Connexin46 mutations linked to congenital cataract show loss
of gap junction channel function
Jay D.
Pal1,
Xiaoqin
Liu1,
Donna
Mackay2,
Alan
Shiels2,
Viviana M.
Berthoud3,
Eric C.
Beyer3, and
Lisa
Ebihara1
1 Department of Physiology and Biophysics, Finch University
of Health Sciences/The Chicago Medical School, North Chicago, Illinois
60064; 2 Department of Ophthalmology and Visual Sciences,
Washington University, St. Louis, Missouri 63110; and
3 Department of Pediatrics, University of Chicago, Chicago,
Illinois 60637
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ABSTRACT |
Human connexin46 (hCx46) forms gap
junctional channels interconnecting lens fiber cells and appears to be
critical for normal lens function, because hCx46 mutations have been
linked to congenital cataracts. We studied two hCx46 mutants, N63S, a
missense mutation in the first extracellular domain, and fs380, a
frame-shift mutation that shifts the translational reading frame at
amino acid residue 380. We expressed wild-type Cx46 and the two mutants
in Xenopus oocytes. Production of the expressed proteins was
verified by SDS-PAGE after metabolic labeling with
[35S]methionine or by immunoblotting. Dual
two-microelectrode voltage-clamp studies showed that hCx46 formed both
gap junctional channels in paired Xenopus oocytes and
hemi-gap junctional channels in single oocytes. In contrast, neither of
the two cataract-associated hCx46 mutants could form intercellular
channels in paired Xenopus oocytes. The hCx46 mutants were
also impaired in their ability to form hemi-gap-junctional channels.
When N63S or fs380 was coexpressed with wild-type connexins, both
mutations acted like "loss of function" rather than "dominant
negative" mutations, because they did not affect the gap junctional
conductance induced by either wild-type hCx46 or wild-type hCx50.
human connexin 46; intercellular communication; lens
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INTRODUCTION |
GAP
JUNCTIONS are membrane specializations containing intercellular
channels that allow the passage of ions and low-molecular-weight molecules between adjacent cells. These channels are made of subunit proteins called connexins that are members of a multigene family with
at least 14 members (16). A gap junction hemichannel, or connexon, is a hexameric assembly of connexins. A gap junction channel
is formed by the docking of two connexons from neighboring cells.
During gap junction channel formation, connexons traffic to the
nonjunctional plasma membrane, where they can reside as functional
hemichannels before the formation of complete channels (7,
11, 21, 26, 28,
37). These hemichannels can be triggered to open in
response to a variety of stimuli such as depolarization or reduction in
external calcium concentration (4, 7,
11).
Mutations in connexins have been linked to several human genetic
diseases. X-linked Charcot-Marie-Tooth disease (CMTX), a demyelinating
peripheral neuropathy, is associated with mutations in connexin32
(Cx32) (1, 3, 30). Hereditary
forms of nonsyndromic deafness are associated with mutations in Cx26,
Cx30, and Cx31 (17, 20, 41).
Hereditary skin disorders have been associated with mutations in Cx26
and Cx31 (23, 31).
Mammalian lens fiber cells contain two connexins, Cx46 and Cx50
(28, 39). Targeted disruption of either of
these connexin genes results in cataracts in mice (15,
40). Moreover, a mutation in Cx50 that leads to loss of
channel function has been identified in the No2 mouse, which develops
severe cataracts (36, 42). Recently,
mutations in the human Cx50 gene have been associated with "zonular
pulverulent" cataracts (2, 34). One of
these mutations (P88S) is a missense mutation that lies within the
second transmembrane domain. We have previously shown that P88S does not form functional gap junctional channels when expressed in Xenopus oocyte pairs and inhibits the function of
coexpressed wild-type Cx50, i.e., it behaves as a dominant negative
(27).
