RAPID COMMUNICATION
Molecular mechanism underlying a Cx50-linked congenital
cataract
J. D.
Pal1,
V. M.
Berthoud2,
E. C.
Beyer2,
D.
Mackay3,
A.
Shiels3, and
L.
Ebihara1
1 Department of Physiology and
Biophysics, Finch University of Health Sciences/The Chicago Medical
School, North Chicago 60064;
2 Department of Pediatrics,
University of Chicago, Chicago, Illinois 60637; and
3 Department of Molecular
Genetics, Institute of Ophthalmology, Washington University School of
Medicine, St. Louis, Missouri 63110
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ABSTRACT |
Mutations in gap
junctional channels have been linked to certain forms of inherited
congenital cataract (D. Mackay, A. Ionides, V. Berry, A. Moore, S. Bhattacharya, and A. Shiels. Am. J. Hum. Genet. 60: 1474-1478, 1997; A. Shiels, D. Mackay,
A. Ionides, V. Berry, A. Moore, and S. Bhattacharya.
Am. J. Hum. Genet. 62: 526-532,
1998). We used the Xenopus oocyte pair
system to investigate the functional properties of a missense mutation
in the human connexin 50 gene (P88S) associated with zonular
pulverulent cataract. The associated phenotype for the mutation is
transmitted in an autosomal dominant fashion.
Xenopus oocytes injected with
wild-type connexin 50 cRNA developed gap junctional conductances of
~5 µS 4-7 h after pairing. In contrast, the P88S mutant
connexin failed to form functional gap junctional channels when paired
homotypically. Moreover, the P88S mutant functioned in a dominant
negative manner as an inhibitor of human connexin 50 gap junctional
channels when coinjected with wild-type connexin 50 cRNA. Cells
injected with 1:5 and 1:11 ratios of P88S mutant to wild-type cRNA
exhibited gap junctional coupling of ~8% and 39% of wild-type
coupling, respectively. Based on these findings, we conclude that only
one P88S mutant subunit is necessary per gap junctional channel to abolish channel function.
connexin 50; gap junction; P88S; dominant negative
inhibition
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INTRODUCTION |
INHERITED CONGENITAL CATARACT is a heterogeneous lens
disorder that is often transmitted as an autosomal dominant Mendelian trait. Autosomal dominant cataract has been mapped to at least 10 separate loci (8). One type of congenital cataract is the zonular pulverulent cataract that, in one case, has been linked to a
proline-to-serine mutation at position 88 in human connexin 50 (Cx50)
(17). Cx50 is a member of the connexin family of gap junctional
proteins and is expressed primarily in the lens, together with Cx46,
where it forms intercellular channels between adjacent fiber cells (6).
These intercellular channels consist of 12 connexins, arranged in two
hexameric connexons or hemichannels, which are located in the plasma
membranes of adjacent cells. The topology of connexins in the plasma
membrane predicts that the P88S mutation lies within the second
transmembrane domain (Fig. 1A).
This proline is conserved throughout the connexin family and is thought
to be involved in the voltage gating of connexins. Suchyna et al. (21)
demonstrated that mutations of this proline in Cx26 prevented the
formation of homotypic (same connexons) gap junctional channels and
caused a reversal of voltage-gating polarity when paired
heterotypically (different connexons) with wild-type Cx26. Here we have
used the paired Xenopus oocyte system to characterize the voltage-dependent gating properties of human Cx50
gap junctional channels and to examine the functional consequences of a
mutation in Cx50 associated with congenital cataract.

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Fig. 1.
A: diagram of predicted connexin
membrane topology indicating location of P88S mutation. Position 88 is
in the 2nd transmembrane domain (TM2). N, amino terminus; TM,
transmembrane domains; E, extracellular domain; CL, cytoplasmic loop;
C, carboxyl terminus. B: immunoblot
detection of connexin 50 (Cx50) protein in
Xenopus oocytes. Membrane-enriched
fractions of wild-type and mutant Cx50 cRNA injected oocytes were
separated on a 9% SDS-polyacrylamide gel and transferred to a
polyvinylidene difluoride membrane. Equal amounts of cRNA were injected
to estimate translational efficiency. Membrane was probed with a
monoclonal anti-Cx50 antibody. Oocytes injected with AS-Cx38
oligonucleotide were used as a negative control, and a bovine lens
homogenate was used as a positive control.
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METHODS |
Wild-type and mutant Cx50 alleles were PCR amplified from an affected
individual of the Ev. family (15, 16). The two primers correspond to codons 1-7 and 428-Stop of the human Cx50 gene with EcoR I linkers. The PCR conditions
were as described previously (17). We sequenced the entire coding
region to determine whether the PCR products encoded the mutant or
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 (3). 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.
