Ilyaskin AV , Korbmacher C , Diakov A .

             vesicle scission involved in clathrin-mediated endocytosis

	Figure 1. Inhibition of endogenous Cx38 and functional expression of human Cx30 in Xenopus laevis oocytes. A, Left panels, representative traces demonstrating the effect of divalent cation removal on whole-cell currents recorded at a holding potential of −60 mV in noninjected oocytes, oocytes injected with antisense oligoDNA against endogenous Cx38 (AS Cx38) or coinjected with AS Cx38 and human Cx30 cRNA (AS Cx38 + Cx30). Bath solution contained 1.8 mM Ca2+ and 1 mM Mg2+ or was nominally free of Ca2+ and Mg2+ and contained 2 mM EDTA for the time intervals (100 s) indicated by open bars (Ca2+Mg2+- Removal). Dashed lines indicate zero current level. Right panels, baseline inward currents in standard Ca2+and Mg2+-containing bath solution (left +), maximum inward currents in the absence of divalent cations (−), and minimum inward currents reached after reapplication of Ca2+ and Mg2+ ions (right +) from similar experiments as shown in left panels (n = 20; N = 2). Lines connect data points from individual oocytes. B, data from the experiments shown in (A) are summarized by subtracting the baseline current values in the presence of divalent cations from the corresponding maximal current value reached after Ca2+ and Mg2+-removal (ΔICa2+Mg2+-Removal). C, summary of baseline current values recorded at the beginning of each experiment in the presence of Ca2+ and Mg2+ in the bath solution. Mean ± SEM and data points for individual oocytes are shown; ∗∗∗p < 0.001; n.s., not significant; Kruskall–Wallis with Dunn’s post hoc test.
	Figure 2. Residual Cx30-mediated cation conductance in the presence of divalent cations (Ca2+, Mg2+) in the extracellular solution.A and B, representative whole-cell current traces recorded in a human Cx30 expressing oocyte (A) or in a control oocyte (B). Oocytes were superfused with standard bath solution (NaCl, open bars) or solution in which Na+ was replaced by NMDG+ (NMDG-Cl, grey bar). Dashed lines indicate zero current level. Continuous holding potential was −60 mV. Before, during, and after exposure to NMDG+ bath solution, voltage step protocols were performed with nine consecutive 2 s voltage steps in 20 mV increments starting with a hyperpolarizing pulse to −120 mV. Insets show overlays of resulting whole-cell current traces obtained at different holding potentials. C and D, Left panels, current data from the final 100 ms portion of each pulse were taken from similar experiments as shown in A and B, respectively, to construct corresponding average I/V curves. Mean and SEM (n = 34; N = 4) are shown. Right panels, reversal potentials obtained from the same experiments summarized in left panels. Individual data points and mean ± SEM are shown. Lines connect data points from individual oocytes. ∗∗∗p < 0.001; paired Student’s t-test. E, Left panel, average I/V plots corrected for endogenous oocyte currents using the data from the experiments summarized in C and D. The average whole-cell current values measured in control oocytes were subtracted from the corresponding individual whole-cell current values measured in oocytes from the same batch expressing Cx30. Right panel, reversal potentials obtained in the same experiments as summarized in the left panel. Individual data points and mean ± SEM are shown. ∗∗∗p < 0.001; paired Student’s t-test. F and G, representative overlays of whole-cell current traces resulting from similar voltage step protocols as described in A and B are shown from an oocyte expressing Cx30 (F) and from a control oocyte (G). For each oocyte current overlays are shown before (−carb) and 1 min after switching to a bath solution containing 1 mM carbenoxolone (+carb). H, current data from the final 100 ms portion of the pulses were taken from similar experiments as shown in F and G. Average I/V plots were constructed using carbenoxolone-sensitive current values (ΔIcarb), which were calculated by subtracting the current values recorded in the presence of carbenoxolone (+carb) from the corresponding current values recorded in its absence (−carb). Mean and SEM (Cx30: n = 21; N = 2; control: n = 27; N = 2) are shown.
