellular domain is a determinant of
compatibility between connexins. J Cell Biol 125: 879 – 892, 1994.
40. Xu X, Berthoud VM, Beyer EC, and Ebihara L. Functional role of the
carboxyl terminal domain of connexin 50 in gap junctional cheannels. J
Membr Biol 186: 101–112, 2002.
41. Young KC and Peracchia C. Carbon dioxide sensitive voltage gating of
connexin32 and connexin32/45 chimeric channels (Abstract). Mol Biol
Cell 13: 351a, 2002.
42. Young KC and Peracchia C. Opposite Cx32 and Cx26 voltage-gating
response to CO2 reflects opposite voltage-gating polarity. J Membr Biol
202: 161–170, 2004.
288 • JUNE 2005 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.32.247 on June 16, 2017
26. Sasaki S, Ishibashi K, Nagai T, and Marumo F. Regulation mechanisms
of intracellular pH of Xenopus laevis oocyte. Biochim Biophys Acta 1137:
45–51, 1992.
27. Spray DC, Harris AL, and Bennett MVL. Equilibrium properties of a
voltage-dependent junctional conductance. J Gen Physiol 77: 77–93,
1998.
28. Spray DC, Harris AL, and Bennett MV. Gap junctional conductance is a
simple and sensitive function of intracellular pH. Science 211: 712–715, 1981.
29. Srivas M, Costa M, Gao Y, Fort A, Fishman GI, and Spray DC.
Voltage dependence of macroscopic and unitary currents of gap junction
channels formed by mouse connexin50 expressed in rat neuroblastoma
cells. J Physiol 517: 673– 689, 1999.
30. Stergiopoulos K, Alvarado JL, Mastroianni M, Ek-Vitorin JF, Taffet
SM, and Delmar M. Hetero-domain interactions as a mechanism for the
regulation of connexin channels. Circ Res 84: 1144 –1155, 1999.
31. Suchyna TM, Xu LX, Gao F, Fourtner CR, and Nicholson BJ.
Identification of a proline residue as a transduction element involved in
voltage gating of gap junctions. Nature 365: 847– 849, 1993.
32. Török K, Stauffer K, and Evans WH. Connexin 32 of gap junctions
contains two cytoplasmic calmodulin-binding domains. Biochem J 326:
479 – 483, 1997.
33. Turin L and Warner AE. Carbon dioxide reversibly abolishes ionic
communication between cells of early amphibian embryo. Nature 270:
56 –57, 1977.
34. Verselis VK, Ginter CS, and Bargiello TA. Opposite voltage gating
polarities of two closely related connexins. Nature 368: 348 –351, 1994.
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