Xenopus oocytes were prepared and
tested as described previously, except that the oocytes were incubated
for 2 days after injection of cRNA and studied 4-7 h after
pairing. To measure the junctional conductance, cell pairs were studied
using the dual two-microelectrode technique described by Spray et al.
(19). Families of junctional currents were generated by applying
transjunctional voltage-clamp steps to ±70 mV from a holding
potential of
40 mV. Changes in junctional conductance during the
experiment were normalized by applying a 5-mV prepulse 1 s before the
initiation of the test pulse. Only cell pairs with resting membrane
potentials more negative than
15 mV were considered for data
analysis. Data acquisition and analysis were conducted using a personal
computer running pCLAMP version 6.0.5 (Axon Instruments).
Microelectrodes were filled with 3 M KCl (pH 7.4) and had resistances
of 0.2-2 M
. The bath solution was modified Barth's solution.
Oocytes were frozen in liquid nitrogen and stored at
80°C
for immunoblot analysis. Plasma membrane-enriched preparations were
resolved on a 9% SDS-polyacrylamide gel and transferred to a
polyvinylidene difluoride membrane (7, 22). The membrane was probed with a Cx50 monoclonal antibody (12), and the bound antibody was detected using nitro blue
tetrazolium/5-bromo-4-chloro-3-indolylphosphate-p-toluidine salt (Promega, Madison, WI) after incubation in alkaline
phosphatase-conjugated goat anti-mouse Ig (Boehringer Mannheim,
Indianapolis, IN).
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RESULTS |
In vitro synthesized cRNAs for wild-type and mutant human Cx50 were
injected into Xenopus oocytes, either
singly or in combination. The expression of wild-type and mutant human
Cx50 in cRNA-injected oocytes was verified by Western blot analysis of
cell membrane-enriched preparations using a monoclonal anti-MP70 (the
sheep homolog of human Cx50) antibody (Fig.
1B) (12). A bovine lens homogenate was used as the positive control. Cells injected with either wild-type or mutant Cx50 exhibited a predominant band with an electrophoretic mobility of ~70 kDa. No immunoreactive bands were detected in oocytes
injected with antisense oligonucleotide to endogenous Cx38 only.
To characterize the voltage-dependent characteristics of human Cx50 gap
junctional channels, Xenopus oocytes
were injected and paired as described previously. Transjunctional
coupling was assayed using the dual two-microelectrode voltage-clamp
technique (19). Endogenous coupling was inhibited by
injecting the cells with Cx38 antisense oligonucleotide (1). Wild-type
human Cx50 efficiently made gap junctional channels (Table
1), and characteristic junctional current
traces are shown in Fig.
2A. The
junctional current decayed to new steady-state levels on application of
depolarizing or hyperpolarizing transjunctional voltage-clamp steps to
potentials greater than ±20 mV. The normalized initial and
steady-state conductance-voltage relationships are shown in Fig.
2B. The solid lines are the best fit
of the steady-state data to a Boltzmann equation with
A = 0.08, Vo = 34 mV,
Gmax = 1.1, and
Gmin = 0.22 for
positive transjunctional voltages and
A =
0.08,
Vo =
32 mV,
Gmax = 1.1, and
Gmin = 0.24 for
negative Vj
values, where
A is the steepness factor,
Vo and Vj are midpoint
and junctional voltage, respectively, and
Gmax and
Gmin are maximum
and minimum conductance, respectively. These findings indicate that
human Cx50 is less voltage sensitive than mouse Cx50
(22).

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Fig. 2.
A: representative family of junctional
current traces recorded from a cell pair injected with wild-type Cx50
cRNA. Cells were initially voltage clamped at 40 mV. Junctional
current traces were recorded in response to transjunctional
voltage-clamp steps between +70 and 70 mV in 10-mV increments.
Changes in junctional conductance
(Gj) during the
experiment were normalized by applying a 5-mV prepulse 1 s before
initiation of test pulse. For this pair, gap junctional conductance was
measured to be 5.02 µS. B:
normalized initial and steady-state conductance-voltage relationship
for Cx50 gap junctional channels. Solid lines are best fit of
steady-state data to a Boltzmann equation with
A = 0.08, Vo = 34 mV,
Gmax = 1.1, and
Gmin = 0.22 for
positive transjunctional voltages and
A = 0.08,
Vo = 32
mV, Gmax = 1.1, and Gmin = 0.24 for negative Vj
values; n = 8 cell pairs.