	Figure 3. Coexpression of Cx30 inhibits αβγENaC currents in a dose-dependent manner.A, representative whole-cell current traces recorded in a human αβγENaC-expressing control oocyte or in oocytes injected with both αβγENaC and different amounts of human Cx30 cRNA (1–4 ng/oocyte). Application of amiloride (Ami, 2 μM) and removal of divalent cations from the bath solution for 60 s (Ca2+Mg2+- Removal) are indicated by corresponding filled and open bars, respectively. Dashed lines indicate zero current level. B, ΔICa2+Mg2+-Removal values were obtained from similar experiments as shown in A and calculated as described in Figure 1. Mean ± SEM and individual data points for each experiment are shown (n = 20, N = 5). C, ENaC-mediated amiloride-sensitive current values (ΔIAmi) were calculated from similar experiments as shown in A by subtracting the baseline current in the presence of amiloride from the current level reached after amiloride washout. Mean ± SEM and data points for individual oocytes are shown; (n = 68, N = 5). D, the relative inhibitory effect of Cx30 on ENaC was calculated according to the following equation: (ΔIAmiΔIAmi(mean,control)−1)×100%, where ΔIAmi is the amiloride sensitive current of an individual oocyte coexpressing Cx30 and ENaC, whereas ΔIAmi(mean,control) is the mean ΔIAmi measured in control oocytes from the same batch but expressing ENaC alone (control). Original data are the same as in C. Mean ± SEM and data points for individual oocytes are shown; ∗∗p < 0.01; ∗∗∗p < 0.001; n.s., not significant; compared with control (markers above the columns) or to another comparison group as indicated; Kruskall–Wallis with Dunn’s post hoc test.
	Figure 4. Carbenoxolone significantly reduces the inhibitory effect of Cx30 on ENaC.A and B, representative whole-cell current traces recorded in oocytes expressing ENaC without (left traces) or with Cx30 (right traces). After cRNA injection oocytes were incubated for 48 h in incubation solution with carbenoxolone (100 μM, A) or without (mock incubation, B). Carbenoxolone was absent from the bath solution during the whole-cell current recordings. Application of amiloride (Ami, 2 μM) and removal of divalent cations from the bath solution for 60 s (Ca2+ Mg2+-Removal) are indicated by corresponding bars. Dashed lines indicate zero current level. C, ΔIAmi values from similar experiments as shown in the representative traces in A and B. Mean ± SEM and data points for individual oocytes are shown; ∗∗∗p < 0.001; n.s., not significant; Kruskall–Wallis with Dunn’s post hoc test (n = 44, N = 4). D, relative inhibitory effect of Cx30 on ENaC calculated from the data shown in C essentially as described in Figure 3. Mean ± SEM and data points for individual oocytes are shown; ∗∗∗p < 0.001; Mann–Whitney test.
	Figure 5. Mutant Cx30 (M34A) with no detectable ion channel activity did not inhibit ENaC.A, representative whole-cell current traces recorded in oocytes expressing ENaC only (upper trace), coexpressing ENaC with C-terminally V5-tagged wild-type Cx30 (ENaC + Cx30, middle trace) or Cx30 with single point mutation M34A (ENaC + Cx30M34A, lower trace). Application of amiloride (Ami, 2 μM) and removal of divalent cations from the bath solution for 60 s (Ca2+ Mg2+-Removal) are indicated by corresponding bars. Dashed lines indicate zero current level. B, ΔICa2+Mg2+-Removal values were obtained from similar experiments as shown in A and calculated as described in Figure 1. Mean ± SEM and individual data points for each experiment are shown; ∗∗∗p < 0.001; n.s., not significant; one-way ANOVA with Bonferroni post hoc test (n = 36, N = 3). C, ΔIAmi were calculated from similar experiments as shown in A as described in Figure 3. Mean ± SEM and data points for individual oocytes are shown; ∗∗∗p < 0.001; n.s., not significant; Kruskall–Wallis with Dunn’s post hoc test. D, relative inhibitory effect of Cx30 on ENaC calculated from the data shown in C essentially as described in Figure 3. Mean ± SEM and data points for individual oocytes are shown; ∗∗∗p < 0.001; n.s., not significant; Kruskall–Wallis with Dunn’s post hoc test. E and F, representative western blots showing cell surface (E) or intracellular (F) expression of C-terminally V5-tagged wild-type Cx30 and mutant Cx30M34A in oocytes from the same batch as used in A. No specific signal was detected with the anti-V5 antibody in oocytes expressing ENaC alone. To validate separation of cell surface proteins from intracellular proteins, blots were stripped and reprobed using an antibody against β-actin. G and H, densitometric evaluation of Cx30 and Cx30M34A expression from three similar blots as shown in E and F. In each blot the density value of the Cx30M34A band was normalized to that of the Cx30 band. Lines connect data points obtained in the same experiment, and mean ± SEM are shown; n.s. not significant; one sample Wilcoxon signed-rank test (N = 3).