Mutations in human Cx46 (hCx46) have also been found in two families
with inherited congenital cataracts (22). One of these is
a missense mutation, N63S, that occurs in the first extracellular domain (E1); the other is a frame-shift mutation, fs380, containing a
single base insertion that shifts the translational reading frame at
amino acid residue 380 (Fig. 1). The
frame-shift causes read-through into the 3'-untranslated region and
introduces an in-frame stop codon 90 nucleotides downstream from the
wild-type stop codon. In the present study, the functional properties
of wild-type human Cx46 and these two congenital cataract-linked hCx46
mutations were examined.

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Fig. 1.
Diagram of the predicted membrane topology of human
connexin46 (hCx46) showing the positions at which the N63S and fs380
mutations occur. N, amino terminus; E1 and E2, 1st and 2nd
extracellular domains; CL, cytoplasmic loop; C, carboxy terminus.
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METHODS |
Cloning of wild-type and mutant human lens connexin DNA.
Wild-type and mutant hCx46 alleles were PCR amplified from affected
individuals from two families with congenital cataract linked to
chromosome 13q as previously described (22). We sequenced the entire coding region to determine whether the PCR products encoded
the mutant or the wild-type allele and to verify that PCR amplification
did not introduce any random errors. The PCR products were then
subcloned into the RNA expression vector SP64TII (9).
Cloning of human lens Cx50 DNA for oocyte expression has been
previously described (27).
In vitro transcription of connexin DNA.
The plasmids were linearized with Sal I, and capped RNAs
were synthesized using the mMessage mMachine SP6 in vitro transcription kit (Ambion, Austin, TX) according to the manufacturer's instructions. The amount of RNA was quantitated by measuring the absorbance at 260 nm.
Expression of connexins in Xenopus oocytes.
Female Xenopus laevis were anesthetized, and a partial
ovariectomy was performed. The frogs were maintained and treated in accordance with National Institutes of Health guidelines. The oocytes
were defolliculated and microinjected with connexin cRNAs and an
oligonucleotide antisense to endogenous Cx38 as previously described
(9). For immunoblot analysis, oocytes were frozen in
liquid nitrogen 15-18 h after injection and stored at
80°C. Plasma membrane-enriched preparations were made by homogenization of
oocytes in 1 ml of homogenization buffer (5 mM Tris · HCl, 5 mM
EDTA, 5 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, pH 8.0) by
repeated passage through a 20-gauge needle. Homogenates were then
centrifuged at 3,000 g for 5 min at 4°C to pellet yolk
granules. The supernatant was then centrifuged at 100,000 g
for 50 min at 40C, and the pellet was resuspended in the
homogenization buffer as previously described (18).
Proteins were resolved by SDS-PAGE on 9% acrylamide gels and
electrotransferred onto Immobilon P (Millipore, Bedford, MA). Membranes
were blocked in 5% nonfat dry milk in Tris-buffered saline (TBS; pH
7.4) and incubated with a rabbit polyclonal antiserum directed against
amino acids 411-416 of rat Cx46 sequence (28) at a
1:500 dilution overnight at 4°C. Membranes were then rinsed several
times in TBS and incubated in peroxidase-conjugated goat anti-rabbit
IgG antibodies (Jackson ImmunoResearch, West Grove, PA) at a 1:4,000
dilution for 1 h at room temperature. After that period of time,
membranes were rinsed several times, and immunoreactive complexes were
detected by enhanced chemiluminescence (ECL, Amersham Life Sciences,
Arlington Heights, IL) according to the manufacturer's directions.
Metabolic labeling studies were performed by coinjection of
[35S]methionine (1 µCi/oocyte; specific activity > 1,000 Ci/mmol, 15 mCi/ml; Amersham) with the connexin cRNA as
previously described (8). Homogenates of whole
oocytes or membrane-enriched preparations were separated by SDS-PAGE on
4-18% acrylamide gels, and radiolabeled proteins were detected by fluorography.
Electrophysiological measurements.
Connexin cRNA-injected oocytes were devitellinized and paired as
previously described (10). To measure gap junctional
conductance, dual two-microelectrode voltage-clamp experiments were
performed using a Geneclamp 500 and an Axoclamp 2A voltage-clamp
amplifier (Axon Instruments, Foster City, CA) (35).
