Expression of KID syndromic mutation Cx26S17F produces hyperactive hemichannels in supporting cells of the organ of Corti

Abstract

Some mutations in gap junction protein Connexin 26 (Cx26) lead to syndromic deafness, where hearing impairment is associated with skin disease, like in Keratitis Ichthyosis Deafness (KID) syndrome. This condition has been linked to hyperactivity of connexin hemichannels but this has never been demonstrated in cochlear tissue. Moreover, some KID mutants, like Cx26S17F, form hyperactive HCs only when co-expressed with other wild-type connexins. In this work, we evaluated the functional consequences of expressing a KID syndromic mutation, Cx26S17F, in the transgenic mouse cochlea and whether co-expression of Cx26S17F and Cx30 leads to the formation of hyperactive HCs. Indeed, we found that cochlear explants from a constitutive knock-in Cx26S17F mouse or conditional in vitro cochlear expression of Cx26S17F produces hyperactive HCs in supporting cells of the organ of Corti. These conditions also produce loss of hair cells stereocilia. In supporting cells, we found high co-localization between Cx26S17F and Cx30. The functional properties of HCs formed in cells co-expressing Cx26S17F and Cx30 were also studied in oocytes and HeLa cells. Under the recording conditions used in this study Cx26S17F did not form functional HCs and GJCs, but cells co-expressing Cx26S17F and Cx30 present hyperactive HCs insensitive to HCs blockers, Ca²⁺ and La³⁺, resulting in more Ca²⁺ influx and cellular damage. Molecular dynamic analysis of putative heteromeric HC formed by Cx26S17F and Cx30 presents alterations in extracellular Ca²⁺ binding sites. These results support that in KID syndrome, hyperactive HCs are formed by the interaction between Cx26S17F and Cx30 in supporting cells probably causing damage to hair cells associated to deafness.

Keywords: cochlea; connexin; gap junction; hemichannel; organ of Corti; syndromic deafness.

FIGURE 1

Expression of Cx26S17F changed the distribution of Cx26 and Cx30 in supporting cells of the organ of Corti in cochlear explants. a, (C). Control cochlea (Cx26^+/floxS17F, (A) showed the typical distribution of Cx26 (green) and Cx30 (red), forming heteromeric GJ plaques, as seen in the magnified views of 1 μm thick of OSC, PC, and DC and ISC (C). b, (D). Expression of cKI Cx26S17F induced by the addition of TAT-Cre (Cx26^+/floxS17F TAT-Cre, (B) changed the distribution of mutant Cx26S17F and Cx30. Magnified views are shown in (D), where GJ plaques are reduced, and both Cxs are retained in the intracellular space in OSC. In addition, the ISC expression of GJCs is totally lost. PC and DC did not show any differences. (E). Diagram of the organ of Corti, showing the relative position of the focal plane used for (F) and (G), respectively (cyan rectangle). (F), (G). Transversal views of representative zones of the cultured organ of Corti [from (A) and (B), respectively]. Scale bars: 50 μm (B), 5 μm (D). Supplementary Figure 6 shows the same images of Cx26 and Cx30 localization in separate color panels.

FIGURE 2

cKI Cx26S17F expression increased the activity of HCs in supporting cells, reduced intercellular GJCs coupling, and affected the morphology of hair cell stereocilia in cochlear explants. (A–C). Rate of DAPI uptake by OSC, PC, and ISC in control (blue) and TAT-Cre (red) treated cochlear explants of Cx26^+/floxS17F mice, shown as box and whiskers plots showing the minimum and maximum values, and the box represents the 25th and 75th percentiles, and the central line marks the median. Intragroup comparisons are presented in blue (control) and red (TAT-Cre). (D) Representative fluorescence recoveries in FRAP experiments show a severe reduction of intercellular communication between supporting cells of cKI Cx26S17F cochlear explants (red) compared to the control (blue). (E) Average calcein recovery rates of experiments are shown in d. (F–I). Representative views of stereocilia in the organ of Corti obtained from cochlear culture from the different conditions, phalloidin stained conjugated with Texas-Red (red, cytoskeleton) and DAPI (blue, nuclei). Statistical analysis, *p < 0.05; ****p < 0.0001 (A–E).