	Figure 6. Extracellular Ca2+modulates the inhibitory effect of Cx30 on ENaC.A and B, representative whole-cell current traces recorded in oocytes expressing ENaC only (A) or coexpressing ENaC with Cx30 (B) incubated for 48 h in solution containing the indicated concentration of Ca2+ ([Ca2+]o = 0.9, 1.8 or 3.6 mM). Application of amiloride (Ami, 2 μM) is indicated by filled bars. Dashed lines indicate zero current level. C, ΔIAmi values from similar experiments as shown in the representative traces (A and B). Mean ± SEM and data points for individual oocytes are shown; ∗∗∗p < 0.001; ∗∗p < 0.01; n.s., not significant; Kruskall–Wallis with Dunn’s post hoc test (n = 24, N = 2). D, relative inhibitory effect of Cx30 on ENaC calculated from the data shown in (C) essentially as described in Figure 3. Mean ± SEM and data points for individual oocytes are shown; ∗∗∗p < 0.001; ∗∗p < 0.01; Kruskall–Wallis with Dunn’s post hoc test.
	Figure 7. Replacing extracellular Ca2+with Ba2+enhances Cx30-mediated inhibition of ENaC.A and B, representative whole-cell current traces recorded in oocytes expressing ENaC alone (left panels) or coexpressing ENaC with Cx30 (right panels) incubated for 48 h in standard incubation solution containing 1.8 mM Ca2+ ([Ca2+]o = 1.8 mM; A) or in incubation solution in which 1.8 mM Ca2+ was replaced with 1.8 mM Ba2+ ([Ba2+]o = 1.8 mM; B). Application of amiloride (Ami, 2 μM) is indicated by filled bars. Dashed lines indicate zero current level. C, ΔIAmi values from similar experiments as shown in the representative traces A and B. Mean ± SEM and data points for individual oocytes are shown; ∗∗∗p < 0.001; n.s., not significant; Kruskall–Wallis with Dunn’s post hoc test (n = 20, N = 2). D, relative inhibitory effect of Cx30 on ENaC calculated from the data shown in C essentially as described in Figure 3. Mean ± SEM and data points for individual oocytes are shown; ∗∗∗p < 0.001; Mann–Whitney test.