Families of junctional currents were generated by applying
transjunctional voltage-clamp steps to ±70 mV in increments of 10 mV
from a holding potential of
40 mV. Changes in junctional conductance
during the experiment were monitored by applying a 5-mV prepulse of 1-s
duration 1 s before the initiation of the test pulse. Data
acquisition and analysis were conducted using a personal computer
running pCLAMP version 6 software. Microelectrodes were filled with 3 M
KCl and had resistances of 0.2-2 M
. The bath solution was
modified Barth's solution.
Gap junctional hemichannel currents in single oocytes injected with
wild-type or mutant hCx46 cRNA were measured using a conventional two-microelectrode voltage-clamp technique. All of the experiments were
performed on oocytes pretreated with antisense Cx38 oligonucleotides to
ensure that the observed currents were caused by the expression of
exogenous connexins (8).
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RESULTS |
Biochemical characterization.
To verify that wild-type and mutant human Cx46 cRNAs were efficiently
translated, oocytes were coinjected with wild-type or mutant hCx46 cRNA
and [35S]methionine. Protein bands of the expected
electrophoretic mobilities were observed in homogenates of oocytes
injected with wild-type hCx46, N63S, or fs380 cRNA (Fig.
2A). No bands of similar
mobility were observed in antisense-injected control oocytes. Similar
results were observed with membrane-enriched preparations (data not
shown). The bands identified in oocytes injected with fs380 cRNA were less intense than those in N63S or wild-type hCx46 cRNA-injected oocytes. Because equal amounts of cRNA were injected into each oocyte,
these results suggest that the fs380 mutant was being either less
efficiently translated or more rapidly degraded, or both.

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Fig. 2.
Expression of wild-type and mutant hCx46 in
Xenopus oocytes. A: detection of radiolabeled
wild-type and mutant hCx46 in Xenopus oocyte homogenates.
Proteins from homogenates of Xenopus oocytes coinjected with
[35S]methionine, antisense Cx38 oligonucleotide (AS), and
15 ng of wild-type hCx46 (WTCx46), N63S mutant, or fs380 mutant cRNA
were resolved on SDS-containing polyacrylamide gels, and the
[35S]methionine-labeled proteins were detected by
fluorography. No bands were detected in oocytes injected with an
antisense Cx38 oligonucleotide. Wild-type hCx46 and N63S mutant
cRNA-injected oocytes showed a prominent band at 66 kDa; a less intense
band was observed in oocytes injected with fs380 mutant cRNA (66 kDa).
A longer exposure of the radiolabeled proteins from the fs380
cRNA-injected oocytes (right) better shows this band.
B: detection of wild-type and mutant hCx46 in
membrane-enriched preparations of Xenopus oocytes by
immunoblotting. Oocytes were injected with antisense Cx38
oligonucleotide (AS), wild-type hCx46 (WTCx46), N63S mutant, or fs380
mutant cRNA, and plasma membrane-enriched preparations were analyzed by
immunoblotting with anti-Cx46 antibodies. A single band of 66 kDa was
detected in oocytes injected with wild-type hCx46 or N63S mutant. No
bands were observed in oocytes injected with antisense Cx38
oligonucleotide or fs380 mutant.
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To further verify that the expressed connexins were transported to the
plasma membrane in Xenopus oocytes, immunoblot analysis of
plasma membrane-enriched preparations was performed (Fig.
2B). A major immunoreactive band of ~66 kDa was detected
by anti-Cx46 antibodies in oocytes injected with either wild-type hCx46
or N63S mutant cRNA. No immunoreactive bands were observed in oocytes injected with cRNA for the fs380 mutation. This failure was expected because the epitope recognized by these antibodies is absent in the
fs380 mutant.
Functional characterization of wild-type and mutant hCx46 in oocyte
pairs.