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We tested the ability of the P88S mutation to induce gap junctional
coupling, either alone or in combination with wild-type Cx50. The
results of these experiments are summarized in Table 1. Expression of
the P88S mutant failed to induce coupling in homomeric oocyte pairs
(same connexins in both cells), and the mutant cRNA also did not cause
the formation of hemichannels that could interact with wild-type Cx50
in a heterotypic manner (different connexins in each member of the cell pair).
The effect of mixing was examined by coinjecting oocytes with different
ratios of mutant to wild-type cRNA while keeping the total amount of
injected cRNA constant. When equal amounts of mutant and wild-type cRNA
were injected into oocytes to simulate the situation in a heterozygous
individual, there was a profound inhibition of gap junctional coupling,
to a level that was comparable with antisense-injected, negative
controls. However, as the ratio of mutant to wild-type cRNA was
reduced, an increase in coupling was noted. When the ratio of mutant to
wild-type cRNA was 1:5 (to obtain one mutant subunit per hemichannel on
average), we noted transjunctional coupling that was 8% of the
wild-type coupling. When the mutant-to-wild-type ratio was further
reduced to 1:11, transjunctional coupling was 39% of the wild-type
value. These findings suggest that the P88S mutant is functioning as a
dominant negative inhibitor of Cx50 gap junctional coupling and that
only one mutant subunit per gap junctional channel is sufficient to abolish channel function.
To test this hypothesis, we used the following equations to represent
the possible combinations of mutant and wild-type connexins in a gap
junction and to model the inhibition of gap junctional coupling by
mutant subunits
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(1)
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(2)
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where
y is the fractional expected current,
n is the number of subunits in the
channel, a is the maximum number of
mutant subunits permissible in a functional channel,
fmut is the
mutant mole fraction, and
fwt is the
wild-type mole fraction. This equation is valid for cases where the
translation efficiencies of the cRNAs are comparable and the connexins
aggregate randomly in the cell membrane. For the case where only one
mutant subunit is required to abolish channel function,
a = 0, and Eq. 2 reduces to
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(3)
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For
the case where two and three mutant subunits are required to abolish
channel function, the resulting equations are
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(4)
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(5)
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In
the derivation of this equation, we assumed the arrangement of the
mutant subunits within the gap junctional channel to be insignificant.
If placement of the mutant connexins is important, then
Eqs. 4 and 5 will underestimate the true amount
of coupling. Figure 3 compares the
inhibition of coupling due to the P88S mutation with the predicted
reduction if one or multiple subunits were necessary to abolish channel
function. The measured inhibition of coupling follows
Eq. 3 and indicates that only one
mutant subunit is necessary to abolish channel function.

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Fig. 3.
Predicted and measured inhibition of gap junctional coupling by P88S.
Theoretical inhibition of current through a dodecameric channel by 1, 2, or 3 subunits, respectively, was modeled by the following functions:
y = (1 x)12,
y = (1 x)12 + 12x(1 x)11,
and y = (1 x)12 + 12x(1 x)11 + 66x2(1 x)10,
where x represents the mutant mole
fraction. Theoretical and measured inhibition of current was normalized
to coupling measured in oocytes injected with wild-type Cx50. Data are
plotted as mean conductance ± SE (µS;
n = 9 for each data point).
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DISCUSSION |
Gap junctions are thought to play an important role not only in lens
homeostasis but in virtually every organ system of the body. Mutations
in connexin genes have been associated with X-linked Charcot-Marie-Tooth disease (9, 14), hereditary nonsyndromic deafness
(10, 11), and congenital heart defect (2, 18). With regard to the lens,
recent studies have shown that disruption of the genes for Cx46 or Cx50
by homologous recombination leads to cataractogenesis in mice (5, 25).
In addition, a missense mutation in the Cx50 gene has been associated
with the mouse No2 cataract (20). In the present study, we investigated
the molecular mechanisms involved in cataractogenesis associated with a
mutation of the human Cx50 gene. Our results indicate that the P88S
mutant interacts with wild-type connexins and can abolish channel
function by virtue of a single mutant subunit per gap junctional
channel. This type of behavior can explain the autosomal dominant
pattern of inheritance observed in this family. Recently, White et al. (24) reported similar findings regarding a Cx26 mutant in a family with
profound deafness inherited as a dominant trait. Thus dominant negative
behavior of connexins may be a general phenomenon to explain autosomal
dominantly inherited diseases due to connexin mutations.
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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 |
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: J. D. Pal, Dept.
of Physiology and Biophysics, FUHS/The Chicago Medical School, 3333 Greenbay Rd., North Chicago, IL 60064 (E-mail:
jdp80134{at}mis.finchcms.edu).
Received 12 December 1998; accepted in final form 21 March 1999.
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