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First published online 14 November 2007
doi: 10.1242/jcs.012237

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The cataract-inducing S50P mutation in Cx50 dominantly alters the channel gating of wild-type lens connexins

¹ Department of Physiology and Biophysics and the Graduate Program in Genetics, State University of New York, Stony Brook, NY 11794, USA
² School of Optometry and Vision Science Program, University of California at Berkeley, Berkeley, CA 94720, USA

View larger version (32K): [in this window] [in a new window]   Fig. 1. Cx50-S50P fails to form functional intercellular channels. (A,B) Immunoblot analysis of oocytes showed equivalent levels of wild-type and mutant connexin expression for all conditions tested. (C,D) Band densitometry quantitatively confirmed that mean protein expression was not significantly changed (P>0.05, Student's t-test). (E) Junctional conductance measurements recorded from Xenopus oocyte pairs injected with wild-type Cx50, Cx46 or S50P transcripts alone or in combination. Cell pairs expressing wild-type Cx50 or Cx46 subunits alone form functional gap junctions with mean conductance values of 26 µS or 20 µS, respectively. Oocytes co-injected with both wild-type and mutant Cx50 transcripts formed channels that displayed a 20% decrease in Gj when compared with homotypic Cx50 gap junctions, a level of coupling significantly higher than that of the background (P<0.05, Student's t-test) but not significantly different from homotypic Cx50 channels (P>0.05, Student's t-test). Similarly, the co-expression of Cx46 and Cx50-S50P subunits did not significantly alter junctional conductance (P>0.05, Student's t-test) as these channels exhibited a mean Gj of 17 µS. Heterotypic channels failed to form functional channels with levels of conductance significantly higher than that of the water injected negative controls (P>0.05, Student's t-test). Cx50-S50P subunits alone failed to produce functional intercellular channels. Data points represent individual conductance measurements. Columns indicate the mean ± s.e.m.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Voltage-gating properties of homotypic wild-type Cx50 channels and channels co-expressing Cx50-S50P and wild-type Cx50 subunits. (A,B) Junctional current was measured and plotted as a function of time, showing that channels co-expressing (B) both wild-type and mutant Cx50 proteins did not visibly alter current decay when compared with (A) homotypic Cx50 channels. (C) Analysis of channel closure kinetics. Representative current traces plotting the initial 250 mseconds of Ij recordings were fit to a monoexponential decay to determine the mean time constant . The co-expression of (C) wild-type and mutant Cx50 transcripts produce a mean value of 52 ms (n=5) a value significantly different (P<0.05, Student's t-test) from the 61 msecond mean channel closure time (n=4) exhibited by the homotypic wild-type channel during an 80 mV potential. Ij measurements recorded during a 120 mV voltage application show a significant decrease in the mean values from 34 mseconds for the (E) homotypic wild-type channel (n=4) to 25 ms shown by the (F) co-injected gap junctions (n=5, P<0.05, Student's t-test).

View larger version (33K): [in this window] [in a new window]   Fig. 3. Voltage-gating properties of homotypic and heteromeric Cx46 channels. (A-C) Ij values recorded from oocyte pairs were plotted as a function of time to reveal that channels expressing (A) wild-type Cx46 alone and mixed channels containing (B) both Cx46 and Cx50-S50P proteins as well as channels expressing (C) wild-type Cx46 and Cx50 subunits displayed altered voltage-gating sensitivity as co-injected pairs appear more responsive at larger voltage applications. Analysis of channel closure kinetics. The initial 250 mseconds of junctional current was fit to a monoexponential decay model to determine the mean time constant . (E,D) Representative Ij decays show that during an +80 mV potential channels containing (E) both Cx46 and Cx50-S50P subunits closed in 101 mseconds a value significantly faster then the 132 mseconds mean value exhibited by the (D) homotypic Cx46 channel (P>0.05, ANOVA). (F) Interestingly, channels co-expressing both wild-type Cx46 and Cx50 closed in 85 mseconds, a rate approximately 16% or 36% faster than the mixed channels containing Cx46 and S50P or homotypic Cx46 gap junctions, respectively. (G,H) Similarly, when a 120 mV Vj was applied, homotypic Cx46 channels closed in 126 mseconds (G), a mean channel closure time significantly slower then the 90 mseconds exhibited by the mixed channels containing wild-type Cx46 and S50P proteins (H) (P>0.05, ANOVA). Heteromeric channels containing both wild-type Cx46 and Cx50 (I), displayed a mean value of 75 mseconds, a closure speed that is significantly faster than either the homotypic Cx46 or heteromeric Cx46 and S50P channel (P>0.05, ANOVA). All means are the sum of four independent experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Comparison of steady-state conductance properties. Equilibrium gating properties were analyzed by plotting normalized junctional conductance against transjunctional voltage and fit to the Boltzmann equation. (A) comparison of homotypic wild-type Cx50 channels (n=4) and mixed gap junctions containing both wild-type and mutant Cx50 subunits (n=6) showed similar voltage sensitivity, as indicated by the overlapping pattern of the two Boltzmann plots. The steady-state reduction in conductance was greater for mixed channels expressing both Cx46 and S50P subunits (n=8) when compared with that of the homotypic Cx46 channel (n=4), indicating an increase in voltage gating sensitivity for heteromeric channels. (B) Equilibrium gating properties displayed modest changes at positive potentials when mixed channels containing both wild-type Cx46 and Cx50 (n=6) were compared with heteromeric channels expressing wild-type Cx46 and S50P. Results are shown as the mean ± s.e.m.

