Volume 8 issue 9 September 2007
The PY-motif of ENaC, mutated in Liddle syndrome, regulates channel internalization, sorting and mobilization from a sub-apical pool
Lu C., Pribanic S., Debonneville A., Jiang C. and Rotin D

Supplementary Figure 1: Poor recycling of internalized ENaC. ENaC-expressing MDCK-I cells were grown to confluency on permeable support (filters) and once polarized, immunostained with anti myc antibodies (red) at 4°C without permeabilization, to visualize only the cell-surface pool of ENaC. Cells were transferred to 37°C for the indicated period of times. Note the rapid internalization of ENaC upon temp shift from 4 to 37°C, and that only a small fraction of cell surface ENaC is recycled back to the plasma membrane (after 30-60 min). This rapid internalization is further discussed below in the Supplementary Discussion.

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Supplementary Figure 2: ENaC labeled with myc-Fab fragments does not recycle. ENaC-expressing MDCK-I cells (non-permeabilized) were labeled at 4°C with either monoclonal myc antibodies or monovalent myc-Fab fragments, washed and incubated at 37°C for the indicated times, to follow the return of recycled ENaC to the cell surface. Data are summaries of 2 experiments each in 4 or 8 replicates.

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Supplementary Figure 3: Replenishment of internalized cell-surface ENaC with channels originating from an intracellular pool. .MDCK-I cells stably expressing ENaC (WT or its PY-motif mutants) were treated with CHX to inhibit synthesis of new channels (and thus the arrival of new channels to the PM), allowed ENaC to internalize until close to its half-life (t½) value (30 min for WT and 120 min for the mutants) and labeled with anti myc antibodies at 4°C. After washing away unbound antibodies, cells were incubated for 5 min at 37°C to determine the amount of ENaC remaining at or recycled back to the cells surface. ie, parallel experiments were performed simultaneously to detect: (i) the amount of cell surface ENaC at the beginning of the experiment (white bars); (ii) the amount of ENaC remaining at the cell surface after CHX treatment (red bars); (iii) the amount of ENaC remaining at the cell surface after rapid internalization with a temp shift from 4 to 37°C (yellow bars); and (iv) the amount of ENaC replenished at the cell surface at the end of the experiment (ie after 5 min incubation at 37°C, blue bars). The experiment was repeated 3 times, each in quadruplicates. Bars are Means ± SD. Asterisk denotes statistically significant difference (P=0.0005, paired t-test).

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Supplementary Figure 4: Ubiquitination of total cellular pool of ENaC by Nedd4-2. (A) Catalytically-inactive Nedd4-2(CS) inhibits ubiquitination of ENaC: MDCK-I cells stably expressing αβγENaC were transiently transfected (or not) with V5-Nedd4-2(CS). αENaC was then immunoprecipitated from total lysates of these cells with antibodies to its tag (HA) and immunoblotted with anti ubiquitin (Ub, top panel) or anti HA (αENaC) antibodies (lower panel). Lowest panel depicts expression of transfected Nedd4-2(CS). (B) The Y618A mutant of ENaC exhibits impaired ubiquitination: MDCK-I cells stably expressing WT-ENaC or the PY motif mutant Y618A, were lysed, αENaC immunoprecipitated with anti HA antibodies and proteins immunoblotted for anti ubiquitin (Ub, top panel) or anti HA (αENaC) (bottom panel) antibodies. Note impairment of ubiquitination in cells overexpressing a catalytically-inactive Nedd4(CS) (A) or expressing the PY motif mutant (Y618A) that cannot efficiently bind Nedd4-2 (B).

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Supplementary Figure 5: cAMP enhances rapid replenishment of ENaC at the plasma membrane. MDCK-I cells stably expressing WT or the PY-motif mutant of ENaC were treated (at 37°C) with CHX to inhibit the biosynthetic pathway (and thus the arrival of new channels to the plasma membrane), as described in the legend to Supplementary Fig S3 above. Once they reached approximately their half-life value (red bars), 8-CPT-cAMP + IBMX (cAMP stimulation) were added to the cells and cells incubated for 30 min at 37°C (blue bars). Myc labeling was performed at the end of the treatment. White bars: myc labeling at 4°C without CHX and cAMP treatment (indicating the relative amount of ENaC at the beginning of the experiment). Orange bars: cAMP treatment alone (without CHX). Bars are mean±SD of 3 separate experiments, each in triplicates.

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Supplementary Movie 1: Internalization of WT-ENaC. Live MDCK-I cells stably expressing WT-ENaC were incubated with CHX, myc-labeled (at room temp, RT), and ENaC internalization (red color) was followed over time using the spinning disc confocal imaging at 37°C. ConA staining of the apical membrane is in green. Arrows point to several ENaC containing vesicles during internalization. Note that these vesicles internalize quickly, coalesce with other ENaC-containing vesicles and then disappear.

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Supplementary Movie 2: Attenuated internalization of Y618A-ENaC. Live MDCK-I cells stably expressing Y618A-ENaC were incubated with CHX, myc-labeled (at RT), and ENaC internalization (red color) was followed over time using the spinning disc confocal imaging at 37°C. ConA staining of the apical membrane is in green. Arrows point to ENaC containing vesicles. Note poor internalization of these vesicles.

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Supplementary Movie 3: cAMP-dependent recycling of ENaC. Live MDCK-I cells stably expressing WT-ENaC (red color) were incubated with CHX, myc-labeled (at RT), and ENaC internalization was followed over 4hrs in the presence of cAMP stimulation using the spinning disc confocal imaging at 37°C. ConA staining of the apical membrane is in green. Colored arrows point to several ENaC containing vesicles during their recycling back to the apical membrane.

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Supplementary Movie 4: Poor recycling of ENaC in the absence of cAMP stimulation. Live MDCK-I cells stably expressing WT-ENaC (red color) were incubated with CHX, myc-labeled (at RT), and ENaC internalization (red color) was followed over 4hrs using the spinning disc confocal imaging at 37°C. ConA staining of the apical membrane is in green. Note that some of the yellow vesicles that are seen after 50-60 min represent channels that have not yet internalized and are moving laterally at the PM.

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Supplementary Discussion: During the course of our studies, we noticed that a rapid temp shift from 4 to 37°C leads to very fast internalization of ENaC from the cell-surface (~80% within 5 min, Suppl. Fig S1), suggesting an exquisite sensitivity of ENaC to temp changes. We believe this re-warming -dependent rapid internalization is not a result of changes in membrane fluidity, since fluid-phase endocytosis was not affected, but instead represents a phenomenon found in some plasma membrane proteins, including ENaC. We utilized this property of ENaC to test its ability to rapidly (within 5 min) replenish its plasma membrane pool. Our work presented here demonstrates that ENaC can indeed rapidly replenish its cell surface pool from a sub-apical (“reserve”) pool, which is not the recycling pool, and that such replenishment is attenuated in the PY motif (Y618A) mutant of ENaC.