Valdez Capuccino JM et al. (2019), The connexin26 human mutation N14K disrupts cyt...

XB-ART-57033

J Gen Physiol 2019 Mar 04;1513:328-341. doi: 10.1085/jgp.201812219.

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The connexin26 human mutation N14K disrupts cytosolic intersubunit interactions and promotes channel opening.

Valdez Capuccino JM , Chatterjee P , García IE , Botello-Smith WM , Zhang H , Harris AL , Luo Y , Contreras JE .

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A group of human mutations within the N-terminal (NT) domain of connexin 26 (Cx26) hemichannels produce aberrant channel activity, which gives rise to deafness and skin disorders, including keratitis-ichthyosis-deafness (KID) syndrome. Structural and functional studies indicate that the NT of connexin hemichannels is folded into the pore, where it plays important roles in permeability and gating. In this study, we explore the molecular basis by which N14K, an NT KID mutant, promotes gain of function. In macroscopic and single-channel recordings, we find that the N14K mutant favors the open conformation of hemichannels, shifts calcium and voltage sensitivity, and slows deactivation kinetics. Multiple copies of MD simulations of WT and N14K hemichannels, followed by the Kolmogorov-Smirnov significance test (KS test) of the distributions of interaction energies, reveal that the N14K mutation significantly disrupts pairwise interactions that occur in WT hemichannels between residue K15 of one subunit and residue E101 of the adjacent subunit (E101 being located at the transition between transmembrane segment 2 [TM2] and the cytoplasmic loop [CL]). Double mutant cycle analysis supports coupling between the NT and the TM2/CL transition in WT hemichannels, which is disrupted in N14K mutant hemichannels. KS tests of the α carbon correlation coefficients calculated over MD trajectories suggest that the effects of the N14K mutation are not confined to the K15-E101 pairs but extend to essentially all pairwise residue correlations between the NT and TM2/CL interface. Together, our data indicate that the N14K mutation increases hemichannel open probability by disrupting interactions between the NT and the TM2/CL region of the adjacent connexin subunit. This suggests that NT-TM2/CL interactions facilitate Cx26 hemichannel closure.

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Species referenced: Xenopus
Genes referenced: gjb2
GO keywords: gap junction channel activity

???displayArticle.disOnts??? autosomal dominant keratitis-ichthyosis-deafness syndrome
???displayArticle.omims??? KERATITIS-ICHTHYOSIS-DEAFNESS SYNDROME, AUTOSOMAL DOMINANT; KIDAD