Oocyte pairs injected with 2-4 ng of wild-type hCx46 cRNA
efficiently developed gap junctional conductances with a mean
junctional conductance of 1.52 µS (Table
1). A representative family of gap
junctional current traces recorded from a pair of Xenopus oocytes expressing wild-type hCx46 is shown in Fig.
3A. The junctional current
showed a time- and voltage-dependent decay to a steady-state value at
transjunctional voltage-clamp steps of ± 20 mV that could be
described by two exponentials. When the steady-state junctional conductance was plotted as a function of voltage, the data could be fit
to a Boltzmann relation with A = 0.18, Vo =
32 mV,
Gj max = 1.0, and
Gj min = 0.19 for positive transjunctional
voltages and A =
0.21, Vo = 30 mV, Gj max = 1.0, and
Gj min = 0.16 for negative transjunctional
voltages (Fig. 3B), where A is the steepness
factor, Gj max and
Gj min are maximum and minimum conductance, and
V0 is the voltage at which the steady-state
junctional conductance, Gj, equals the midpoint between Gj max and
Gj min.

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Fig. 3.
Voltage dependence of wild-type hCx46 gap junctional
channels. A: representative family of gap junctional current
traces recorded from a cell pair injected with wild-type hCx46 cRNA
using the dual whole cell voltage-clamp technique. Junctional currents
were elicited by a series of 12-s duration transjunctional voltage
steps between 70 and +70 mV in 10-mV increments from a holding
potential of 40 mV. Changes in junctional conductance
(Gj) during the experiment were normalized by
applying a 5-mV prepulse of 1-s duration 1 s before the initiation
of the test pulse. B: normalized steady-state junctional
conductance vs. voltage relationship for wild-type hCx46 gap junctional
channels. Steady-state junctional conductances obtained at the
different transjunctional voltage steps were normalized to the values
obtained at transjunctional voltages of ± 5 mV, and these
calculated values were plotted against the transjunctional voltage. The
solid lines are the best fit of the steady-state data to a Boltzmann
equation with A = 0.18, Vo= 32
mV, Gj max = 1.0, and
Gj min = 0.19 for positive
transjunctional voltages and A = 0.21,
Vo= 30 mV, Gj max = 1.0, and Gj min = 0.16 for negative
transjunctional voltages, where A is the steepness factor,
Gj max and Gj min
are the maximum and minimum conductances, and Vo
is the voltage at which Gj equals the midpoint
between Gj max and
Gj min. The data are presented as means ± SE (n = 5).
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The ability of mutant human Cx46 to induce cell-to-cell coupling was
also examined using the Xenopus oocyte pair assay. In contrast to wild-type hCx46 cRNA-injected oocytes, oocyte pairs injected with 2-4 ng cRNA for N63S or fs380 mutants failed to show
significant gap junctional coupling compared with oocytes injected with
Cx38 antisense oligonucleotides alone (Table 1).
To examine whether the hCx46 mutants exerted a dominant negative effect
on wild-type hCx46 channel activity, gap junctional currents were
recorded from oocyte pairs coinjected with equal amounts of mutant and
wild-type hCx46 cRNAs. In oocyte pairs coinjected with 0.8 ng of N63S
plus 0.8 ng of wild-type hCx46 cRNA, the mean gap junctional
conductance was reduced to 35% of the conductance in oocyte pairs
injected with 1.6 ng of wild-type hCx46 cRNA alone (Table
2). Similarly, gap junctional conductance
was reduced to 58% in oocyte pairs coinjected with fs380 plus
wild-type hCx46 cRNAs (Table 2). This reduction in gap junctional
conductance is most likely due to the decreased amount of wild-type
hCx46 cRNA in oocyte pairs coinjected with mutant plus wild-type cRNAs and suggests that both Cx46 mutations act like loss-of-function mutations without strong dominant negative inhibition.