	Figure 8. Inhibition of ENaC currents by Cx30 is due to decreased ENaC expression at the cell surface.A, the following groups of oocytes were used: ENaC (injected with 0.6 ng/subunit/oocyte of ENaC cRNA); ENaC+Cx30 (injected with 0.6 ng/subunit/oocyte of ENaC cRNA and 2 ng/oocyte of Cx30 cRNA); 1/3 ENaC (injected with 0.2 ng/subunit/oocyte of ENaC cRNA). To detect channel expression at the cell surface, FLAG-tagged βENaC was coexpressed with wild-type α- and γENaC. ΔIAmi values of individual oocytes were normalized to the mean ΔIAmi measured in oocytes from the corresponding ENaC group. Mean ± SEM and data points for individual oocytes are shown; ∗∗∗p < 0.001; n.s. not significant; Kruskall–Wallis with Dunn’s post hoc test (n = 23, N = 3). B, in parallel with the ΔIAmi measurements shown in A, ENaC-βFLAG surface expression was detected as chemiluminescence signal in relative light units (RLU) using oocytes from the same batch. Control oocytes not expressing ENaC were used to determine the nonspecific background chemiluminescence. Mean ± SEM and data points for individual oocytes are shown; ∗∗∗p < 0.001; Kruskall–Wallis with Dunn’s post hoc test (n = 70, N = 3). C–E, representative western blots showing whole-cell expression of αENaC (C), βENaC (D), or γENaC (E) in oocytes expressing ENaC alone (ENaC) or coexpressing ENaC and Cx30. Specific bands for full-length α-, β-, and γENaC at ∼95 kDa (C–E) and for cleaved γENaC at ∼74 kDa (E) were not detected in control oocytes not expressing ENaC. F–H, densitometric evaluation of full-length bands for αENaC (F) and βENaC (G) and of full-length and cleaved bands for γENaC (H) from similar blots as shown in (C–E). Density values were normalized in each blot to the signal of αENaC (F), βENaC (G), or γENaC (H) bands obtained from oocytes expressing ENaC alone. Lines connect data points obtained in the same experiment, and mean ± SEM are shown; n.s. not significant; one sample Wilcoxon signed-rank test (N = 7). I, in parallel experiments to those shown in (C–H), ΔIAmi was measured to confirm the inhibitory effect of Cx30 on ENaC in these batches of oocytes. Each ΔIAmi value was normalized to the mean ΔIAmi obtained in oocytes expressing ENaC alone. Mean ± SEM and individual data points for each experiment are shown; ∗∗∗p < 0.001; Mann–Whitney test (n = 81, N = 7).
	Figure 9. Cx30 stimulates ENaC internalization.A and B, representative whole-cell current traces are shown from oocytes expressing wild-type α- and γENaC together with a mutant βENaC subunit carrying a single-point mutation (S520C) without Cx30 (ENaC-βS520C, A) or with Cx30 coexpression (ENaC-βS520C+Cx30, B). To determine ΔIAmi at different time points, oocytes were repeatedly clamped for a short period of time at a holding potential of −60 mV and current traces were recorded as illustrated. Oocytes were unclamped during the remaining time of the recording to minimize sodium loading of the oocytes. Impaling microelectrodes were not removed from the oocyte until the end of the experiment. Application of amiloride (Ami, 2 μM) during each current measurement is indicated by filled bars. Dashed lines indicate zero current level. After determining initial ΔIAmi, oocytes were exposed to MTSET for 5 min by exchanging the bath solution from ND96 with 2 μM amiloride to a solution containing in addition 1 mM MTSET (application of MTSET is indicated by arrows). Before the second current measurement (time 0 min) MTSET was washed out with ND96 containing 2 μM amiloride. ΔIAmi was determined every 10 min as indicated (min: 10, 20, …, 70). Between the measurements, oocytes were maintained in ND96 with 2 μM amiloride. At the end of the recordings, oocytes were exposed to MTSET for a second time. C, summary of ΔIAmi values from similar experiments as shown in the representative traces A and B. Mean ± SEM current values for ENaC-βS520C oocytes (black line and symbols; n = 11; N = 3) or ENaC-βS520C+Cx30 oocytes (red line and symbols; n = 12; N = 3). D, data shown in C were normalized as ratio of ΔIAmi measured at different time points to ΔIAmi measured at time 0 min after incubation with MTSET (ΔIAmi/ΔIAmi (0 min)). ∗∗p < 0.01; ∗p < 0.05; n.s., not significant; ENaC-βS520Cversus ENaC-βS520C+Cx30; two-way ANOVA with Bonferroni post hoc test. E and F, linear regression analysis of ΔIRetrievalversus ΔIAmi (0 min) in oocytes expressing ENaC alone (E) or coexpressing ENaC together with Cx30 (F). Each point represents an individual measurement from the same experiments summarized in C and D. Calculated linear regressions with proportionality coefficients (k) are depicted by dashed lines. For better comparison, the diagonal dashed lines depict the hypothetical linear regression with k = 1. G, normalized ΔIRetrieval (ΔIRetrieval/ΔIAmi (0 min)) calculated from the same experiments shown in E and F. ∗∗p < 0.01; Student’s t-test.