FIGURE 3

Mutant Cx26S17F co-localizes with Cx30 and changes the distribution of heteromeric HCs and GJCs in HeLa cells. (A). Strong co-localization of Cx26WT (green) and Cx30 (red) in GJ plaques. (B). Zoom of GJ plaques observed in (A). (C). Strong co-localization of Cx26S17F/Cx30 was observed mainly in intracellular compartments and occasionally in a few small plaques in the appositional zone. (D,E) Zoom of small GJ plaques (D) and intracellular (E) co-localization between Cx26S17F/Cx30. (F,G) Relative expression of each Cx by subcellular area in (F) cells expressing only Cx26WT, Cx26S17F, or Cx30 (homomeric configuration) and (G) cells co-expressing Cx26WT with Cx30 (blue) or Cx26S17F (red). (H). GJ size index between neighbor cells expressing one Cx type or co-expressing Cx26WT with Cx30 or Cx26S17F. Mean values, SEM, and p values are indicated in Table 8. (I, J). PLA (red) shows an association between Cx30 (green) with Cx26WT (no tag) (I), and Cx26S17F (J). Highlighted zone in (I) is shown in lateral panels 1) PLA, 2) Cx30, and 3) DAPI. Scale bar: 10 µm. Intracellular signal in the highlighted square in (J) is shown in 1) PLA, 2) Cx26S17F/Cx30 and 3) DAPI; and small GJCs are shown in a highlighted ellipse in 4) PLA 5) Cx26S17F/Cx30 and 6) DAPI. Scale bar: 3 µm. Scale bar: 10 µm (A,C,I,J), 4 µm (j3 and 6) and 2 µm (b, d, e and i3). Statistical analysis, *p < 0.05; **p < 0.005, ***p < 0.001; ****p < 0.0001 (H). N = 7. Supplementary Figure S7 shows the same images of Cx26 and Cx30 localization in separate color panels and Supplementary Figure 8 show positive and negative controls of PLA experiments.

FIGURE 4

Expression of Cx26S17F impairs GJ functionality of Cx30. (A, B). Co-expression of Cx26S17F/Cx30 strongly reduces gap junctional communication. (A) Representative fluorescence recovery of the bleached cell over time in FRAP experiments. (B) Quantification of total recovery rate. ***p < 0.001. Cx26 WT n = 2; Cx30 n = 2; Cx26WT/Cx30 n = 4; Cx26S17F n = 4.

FIGURE 5

Heteromeric hemichannels formed by Cx26S17F/Cx30 induce strong currents in Xenopus oocytes. (A,B). HC currents in oocytes co-expressing Cx30 (A) or Cx26S17F (B). Representative membrane currents in response to depolarizing voltage steps from a holding potential of -10 mV and stepped in 20 mV increments from −60 mV to +60 mV. (C, D). HCs currents in oocytes co-expressing Cx26WT/Cx30 (C) or Cx26S17F and Cx30 (D). (E). Graph representing the current/voltage relationship obtained from HC macroscopic currents. Data points represent mean ± SEM. n = 3.

FIGURE 6

Heteromeric hemichannels formed by Cx26S17F/Cx30 have a hyperactive function and are insensitive to the Cx HC blockers La³⁺ and Ca²⁺. (A). Consolidated data of dye uptake experiments. HeLa cells transfected with indicated Cx-constructs were bathed in Hanks solution containing 1 µM Etd, and 1.26 mM external Ca²⁺, 0 mM (nominal divalent cationic fee solution, DCFS), or DCFS plus 100 µM LaCl₃ (DCFS + La³⁺). (B). Representative Etd uptake in HeLa cells in basal, divalent cation-free solution (DCFS), and in DCFS +100 µM La³⁺. (C). Relative changes in dye-uptake rate when [Ca²⁺]_e is reduced. (D). Relative reduction of dye uptake after application of the HC blocker La³⁺. (E). Normalized Ca²⁺ influx increment with respect to Ca²⁺ uptake rate at 10 μM. Statistical analysis, Mann-Whitney test (A), *p < 0.05; **p < 0.005, ***p < 0.001; ****p < 0.0001 (A–E). n = 13.

FIGURE 7

Expression of Cx26S17F/Cx30 increases cell damage and cell death in HeLa cells. (A–D). Positive signal of annexin V at the membrane (blue, and (B) and PI [nuclear red stain, and (D)] at the nucleus of cells co-transfected with Cx26S17F (green, and (C) and Cx30-mCherry [cytoplasmic and membrane red signal, and (D)]. Dotted ellipse demarks a co-transfected cell with annexin V at the membrane, while the cell demarked with the white arrowhead is also co-expressing the two Cxs but it is in good shape. The cell at the bottom, demarked with a cyan arrowhead shows PI positive signal and co-expression of both Cxs, and rounded morphology as observed in dead cells. (E). Percentage of transfected HeLa cells positive for Annexin V signal, indicative of cell damage and death. Statistical analysis, *p < 0.05. (F). Percentage of transfected HeLa cells positive for PI, indicative of necrosis. Supplementary Figure 9 shows expression of annexin V and PI in cells expressing Cx26WT/Cx30.

FIGURE 8

Possible new interactions in the mutant heteromeric HCs alters regulation by extracellular Ca²⁺. (A,B). Molecular dynamic simulation of a heteromeric HC composed by Cx26 or Cx26S17F and Cx30 (A), according to the most frequent combination (stoichiometry) of each monomer in the heteromeric hemichannel (B), as described in Naulin et al., 2020, which is Cx26-Cx26-Cx30-Cx30-Cx26-Cx30. Colors indicate Cx26 or Cx26S17F in green and Cx30 in grey. (C,D). Zoom of the interaction network around parahelix highlighted in (A). The residues forming salt bridges are shown in sticks with the corresponding label. In the top of the figure the interaction between Asp50 of chain D (Cx30) with Lys61 of the next chain (Cx26), which has been proposed to be associated with the stabilization of open state, appears in Cx26S17F/Cx30 HCs (D), and does not occur in Cx26WT/Cx30 HCs (C).