To test whether wild-type hCx46 and/or mutant hCx46 were able to form
heterotypic gap junctional channels with each other or with another
lens-specific connexin, hCx50, oocytes injected with the cRNAs coding
for these connexins were paired and the gap junctional conductance was
determined (Table 2). Large gap junctional conductances were recorded
when hCx46 cRNA-injected oocytes were paired with Cx50 cRNA-injected
oocytes, suggesting the formation of heterotypic gap junctional
channels. In contrast, when N63S or fs380 cRNA-injected oocytes were
paired with Cx50 or hCx46 cRNA-injected oocytes, no significant
coupling was observed.
To test whether the hCx46 mutations could interact with wild-type Cx50
in a dominant negative manner, oocytes coinjected with N63S or fs380
cRNA and Cx50 cRNA were paired with Cx50 cRNA-injected oocytes and
tested for electrical coupling. Expression of N63S or fs380 did not
inhibit the development of Cx50-induced junctional conductances (Table
2).
Hemi-gap junctional currents formed from wild-type and mutant
hCx46.
To test the ability of wild-type and mutant hCx46 to form functional
hemi-gap junctional channels in the nonjunctional plasma membrane,
single oocytes injected with these connexin cRNAs were studied using a
conventional two-electrode voltage-clamp technique. Oocytes injected
with wild-type hCx46 cRNA developed large outward currents that
activated at potentials positive to
20 mV (Fig. 4, A, left, and
B, open circles), which closely resembled the rat
Cx46 hemi-gap junctional currents previously described
(11). Oocytes injected with N63S cRNA developed outward
currents at potentials more positive than 0 mV, whose amplitudes were
much smaller than those observed in oocytes injected with similar
amounts of wild-type hCx46 cRNA (Fig. 4, A,
middle, and B, solid circles). In
contrast, no detectable hemi-gap junctional currents were observed in
oocytes injected with cRNA for fs380 (Fig. 4A,
right) or antisense-treated control oocytes (not shown).

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Fig. 4.
Voltage dependence of hemi-gap junctional channels formed
by wild-type or mutant hCx46. A: comparison of nonjunctional
currents elicited in Xenopus oocytes injected with equal
amounts of wild-type hCx46 (left), N63S mutant
(middle), or fs380 mutant (right) cRNA when cells
were pulsed for 24 s to potentials between 40 and +30 mV in
10-mV increments from a holding potential of 40 mV. B:
current-voltage relationship for wild-type hCx46 and N63S mutant
hemi-gap junctional channels. Oocytes were injected with 0.92 ng of
wild-type hCx46 ( ) or N63S mutant ( ) cRNA and
kept in modified Barth's solution containing 0.7 mM calcium and 0.8 mM
magnesium during the recordings. Currents elicited using the paradigm
described in A were measured at the end of the depolarizing
pulse and plotted as a function of transmembrane potential. Each point
in the curve represents the mean ± SE (n = 6).
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Because it has been previously shown that reducing extracellular
calcium results in a dramatic increase in the magnitude
(7, 11) of hemi-gap junctional currents
(29), we tested whether calcium had a similar effect on
N63S mutant hemi-gap junctional channels. Hemi-gap junctional-channel
currents were recorded from N63S cRNA-injected oocytes bathed in normal
(0.7 mM) or zero added calcium solution. Reducing extracellular calcium
from 0.7 mM to ~1 µM in the presence of 0.8 mM magnesium resulted
in a large increase in the amplitude of the N63S-induced current (Fig.
5). It also shifted the threshold of
activation from +10 mV to
20 mV and accelerated the time course of
activation (data not shown).

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Fig. 5.
Effects of external calcium on N63S hemi-gap junctional
currents. Nonjunctional currents were recorded from oocytes injected
with N63S mutant cRNA using the same pulse protocol as described in
Fig. 4 while the oocytes were bathed in external solutions containing
0.7 mM calcium (A) or zero added calcium (B).