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	Figure 1. The NT mutation N14K decreases Ca2+ sensitivity and slows deactivation kinetics of Cx26 hemichannels. (A) Current traces elicited by a voltage pulse from −80 to 0 mV from oocytes expressing hCx26 WT hemichannels in the presence of the indicated Ca2+ concentrations. Normalized deactivation tail currents are shown in the inset for extracellular Ca2+ concentrations at 1.8 (black), 0.5 (blue), and 0.01 (red) mM. (B) Current traces elicited under the same conditions as A in oocyte expressing hCx26 N14K mutant hemichannels. Normalized deactivation tail currents are shown in the inset for identical Ca2+ concentrations as in A. (C) Ca2+ dose–response curve for oocytes expressing hCx26 WT (red) or hCx26 N14K (blue) determined from the peak tail currents after a voltage pulse from −80 to 0 mV. The solid line represents the best fit of the data to a Hill equation. The data points represent mean ± SEM (error bars) of at least five independent measurements.
	Figure 2. The N14K mutation causes channels to open at large negative voltages, unlike WT channels. (A) Representative hemichannel currents recorded from oocytes expressing hCx26 WT (top) and N14K mutant (bottom) protein obtained at 1.8 mM Ca2+. Cells were held at −80 mV, and voltage steps in 10-mV increments were applied for 20 s ranging from −100 to 70 mV. (B) Current–voltage relationship curves obtained from the peak tail currents for hCx26 WT (red) and mutant N14K (blue) hemichannels. The solid lines represent the best fit of the data to a Boltzmann equation. The data points represent mean ± SEM (error bars) of at least five independent measurements.
	Figure 3. Deactivation kinetics in extracellular Ba2+ are significantly slower in N14K hemichannels compared with WT. (A) Current traces elicited by a voltage pulse from −80 to 0 mV from oocytes expressing hCx26 hemichannels in the indicated Ba2+ concentration. (B) Current traces elicited under the same conditions as A in oocytes expressing N14K mutant hemichannels. (C) Dose–response relation of hCx26 WT and N14K mutant for Ba2+ determined from the peak tail currents after voltage pulses from −80 to 0 mV. The solid line represents the best fit of the data to a Hill equation (left graph). Deactivation time constants as a function of Ba2+ concentrations (right graph). The solid lines correspond to linear fits to the data. The data points represent mean ± SEM (error bars) of at least three independent measurements.
	Figure 4. N14K mutant hemichannels display higher open probability than WT Cx26 hemichannels. Single-channel conductance and open probability were determined from data analysis of single-channel recordings performed in oocytes expressing WT Cx26 and N14K hemichannels. (A) Representative single-channel traces for a WT Cx26 or a N14K hemichannel obtained at 20, −20, −40, and −60 mV. The black dashed line indicates the closed state, and the red dashed line indicates the full open state (O). (B) Graph depicts the plot of the open probability (Po) at different voltages (mV) for WT Cx26 (squares) and N14K mutant (circles) hemichannels. The data points represent mean ± SEM (error bars); n = 10.
	Figure 5. Double mutant N14K/H100A hemichannels have gating properties similar to N14K mutant hemichannels. (A) Deactivation time constants as a function of Ba2+ concentration obtained from the maximum peak tail current elicited after a depolarizing step from −80 to 0 mV for WT (red), N14K (blue), H100A (green), and N14K/H100A (black). The solid lines correspond to linear fits to the data. The data points represent mean ± SEM (error bars) of at least three independent measurements (left graph). Representative traces of deactivation currents for WT and mutants after a depolarizing step in presence of 5 mM Ba2+ (right panel). All traces were normalized to a value of 1, therefore the ordinate scale lacks units.
	Figure 6. N14K mutant and N14K/H100A double mutant hemichannels display similar open channel probability. (A) Representative single channel for a N14K/H100A hemichannel obtained at 20, −20, −40, and −60 mV. (B) Graph depicts the plot of the open probability (Po) at different voltage (mV) for WT Cx26 (squares), Cx26N14K/H100A (triangles), and N14K mutant (circles) hemichannels. The data points represent mean ± SEM (error bars); n = 10.
	Figure 7. Pairwise GB energy interaction. The pairwise energy distribution of the 29 pairs with SD larger than 0.1 kcal/mol and P value <10−16 from the KS test. The top five pairs with the largest mean energy differences are marked by boxes. Two subunits from the Cx26 hemichannel structural model display residues at regions of interest. The raw data are provided in Table S1.
	Figure 8. Mutant cycle analysis indicates that NT residues are thermodynamically coupled with adjacent residues at the TM2/CL border. (A) Scheme for mutant cycle analysis of WT and mutant hemichannels and image showing the distance between N14 and H100 in the WT channel (top). Graph shows the voltage dependence for oocytes expressing hCx26 WT (red), N14K (blue), and H100A (green) mutants and N14K/H100A (black) double mutant hemichannels. The solid lines represent the best fit of the data to a Boltzmann equation. (B) Same representations as A for mutants N14K, E101A, and N14K/E101A. The data points represent mean ± SEM (error bars) of at least five independent measurements.
	Figure 9. Correlation analysis (Cij) of α carbon motion. The pairwise absolute correlation distribution of 25 pairs with P < 0.005 from the KS test between WT and N14K mutant. Statistical details: Because there are 226 residues in each subunit, the total number of unique residue pairs between neighboring subunits are 226 ⋅ 226 = 51,076. As for the pairwise interaction analysis (Fig. 7), we first pulled the data from all systems, all replicas, and all six subunits together. We then filtered out the α carbon pairs between neighboring subunits that had either mean absolute Pearson correlation value <0.1 in both systems or the SD of absolute correlation value ≤0.1. Next, 8,389 of 51,076 pairs were subjected to the KS test between WT and N14K mutant using data from all replicas and all subunits. The absolute correlation value distribution of all 25 pairs that had P value <0.005 from the KS test are shown in the figure. The top five pairs that showed the largest change in mean difference in correlation are highlighted in boxes and shown on the protein structure. The raw data are provided in Table S2.

References [+] :

Bargiello, Gating of Connexin Channels by transjunctional-voltage: Conformations and models of open and closed states. 2018, Pubmed