Crouthamel OE et al. (2023), Increased Hemichannel Activity Displayed by a C...

XB-ART-59614

Int J Mol Sci 2023 Jan 22;243:. doi: 10.3390/ijms24032222.

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Increased Hemichannel Activity Displayed by a Connexin43 Mutation Causing a Familial Connexinopathy Exhibiting Hypotrichosis with Follicular Keratosis and Hyperostosis.

Crouthamel OE, Li L, Dilluvio MT, White TW.

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Mutations in the GJA1 gene that encodes connexin43 (Cx43) cause several rare genetic disorders, including diseases affecting the epidermis. Here, we examined the in vitro functional consequences of a Cx43 mutation, Cx43-G38E, linked to a novel human phenotype of hypotrichosis, follicular keratosis and hyperostosis. We found that Cx43-G38E was efficiently translated in Xenopus oocytes and localized to gap junction plaques in transfected HeLa cells. Cx43-G38E formed functional gap junction channels with the same efficiency as wild-type Cx43 in Xenopus oocytes, although voltage gating of the gap junction channels was altered. Notably, Cx43-G38E significantly increased membrane current flow through the formation of active hemichannels when compared to wild-type Cx43. These data demonstrate the association of increased hemichannel activity to a connexin mutation linked to a skeletal-cutaneous phenotype, suggesting that augmented hemichannel activity could play a role in skin and skeletal disorders caused by human Cx43 mutations.

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Species referenced: Xenopus laevis
Genes referenced: gja1
GO keywords: gap junction [+]

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	Figure 2. The Cx43-G38E mutation forms active connexin hemichannels. Single oocytes were clamped at –40 mV and subjected to voltage pulses ranging from –30 to +60 mV in 10 mV steps. (A) H2O and (B) wild-type Cx43 injected oocytes displayed negligible membrane currents. (C) Cx43-G38E expressing oocytes had much larger hemichannel currents than wild-type Cx43. (D) Peak currents from each pulse were plotted as a function of membrane voltage. Peak currents in wild-type Cx43 (filled circles), or H2O injected (open triangles) oocytes were negligible at all tested voltages. Cx43-G38E (open circles) expressing cells exhibited significantly increased peak currents at all voltages compared to either control or wild-type Cx43 oocytes. Data are the mean ± SE.
	Figure 3. Patch clamp recordings from Xenopus oocytes injected with wild-type Cx43, or mutant Cx43-G38E RNAs. (A) Cell-attached patches obtained from wild-type Cx43 injected cells failed to display any single hemichannel activity between –70 and +70 mV. (B) Cx43-G38E expressing oocytes displayed single hemichannel currents that showed transitions to the fully closed state, or sub-conductance states.
	Figure 4. Localization of Cx43-G38E in transfected HeLa cells. (A–C) HeLa cells (DAPI stain, blue) transfected with wild-type Cx43 displayed a strong Cx43 antibody labeling (red) that concentrated in linear arrays of plaques at cell-to-cell interfaces (white arrowheads) and correlated with GFP fluorescence (green). (D–F) HeLa cells transfected with the Cx43-G38E mutation showed similar punctate Cx43 staining at cellular interfaces. Scale bar = 5 µm.
	Figure 1. Expression of wild-type and mutant connexins in Xenopus oocytes. (A) Western blots probed with an anti-Cx43 antibody showed a prominent 43 kD band in protein samples derived from oocytes injected with wild-type Cx43, or mutant Cx43-G38E RNAs. Oocytes injected with water as a negative control showed no Cx43 signal. (B) Re-probing of the blots with an antibody against β-tubulin showed confirmed equivalent loading of total protein in all three samples. (C) Analysis of band density revealed that mutant Cx43-G38E was translated at levels ~1.6-fold higher than wild-type Cx43 (p < 0.05, t-test) Data are plotted as the mean ± SE and are derived from five independent experiments.
	Figure 5. Cx43-G38E forms gap junction channels with altered voltage gating. (A) Gap junctional conductance was measured between Xenopus oocyte pairs injected with water (n = 16), wild-type Cx43 (n = 17), or mutant Cx43-38E (n = 22). Both wild-type and mutant Cx43 expressing cells were coupled at significantly greater values than the water injected controls (p < 0.05, one way ANOVA), but were not significantly different from each other (p > 0.05, t-test). (B) Wild-type Cx43 gap junction channels had junctional currents that decreased asymmetrically at higher trans-junctional potentials. (C) Junctional currents between cell pairs expressing Cx43-G38E displayed a more symmetrical decline. (D) Steady-state voltage gating of wild-type Cx43 (filled circles) showed an asymmetric decline in steady state conductance at increasing values of transjunctional voltage. Cell pairs expressing Cx43-G38E (open circles) had a more symmetrical reduction in conductance. Solid lines are Boltzmann fits of the data whose parameters are listed in Supplemental Table S1. Data are plotted as the mean ± SE.

References [+] :

Barrio, Gap junctions formed by connexins 26 and 32 alone and in combination are differently affected by applied voltage. 1991, Pubmed, Xenbase