View larger version (17K): [in this window] [in a new window]   Fig. 5. Gating analysis of heterotypic wild-type and co-injected channels. Gj measurements recorded from oocyte paired to form heterotypic wild-type or coinjected gap junctions. (A) Cell pairs expressing wild-type Cx46 and Cx50 subunits form functional gap junctions with mean conductance values of 22 µS, a level of coupling significantly higher than that of the water-injected negative control (P<0.05, Student's t-test). Co-injected heterotypic channels that expressed both wild-type and mutant Cx50 transcripts formed channels that displayed a 50% decrease in Gj when compared with wild-type heterotypic gap junctions, a level of coupling significantly higher than that of the background (P<0.05, Student's t-test). Data points represent individual conductance measurements. Columns indicate the mean ± s.e.m. (B,C) Junctional currents recorded from oocyte pairs were plotted as a function of time to compare heterotypic gap junctions expressing (B) wild-type Cx46 and Cx50 subunits and (C) heterotypic channels containing wild-type Cx46 and wild-type Cx50 and S50P mutant proteins. Representative Ij decays revealed that co-injected pairs appear more responsive at greater depolarizing voltage applications as well as more asymmetric than heterotypic channels comprising wild-type lens fiber connexins. Comparison of steady-state conductance properties. Equilibrium gating properties were analyzed by plotting normalized junctional conductance against transjunctional voltage and fit to the Boltzmann equation. (D) The steady-state reduction in conductance was greater for channels expressing both wild-type Cx50 and Cx50-S50P subunits (n=6) when compared with that of the heterotypic wild-type channel (n=5), indicating an increase in voltage-gating sensitivity for heterotypic channels containing the mutant protein.

View larger version (92K): [in this window] [in a new window]   Fig. 6. Immunofluorescent imaging of Cx50-S50P in vitro and in vivo. (A,B) Transiently transfected HeLa cells expressing (A) Cx50-S50P or (B) wild-type Cx46 proteins were immunostained and examined by fluorescence microscopy. Merged images taken at x100 (A) and x60 (B,C,D) exhibit Cy2 (green) and/or Cy3 (red) staining of connexins and DAPI staining of cell nuclei (blue). The images showed that Cx50-S50P alone fails to correctly localize to the cell membrane, instead showing the accumulation of connexin subunits in subcellular compartments surrounding the nucleus (A). Wild-type Cx46 was efficiently translated and localized to the membrane specifically at areas of cell to cell apposition (B, arrow). (C,D) HeLa cells cotransfected with both Cx50-S50P (C) and wild-type Cx46 (D) showed that in the presence of Cx46, S50P subunits were colocalized to the membrane and form junctional plaques with at areas of cell-cell contact. (E,F) Fluorescence images of Cx50-S50P immunostaining in the bow region of homozygous mutant lenses (E) with Cx46 (Cx50(S50P/S50P)–Cx46(+/+)) and (F) without endogenous Cx46 (Cx50(S50P/S50P)–Cx46(–/–)). These lenses taken from mice on postnatal day 7 showed more punctate Cx50 staining (green) which extended deeper into the lens differentiating fibers in the presence of wild-type Cx46 proteins. Frozen Lens sections were co-stained with Rhodamine-phalloidin (red) and DAPI (blue). Bars, 5 µm (A-D), 40 µm (E-F).

View larger version (57K): [in this window] [in a new window]   Fig. 7. Description of embryonic and adult lens phenotypes produced by mixing Cx50-S50P and wild-type lens fiber connexins. This model describes the embryonic and postnatal phenotypes of various mouse lenses created by breeding heterozygous mutant (Cx50(S50P/+)–Cx46(+/+)) and double knockout (Cx50(–/–)–Cx46(–/–)) mice. The drawing explains the proposed mechanism behind development of each unique phenotype based on the electrophysiological interactions documented herein.