A mutant connexin50 with enhanced hemichannel function leads to cell death - PubMed (original) (raw)
A mutant connexin50 with enhanced hemichannel function leads to cell death
Peter J Minogue et al. Invest Ophthalmol Vis Sci. 2009 Dec.
Abstract
Purpose: To determine the consequences of expression of a novel connexin50 (CX50) mutant identified in a child with congenital total cataracts.
Methods: The GJA8 gene was directly sequenced. Formation of functional channels was assessed by the two-microelectrode voltage-clamp
Method: Connexin protein levels and distribution were assessed by immunoblot analysis and immunofluorescence. The proportion of apoptotic cells was determined by flow cytometry.
Results: Direct sequencing of the GJA8 gene identified a 137 G>T transition that resulted in the replacement of glycine by valine at position 46 of the coding region of CX50 (CX50G46V). Both CX50 and CX50G46V induced gap junctional currents in pairs of Xenopus oocytes. In single Xenopus oocytes, CX50G46V induced connexin hemichannel currents that were activated by removal of external calcium; their magnitudes were much higher than those in oocytes injected with similar amounts of CX50 cRNA. When expressed in HeLa cells under the control of an inducible promoter, both CX50 and CX50G46V formed gap junctional plaques. Induction of CX50G46V expression led to a decrease in the number of cells and an increase in the proportion of apoptotic cells. CX50G46V-induced cell death was prevented by high concentrations of extracellular calcium ions.
Conclusions: Unlike previously characterized CX50 mutants that exhibit impaired trafficking and/or lack of function, CX50G46V traffics properly to the plasma membrane and forms functional hemichannels and gap junction channels; however, it causes cell death even when expressed at minute levels. The biochemical results indirectly suggest a potential novel mechanism by which connexin mutants could lead to cataracts: cytotoxicity due to enhanced hemichannel function.
Figures
FIGURE 1
Identification of a novel mutation of CX50 in a patient with total cataract. (A, B) Chromatograms show the sequence through the region of the GJA8 gene containing nucleotides 132–141 of the coding region from an unaffected (A) and from the affected (B) individual. (C) Diagram showing the predicted localization of the G46V missense mutation within the topology of CX50 in the plasma membrane.
FIGURE 2
CX50G46V forms large hemichannel currents in Xenopus oocytes that can be blocked by extracellular calcium ions. (A) Comparison of hemichannel currents in control oocytes (upper panel) or oocytes injected with equal amounts of CX50 (middle panel) or CX50G46V (lower panel) cRNA. Currents were elicited in response to voltage clamp steps between −80 and +30 mV in 10 mV increments from a holding potential of −80 mV. (B) Steady-state current-voltage relationships in control, CX50 and CX50G46V cRNA-injected oocytes. Each point in the curve represents the mean ± SEM. Recordings were performed in oocytes kept in modified Barth’s solution containing zero added calcium and 1.0 mM magnesium. (C) Current traces recorded from a CX50G46V cRNA-injected oocyte before and after application of calcium. In the presence of zero added external calcium, a large current was observed that was mostly activated at a holding potential of −40 mV and tended to close on application of large positive and negative voltage clamp steps. Application of 1 mM Ca2+ to the bathing media caused a marked reduction in current amplitude which was more pronounced at negative potentials. The sequence of voltage clamp steps is shown above the currents. The dashed line represents zero current level.
FIGURE 3
Immunodetection of wild type and mutant CX50 in HeLa cells. (A) Aliquots from homogenates of untransfected HeLa cells and HeLa cells transfected with wild type CX50 or CX50G46V without induction and after induction with 1 µM ponasterone A for 48 hours were subjected to immunoblotting using anti-CX50 antibodies. No CX50 protein bands were detected in untransfected HeLa cells (HeLa). HeLa cells transfected with wild type or mutant CX50 (HeLa-CX50ind or HeLa-CX50G46Vind, respectively) showed a low level of CX50 protein expression before induction, but the CX50 protein levels increased markedly in both transfected cells after induction. The migration positions of the molecular mass standards are indicated. (B, C) HeLa-CX50ind and HeLa-CX50G46Vind cells were fixed 48 h after induction and subjected to immunofluorescence using anti-CX50 antibodies. Both HeLa-CX50ind (B) and HeLa-CX50G46Vind (C) showed a significant number of gap junctional plaques. Bar, 18 µm.
