Nielsen BS et al. (2019), Structural determinants underlying permeant dis...

XB-ART-56328

J Biol Chem 2019 Nov 08;29445:16789-16803. doi: 10.1074/jbc.RA119.007732.

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Structural determinants underlying permeant discrimination of the Cx43 hemichannel.

Nielsen BS , Zonta F , Farkas T , Litman T , Nielsen MS , MacAulay N .

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Connexin (Cx) gap junction channels comprise two hemichannels in neighboring cells, and their permeability is well-described, but permeabilities of the single Cx hemichannel remain largely unresolved. Moreover, determination of isoform-specific Cx hemichannel permeability is challenging because of concurrent expression of other channels with similar permeability profiles and inhibitor sensitivities. The mammalian Cx hemichannels Cx30 and Cx43 are gated by extracellular divalent cations, removal of which promotes fluorescent dye uptake in both channels but atomic ion conductance only through Cx30. To determine the molecular determinants of this difference, here we employed chimeras and mutagenesis of predicted pore-lining residues in Cx43. We expressed the mutated channels in Xenopus laevis oocytes to avoid background activity of alternative channels. Oocytes expressing a Cx43 hemichannel chimera containing the N terminus or the first extracellular loop from Cx30 displayed ethidium uptake and, unlike WT Cx43, ion conduction, an observation further supported by molecular dynamics simulations. Additional C-terminal truncation of the chimeric Cx43 hemichannel elicited an even greater ion conductance with a magnitude closer to that of Cx30. The inhibitory profile for the connexin hemichannels depended on the permeant, with conventional connexin hemichannel inhibitors having a higher potency toward the ion conductance pathway than toward fluorescent dye uptake. Our results demonstrate a permeant-dependent, isoform-specific inhibition of connexin hemichannels. They further reveal that the outer segments of the pore-lining region, including the N terminus and the first extracellular loop, together with the C terminus preclude ion conductance of the open Cx43 hemichannel.

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Species referenced: Xenopus laevis
Genes referenced: gja1 gjb1 gjb2
GO keywords: gap junction [+]

