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Am J Physiol Cell Physiol 279: C596-C602, 2000;
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Vol. 279, Issue 3, C596-C602, September 2000

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega . 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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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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  		Table 1.   Gap junctional conductance of wild-type and mutant human Cx46 expressed in Xenopus oocyte pairs



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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).

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.

                              
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  		Table 2.   Effect of heteromeric and heterotypic mixing of mutant and wild-type lens connexins

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 (open circle ) 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).

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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

This work was supported by National Eye Institute Grants EY-10589, EY-12284, and EY-08368.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 279(3):C596-C602
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