	Figure 10. In oocytes coexpressing ENaC and Cx30, the stimulatory effect of chymotrypsin on ENaC currents is similar to that in oocytes expressing ENaC alone. A and B, Left panels, representative whole-cell current traces recorded in an oocyte expressing ENaC alone (A) and in an oocyte coexpressing ENaC and Cx30 (B). Application of amiloride (Ami, 2 μM) or chymotrypsin (2 μg/ml) is indicated by filled and gray bars, respectively. Dashed lines indicate zero current level. Right panels, ΔIAmi values obtained from similar experiments as shown in the representative traces (left panels) before (−) and after (+) chymotrypsin application. Lines connect data points obtained in an individual oocyte; ∗∗∗p < 0.001; Wilcoxon matched-pairs signed-rank test (n = 14, N = 2). C, summary of the individual data shown in (A and B) normalized as relative stimulatory effect of chymotrypsin on ΔIAmi. Mean ± SEM and data points for individual oocytes are shown; n.s., not significant; Student’s ratio t-test.
	Figure 11. Coexpression of Cx30 has no apparent effect on the single-channel current amplitude and open probability of ENaC.A and B, representative continuous single-channel current recordings in an outside-out patch of an ENaC expressing oocyte (A) or an ENaC+Cx30 coexpressing oocyte (B) obtained at a holding potential of −70 mV. Application of amiloride (2 μM) is indicated by filled bars. The current level at which all channels are closed (C) was determined in the presence of amiloride. The channel open levels are indicated by (1) and (2). Single-channel binned current amplitude histograms were obtained by analyzing an 85 s portion of the current trace indicated by a horizontal line and were used to calculate single-channel current amplitude (i) and PO (app) values. C and D, summary of data from similar experiments as shown in A and B. Mean ± SEM and data points for individual oocytes are shown; n.s., not significant; Mann–Whitney test (n = 7, N = 4).
	Figure 12. Intact C-termini of β- and γENaC are necessary for the inhibitory effect of Cx30 on ENaC.A, normalized ΔIAmi values are shown from oocytes expressing wild-type ENaC (αβγ), truncated αENaC (P619X), βENaC (R566X), or γENaC (K576X) with corresponding wild-type ENaC subunits (αTβγ, αβTγ or αβγT) or triple-truncated ENaC (αTβTγT) in each case with or without Cx30. Individual ΔIAmi values were normalized to the mean ΔIAmi obtained in matched oocytes from the same batch expressing wild-type ENaC without Cx30. Mean ± SEM and data points for individual oocytes are shown; ∗∗∗p < 0.001; ∗∗p < 0.01; n.s. not significant; Kruskall–Wallis with Dunn’s post hoc test (67 ≤ n ≤ 153, 5 ≤ N ≤ 7). B, relative inhibitory effect of Cx30 on wild-type (αβγ) or truncated (αTβγ, αβTγ, αβγT or αTβTγT) ENaC calculated as described in Figure 3 using original data shown in A. ΔIAmi obtained in oocytes expressing wild-type (αβγ) or truncated (αTβγ, αβTγ, αβγT, or αTβTγT) ENaC together with Cx30 was normalized to the corresponding mean ΔIAmi recorded in matched control oocytes from the same batch expressing wild-type (αβγ) or truncated (αTβγ, αβTγ, αβγT or αTβTγT) ENaC alone. Mean ± SEM and data points for individual oocytes are shown; ∗∗∗p < 0.001; n.s., not significant; Kruskall–Wallis with Dunn’s post hoc test.