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	Fig. 1. Homology model of Cx43 and alignment of the predicted pore-lining region of Cx43, Cx30 and Cx26. A: Image depicting a homology model of a Cx43 gap junctional channel (AA 1-244), based on the Cx26 gap junction structure. The predicted pore-lining region is highlighted and colored in the typology drawing as well as the in structure of the Cx43 monomer. B: Alignment of the predicted pore-lining region of Cx43, Cx30 and Cx26. The residues of Cx43 selected for mutagenesis are underscored and highlighted in bold.
	Fig. 2. Mutagenesis of select pore-lining residues does not alter Cx43 hemichannel ion conductance. A: Illustrates the predicted pore-lining region of a Cx43 monomer with the select predicted pore-lining residues that were mutated individually to the aligned amino acid in Cx30, marked in pink. B: I-V curves from uninjected-, Cx30-, Cx43-, and Cx43 mutant-expressing oocytes (n=17, n=9, n=11 and n≥7 for the mutations, respectively). C: Summarized membrane currents obtained at -60 mV. D: Ethidium uptake in uninjected-, Cx30-, Cx43-, and Cx43 mutant-expressing oocytes (all n=4). E: Western blot showing oocyte surface expression of Cx43 or the 11 Cx43 mutated constructs. Data in I/V curves and in the bar graphs are presented as mean ± SD. Statistical significance of DCFS-induced hemichannel activity within each group was tested using repeated measures two-way ANOVA (current, interaction: F(26,208) = 46.9, P<0.001; construct: F(13,104) = 68.3, P<0.001; test solution: F(2,208) = 55.0, P<0.001 and Eth, interaction: F(26,84) = 13.2, P<0.001; construct: F(13,42) = 4.2, P<0.001; test solution: F(2,84) = 184.7, P<0.001) with Dunnett’s post hoc test (against control). ; P < 0.01,*; P < 0.001.
	Fig. 3. Cx43 chimera with the predicted pore-lining region of Cx30 display no ion conductance or ethidium uptake. A: Alignment of the N-terminus (NT), transmembrane region 1 (TM1), and extracellular loop 1 (E1) of Cx30 and Cx43 with the predicted pore-lining residues underlined in the amino acid sequence as well as an illustration of Cx43 chimera containing the predicted pore-lining region of Cx30. Western blot of oocyte surface expression of Cx43 and Cx4330 in the right lower corner. B: I-V curves from uninjected-, Cx30-, Cx43-, and Cx4330 -expressing oocytes (n=10, n=8, n=8, and n=8, respectively). C: Summarized membrane currents obtained at -60 mV. D: Ethidium uptake from uninjected-, Cx30-, Cx43-, and Cx4330-expressing oocytes (all n=4). Data in I/V curves and in the bar graphs are presented as mean ± SD. Statistical significance of DCFS-induced hemichannel activity within every group was tested using repeated measures two-way ANOVA (current, interaction: F(8,74) = 214.8, P<0.001; construct: F(4,37) = 54.1, P<0.001; test solution: F(2,74) = 254.4, P<0.001 and Eth, interaction: F(6,24) = 7.2, P<0.001; construct: F(3,12) = 30.0, P<0.001; test solution: F(2,24) = 38.5 P<0.001) with Dunnett’s post hoc test (against control). ***; P < 0.001.
	Fig. 4. Cx43 chimeras containing the N-terminus or the extracellular loop 1 of the Cx30 display DCFS activated membrane currents. A: Alignment of the NT-, TM1-, and E1 of Cx30 and Cx43 with the predicted pore-lining residues underlined in the amino acid sequence as well as an illustration of Cx43 chimeras containing the NT, TM1, or the E1 from Cx30. Western blot of oocyte surface expression of Cx43, Cx4330NT, Cx4330TM1, and Cx4330E1 in the right lower corner. B: Summarized I/V curves from uninjected-, Cx30-, Cx43-, Cx4330NT-, Cx4330TM1- and Cx4330E1-expressing oocytes (n=11, n=7, n=9, n=13, n=11 and n=15, respectively): Summarized membrane currents obtained at -60 mV. D: Ethidium uptake in uninjected-, Cx30-, Cx43-, Cx4330NT-, Cx4330TM1- and Cx4330E1-expressing oocytes (all n=5). Data in I/V curves and in the bar graphs are presented as mean ± SD. Statistical significance of DCFS induced hemichannel activity within each group was tested using repeated measures two-way ANOVA (current, interaction: F(10,120) = 137.5, P<0.001; construct: F(5,60) = 92.8, P<0.001; test solution: F(2,120) = 315.5, P<0.001 and Eth, interaction: F(10,48) = 20.3, P<0.001; construct: F(5,24) = 6.2, P<0.001; test solution: F(2,48) = 182.8 P<0.001) with Dunnett’s post hoc test (against control) and marked by asterisks above the bar. ***; P < 0.001.
	Fig. 5. Combination of C-terminal truncation of Cx43 (M257) with the incorporation of both the Nterminus and the first extracellular loop of Cx30 enhances the DCFS-activated membrane currents. A: Alignment of the NT-, TM1-, and E1 of Cx30 and Cx43 with the predicted pore-lining residues underlined in the amino acid sequence as well as an illustration of the Cx43M257 chimera containing the NT and the E1 from Cx30. B: Summarized I/V curves from uninjected-, Cx30-, Cx43-, and Cx43M25730NT-E1-expressing oocytes (n=9, n=9, n=9, n=10, and n=11, respectively). C: Summarized membrane currents obtained at -60 mV. D: Ethidium uptake in uninjected-, Cx30-, Cx43-, and Cx43M25730NT-E1-expressing oocytes (all n=5). Data in I/V curves and in the bar graphs are presented as mean ± SD. Statistical significance of DCFS-induced hemichannel activity within every group was tested using repeated measures two-way ANOVA (current, interaction: F(6,64) = 20.9, P<0.001; construct: F(3,32) = 16.5, P<0.001; test solution: F(2,64) = 51.5, P<0.001 and Eth, interaction: F(6,32) = 64.5, P<0.001; construct: F(3,16) = 43.3, P<0.001; test solution: F(2,32) = 194.6, P<0.001) with Dunnett’s post hoc test (against control) and marked by asterisks above the bars, when significant. ***; P < 0.001.
	Fig. 6. Molecular dynamics of Cx30, Cx43 and Cx4330NT-E1 hemichannels. A-B: Side (A) and extracellular (B) view of the Cx30 model. The hemichannel is inserted in a phospholipid bilayer and simulations are performed in explicit solvent environment. Each of the six connexins composing the hemichannel are represented with different colors. K+ is blue and Clis red. In panel A, which represents a single snapshot of the simulation, it is possible to notice the accumulation of K+ in the extracellular part of the channel. C: Cx30 hemichannel embedded in the phospholipid bilayer. The proteins are represented according to their predicted solvent accessible surfaces. Two connexins are removed for showing the internal part of the channel. D: The graphs show the K+ density along the Z axis of the different hemichannels during the molecular dynamics simulations. The Z positions are centered on the midpoint of mass of each hemichannel, and the graphs are reported in register with the Cx30 hemichannel represented in panel C. The two orange dotted lines show the position of residues 42 and 49 in Cx30, which corresponds to 43 and 50 for Cx43 and Cx4330NT-E1. It is evident how K+ accumulates in the extracellular vestibule of Cx30 and Cx4330NT-E1, compared with Cx43. E-F: The position of acidic residues facing the pore in the E1 region for Cx30 (E) and Cx43 (F). For clarity, we report the position in one or two connexin polypeptides only.
	Fig. 7. Permeant-dependent isoform-specific inhibition. A-E (left panels): Cx26- and Cx30-mediated currents were obtained in DCFS with the indicated inhibitor concentrations (n=6-11). Membrane currents were recorded in DCFS from +60 to -140 mV in steps of 20 mV from a holding potential of -30 mV and the currents recorded at -120 mV were used for the inhibitor sensitivity curves upon normalization to the current obtained in the absence of inhibitor or peptide. A-E (right panels): Ethidium uptake was determined in Cx30- and Cx43-expressing oocytes after 40 minutes exposure to DCFS with drug concentrations indicated (n=3-6). For gap27 an additional exposure time of 1 hour (preincubated) were performed with 100 µM and 500 µM (n=3). Uninjected oocyte background uptake was subtracted, and the hemichannel-mediated ethidium uptake was normalized to the control uptake in the absence of inhibitor. Data are presented as mean ± SD.

References [+] :

Abbaci, Advantages and limitations of commonly used methods to assay the molecular permeability of gap junctional intercellular communication. 2008, Pubmed