FIGURE 4
Induction of CX50G46V protein expression decreased the number of cells. (A–D) Phase-contrast photomicrographs from cultures of HeLa-CX50ind (A, B) and HeLa- CX50G46Vind (C, D) that were left untreated (A, C) or that were induced with 1 µM ponasterone A (B, D). (E) Graph shows the quantitation of these data as mean ± S.E.M. of the number of cells/mm2 for uninduced (−) and induced (+) cultures of HeLa-CX50ind (CX50) and HeLa-CX50G46Vind (CX50G46V). While the number of cells remaining in the culture was dramatically decreased 96 h after treatment of HeLa-CX50G46Vind cells with ponasterone A, the cell density in HeLa-CX50ind cells was unaffected. Bar, 111 µm.
FIGURE 5
Expression of CX50G46V protein decreased cell viability/survival. Graph represents the cell viability of HeLa-CX50ind (WT) and HeLa-CX50G46Vind (G46V) that were left untreated (black bars) or that were treated with 1 µM ponasterone A (gray bars) for 72 h. Cell viability was determined using the MTS assay in which the absorbance at 490 nm is proportional to the number of viable cells.
FIGURE 6
Expression of CX50G46V protein increased the proportion of apoptotic cells. Graphs represent the results of cell cycle analysis of propidium iodide-stained HeLa-CX50ind (A, B) and HeLa-CX50G46Vind (C, D) cells that were left uninduced (A, C) or were induced by treatment with 1 µM of ponasterone A for 48 hours (B, D). This analysis revealed an increase in the sub-G1 fraction after induction of CX50G46V protein expression by treatment with 1 µM ponasterone A for 48 h.
FIGURE 7
Expression of CX50G46V induced hemichannel activity-dependent oocyte death. Oocytes were injected with similar amounts of CX50 or CX50G46V cRNA and incubated in modified Barth’s solution containing 0, 1 or 3 mM Ca2+ overnight at 18°C. (A) Oocytes injected with CX50G46V cRNA showed obvious blebbing and discoloration (arrows) when incubated in modified Barth’s solution containing 1 mM Ca2+ for 17 hours (lower left panel). In contrast, oocytes injected with CX50 cRNA or oligonucleotide antisense to the endogenous Xenopus CX38 (Control) showed no apparent detrimental changes when studied under identical conditions (upper panels). The rate of cell death of the CX50G46V cRNA-injected oocytes was significantly reduced by increasing the external calcium concentration from 1 to 3 mM (lower right panel). (B) Graphs represent the number of surviving oocytes at different times following injection of cRNA in media containing 0, 1 or 3 mM Ca2+. Oocytes were scored for cell viability based on appearance as illustrated in A.
References
- Goodenough DA. The crystalline lens. A system networked by gap junctional intercellular communication. Semin Cell Biol. 1992;3:49–58. - PubMed
- Musil LS, Beyer EC, Goodenough DA. Expression of the gap junction protein connexin43 in embryonic chick lens: molecular cloning, ultrastructural localization, and post-translational phosphorylation. J Membr Biol. 1990;116:163–175. - PubMed
Publication types
MeSH terms
Substances
Grants and funding
- R01 EY010589-14A2/EY/NEI NIH HHS/United States
- 068083/WT_/Wellcome Trust/United Kingdom
- R01EY10589/EY/NEI NIH HHS/United States
- R01 EY008368/EY/NEI NIH HHS/United States
- R01EY08368/EY/NEI NIH HHS/United States
- R01 EY010589/EY/NEI NIH HHS/United States
- WT_/Wellcome Trust/United Kingdom
- R01 EY008368-19/EY/NEI NIH HHS/United States
LinkOut - more resources
Full Text Sources
Medical
Miscellaneous