A conserved arginine/lysine-based motif promotes ER export of KCNE1 and KCNE2 to regulate KCNQ1 channel activity

Figures

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  Figure 1.. The C-terminus of KCNE2 regulates ER export of the protein to the plasma membrane in HEK293 cells. (a) Topology diagrams of modified KCNQ1 (Q1) and KCNE (KCNE2 (E2) or KCNE1 (E1)) subunits. KCNQ1 were tagged with a Myc-epitope in the middle of S1–S2 linker or at its C-terminus. KCNE2 or KCNE1 was fused with an EGFP to its C-terminus or tagged with a HA-epitope at its N-terminus. (b) Confocal images of HEK293 cells co-transfected with indicated E2* (WT and mutant E2)-EGFP and ER-TagRFP. The merged images show the combination. The scale bar is 10 μm. The right column shows the pixel intensity profiles of crossed sections indicated by the white line.

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  Figure 2.. A specific C-terminal sequence proximal to the transmembrane domain of KCNE2 is sufficient for ER export. (a) C-terminal truncations of KCNE2 are displayed, where the dashed lines represent the deleted amino acids. (b) Subcellular localization of the KCNE2 truncations that were expressed as EGFP fusion proteins in HEK293 cells. (c) Immunofluorescence images of HEK293 cells transfected with HA-E2* as indicated. The cells were immunostained with mouse anti-HA primary antibody and goat anti mouse Alexa Fluor 488-conjugated second antibody under nonpermeabilized conditions. The scale bar is 10 μm. (d) Statistics of the relative surface fluorescence intensities of HA-E2* were plotted as indicated. n = 70, 80, 80 for E2, E2ΔC, E2(KSKR). *** p < 0.001, N.S. represents no significance (Student’s t-test).

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  Figure 3.. ER export of KCNE2 is dependent on the positively charged residues in the proximal C-terminus. (a) C-terminal mutations of KCNE2 are displayed, where the dashed lines represent the deleted amino acids and the red represents the amino acids mutated to alanine. (b) Confocal images of the KCNE2 mutations that were expressed as EGFP fusion proteins in HEK293 cells.

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  Figure 4.. Forward trafficking of KCNE2 is essential for the regulation of KCNQ1 activity. (a) Current traces of Q1 and Q1/E2* expressed in HEK293 cells. The voltage protocol was placed at the bottom. All the currents were shown as current density in pA/pF. (b) Statistics of the current densities at 60 mV as indicated. n = 15, 15, 15, 14 for Q1, Q1/E2, Q1/E2ΔC, Q1/E2(KSKR). (c) Immunofluorescence images of HEK293 cells transfected with Myc-Q1 or Myc-Q1/E2*. The cells were immunostained with mouse anti-Myc primary antibody and goat anti mouse Alexa Fluor 488-conjugated second antibody under nonpermeabilized conditions. The scale bar is 10 μm. (d) Statistics of the relative surface fluorescence intensities of Myc-Q1 were plotted as indicated. n = 62, 61, 76, 68 for Q1, Q1/E2, Q1/E2ΔC, Q1/E2(KSKR). *** p < 0.001, ** p < 0.01, N.S. represents no significance (Student’s t-test). (e) Immunofluorescence images of HEK293 cells transfected with HA-E2* or HA-E2*/Q1. The cells were detected with mouse anti-HA antibody and goat anti mouse Alexa Fluor 488-conjugated second antibody under nonpermeabilized conditions. The scale bar is 10 μm. (f) Normalized fluorescence intensities of HA-E2* for the…

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  Figure 5.. The conserved RSKK sequence in the C-terminus of KCNE1 is required for its efficient ER export. (a) C-terminal mutations of KCNE1 are displayed, where the dashed lines represent the deleted amino acids and the red represents the amino acids mutated to alanine. (b) Confocal images of HEK293 cells co-transfected with indicated E1* (WT and mutant E1)-EGFP and ER-TagRFP. The merged images show the combination. The scale bar is 10 μm. The right column shows the pixel intensity profiles of crossed sections indicated by the white line. (c) Subcellular localization of WT and mutant KCNE1 that were expressed as EGFP fusion proteins in HEK293 cells. The scale bar is 10 μm. (d) Immunofluorescence images of HEK293 cells transfected with HA-E1* as indicated. The cells were immunostained with mouse anti-HA primary antibody and goat anti mouse Alexa Fluor 488-conjugated second antibody under nonpermeabilized conditions. The scale bar is 10 μm. (e) Statistics of the relative surface fluorescence intensities of HA-E1* were plotted as indicated. n = 97, 86, 83 for E1, E1ΔC, E1(RSKK). *** p < 0.001, N.S. represents no significance (Student’s t-test).

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  Figure 6.. Role of the conserved sequence (RSKK) in determining the function of Q1/E1 complex. (a) Current traces of Q1 and Q1/E1* expressed in HEK293 cells. The voltage protocol was placed at the bottom. All the currents are shown as current density in pA/pF. (b) G-V curves of Q1 and Q1/E1*. Solid lines were fitted by single Boltzmann function. n = 7, 7, 6, 6 for Q1, Q1/E1, Q1/E1ΔC, Q1/E1(RSKK). (c) Immunofluorescence images of HEK293 cells transfected with HA-E1, HA-E1ΔC or HA-E1ΔC/Q1. The cells were detected with mouse anti-HA antibody and goat anti mouse Alexa Fluor 488-conjugated IgG under nonpermeabilized conditions. The scale bar is 10 μm. (d) Normalized fluorescence intensities of HA-E1* for the transfections of HA-E1, HA-E1ΔC or HA-E1ΔC/Q1. n = 149, 137, 141 for E1, E1ΔC, E1ΔC/Q1. *** p < 0.001 (Student’s t-test).