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DISCUSSION |
In the present study, we have demonstrated that wild-type human
Cx46 forms both gap junctional channels in paired Xenopus oocytes and hemi-gap junctional channels in single oocytes. In contrast, neither of the two cataract-associated hCx46 mutants was able
to form intercellular channels when expressed in paired Xenopus oocytes. Moreover, the hCx46 mutants were impaired
in their ability to form hemi-gap junctional channels. Thus it is likely that people carrying these hCx46 mutations develop cataracts as
a consequence of reduced intercellular communication.
Lens homeostasis and the maintenance of transparency depend on an
internal circulatory system composed of continuously circulating currents that flow around and through the avascular lens
(32). Ion channels, water channels,
Na+-K+ pumps, and gap junctional channels have
been implicated in the generation and regulation of these continuously
circulating currents (24). Mutations that disrupt this
circulation may cause cataracts. Mutations in the lens major intrinsic
protein (MIP), whose only known role in the lens is that of a water
channel (38), lead to the development of cataracts in mice
(33). Similarly, mutations in connexin genes that would
also affect the lens circulatory system by reducing intercellular
communication lead to the development of cataracts.
In the N63S mutation, an asparagine is replaced by a serine at position
63. This asparagine in the first extracellular domain (E1) of hCx46 is
highly conserved in all connexins and is flanked by two highly
conserved cysteines (C61 and C65) that are required for formation of
gap junctional channels (5, 14). An amino acid change at position 63 might cause a substantial alteration in the
conformation of E1, a domain that is involved in docking of connexons.
It is thus not surprising that the N63S mutant was unable to form gap
junctional channels even though it was able to form functional
hemichannels in the nonjunctional plasma membrane of oocytes. However,
the size of the N63S hemi-gap junctional currents was reduced compared
with wild-type hCx46. These observations could result from a decrease
in single-channel conductance, an increased sensitivity to block by
external divalent cations, or a reduced number of functional
hemichannels. Further studies may distinguish among these possibilities.
The fs380 mutation that changes the sequence of the carboxy terminus of
hCx46 did not form functional gap junctional channels in oocyte pairs
and did not form open hemi-gap junctional channels in single oocytes at
an extracellular calcium concentration >10
6 M. In
contrast, engineered alterations in the carboxy-terminal regions of
other connexins (including truncations, missense mutations, and
addition of epitope tags) do not prevent the formation of functional
channels (13, 19, 25). This
difference may be explained by decreased translation and/or enhanced
degradation, because reduced amounts of radioactive fs380 protein were
detected in oocytes coinjected with [35S]methionine and
fs380 cRNA compared with the amounts detected for either N63S or
wild-type hCx46 protein. Several missense or truncated carboxy-terminal
Cx32 mutants linked to CMTX (4, 6) have also
been shown to cause loss or reduction in function.
The current results differ significantly from the results obtained with
a Cx50 mutant (P88S) identified in patients with hereditary cataracts
(27). Whereas the Cx50 mutant P88S failed to form gap
junction channels when expressed by itself, it acted like a dominant
negative inhibitor when coexpressed with wild-type Cx50, because it
substantially reduced gap junctional conductance below the values
expected from expression of wild-type channels. In contrast, the
mutants characterized in the present study (N63S and fs380) did not
affect the gap junctional conductance induced by either wild-type Cx46
or wild-type Cx50; both mutations acted like loss-of-function rather
than dominant negative mutations. These results suggest the existence
of a limiting lower level of coupling that is required for normal lens
function. A reduction in the level of coupling below this critical
value (due to "loss of function" of one Cx46 allele) would be
sufficient for induction of cataractogenesis.
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ACKNOWLEDGEMENTS |
This work was supported by National Eye Institute Grants EY-10589,
EY-12284, and EY-08368.
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FOOTNOTES |
Address for reprint requests and other correspondence: L. Ebihara, Dept. of Physiology and Biophysics, Finch Univ. of Health Sciences/The Chicago Medical School, 3333 Green Bay Rd., North Chicago,
IL 60064 (E-mail: Lisa.Ebihara{at}finchcms.edu).
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.
Received 2 November 1999; accepted in final form 15 March 2000.
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