	Figure 13. C-termini of ENaC subunits have overlapping PPPxY- and YxxΦ-motifs.A, schematic representation of α, β, and γENaC illustrating the extracellular loop, two transmembrane domains (black rectangles), and intracellular N- and C-termini. The position within the C-terminus of each subunit at which a truncation mutation was introduced and the localization of the highly conserved PPPxYxxL sequence are indicated by asterisks. B, primary sequence alignment of the corresponding C-terminal regions of human αβγENaC. Highly conserved amino acid residues belonging to PPPxY- and YxxΦ-motifs are highlighted in bold.
	Figure 14. ENaC inhibition by Cx30 does not involve a Nedd4-2-dependent mechanism.A, ΔIAmi values obtained in oocytes expressing wild-type ENaC (αβγ), mutant ENaC (αβγP623A-P625A, αβγY627A, αY644AβY620AγY627A), or coexpressing wild-type ENaC together with xNedd4-CS (αβγ + xNedd4-CS) in each case with (+) or without (−) additional coexpression of Cx30 were normalized as described in Figure 12A. Mean ± SEM and data points for individual oocytes are shown; ∗∗∗p < 0.001; ∗∗p < 0.01; Kruskall–Wallis with Dunn’s post hoc test (32 ≤ n ≤ 185, 3 ≤ N ≤ 4). B, relative inhibitory effect of Cx30 on wild-type ENaC (αβγ), mutant ENaC (αβγP623A-P625A, αβγY627A, αY644AβY620AγY627A), or wild-type ENaC coexpressed with xNedd4-CS (αβγ + xNedd4-CS) calculated as described in Figure 12B using original data shown in A. Mean ± SEM and data points for individual oocytes are shown; ∗p < 0.05; ∗∗∗p < 0.001; n.s., not significant; Kruskall–Wallis with Dunn’s post hoc test.
	Figure 15. Inhibition of clathrin-mediated endocytosis significantly decreases the inhibitory effect of Cx30 on ENaC.A and B, representative whole-cell current traces recorded in oocytes expressing ENaC alone (left panels) or coexpressing ENaC with Cx30 (right panels) incubated for 1 h in standard incubation solution containing 0.02% DMSO (A; mock incubation) or 30 μM Pitstop-2 with 0.02% DMSO (B; Pitstop-2 was dissolved in DMSO to prepare a stock solution). Application of amiloride (Ami, 2 μM) is indicated by filled bars. Dashed lines indicate zero current level. C, ΔIAmi values from similar experiments as shown in the representative traces in A and B. Mean ± SEM and data points for individual oocytes are shown; ∗∗∗p < 0.001; Kruskall–Wallis with Dunn’s post hoc test (n = 36 for each group, N = 3). D, relative inhibitory effect of Cx30 on ENaC calculated from the data shown in C essentially as described in Figure 3. DMSO, dimethyl sulfoxide. Mean ± SEM and data points for individual oocytes are shown; ∗∗∗p < 0.001; Mann–Whitney test.
	Figure 16. YxxΦ-motif of β- and γENaC is critically involved in Cx30-mediated ENaC inhibition.A, ΔIAmi values obtained in oocytes expressing wild-type (αβγ), truncated (αβγK626X, αβγA635X), or mutant (αβγL630D, αβL623Dγ, αβL623DγL630D) ENaC with or without Cx30 were normalized as described in Figure 12A. Mean ± SEM and data points for individual oocytes are shown; ∗∗∗p < 0.001; ∗∗p < 0.01; n.s., not significant; Kruskall–Wallis with Dunn’s post hoc test (30 ≤ n ≤ 172, 3 ≤ N ≤ 4). B, relative inhibitory effect of Cx30 on wild-type (αβγ), truncated (αβγK626X, αβγA635X), or mutant (αβγL630D, αβL623Dγ, αβL623DγL630D) ENaC calculated as described in Figure 12B using original data shown in (A). Mean ± SEM and data points for individual oocytes are shown; ∗∗∗p < 0.001; n.s., not significant; Kruskall–Wallis with Dunn’s post hoc test.

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