Structural determinants at KCNE4 position 145 govern Kv1.3 channel function

Figure 1.. Human KCNE4 145D/E polymorphism. (A) Presence of human KCNE4 reduces Kv1.3 currents. HEK293 cells were transfected with hKv1.3 without or with (+) hKCNE4. Currents were elicited with 250-ms-long pulses from −80 mV to +60 mV in 10-mV steps. (B) Normalized peak current density of hKv1.3 in the absence or presence (+) of hKCNE4 at +60 mV. KCNE4 accelerates slow C-type inactivation of Kv1.3. (C) Schematic cartoon of the KCNE4 regulatory subunit with the arrowhead indicating the distal location of the rs12621643 (145D/E) polymorphism. (D) KCNE4 with predicted sequence disorder (top), residue charge (middle), and TMD (orange box) and 145 polymorphism location sequence (bottom). Blue represents basic (positively charged) residues, and red represents acidic (negatively charged) residues.

Figure 2.. KCNE4 145D/E polymorphism differentially decreases Kv1.3 currents. HEK293 cells were transfected with Kv1.3 in the absence or presence (+) of different variants of KCNE4 (+145D, +145E, and +145A). (A) Representative currents of Kv1.3. Currents were elicited by 250-ms-long pulses from −80 mV to +60 mV in 10-mV steps and tail currents measured at −40 mV. (B) Peak current density was measured at +60 mV for Kv1.3 either without (1,245 ± 133 pA/pF, n = 10) or with different KCNE4 mutants (+145D [732 ± 133 pA/pF, n = 13], +145E [266 ± 32 pA/pF, n = 19], and +145A [290 ± 66 pA/pF, n = 8]). Statistical analysis: Welch’s ANOVA test (P < 0.0001) and unpaired t test with Welch’s correction. Specific P values were as follows: *P < 0.05 (0.0123, Kv1.3 vs. +145D); **P < 0.01 (0.0038, +145D vs. +145E; 0.0077, +145D vs. +145A); ***P < 0.001 (<0.0001, Kv1.3 vs. +145E; <0.0001, Kv1.3 vs. +145A). (C) IV relationship for Kv1.3 currents in the absence or presence of different KCNE4 variants. (D) GV plot of Kv1.3 tail currents measured at −40 mV. Black circles, Kv1.3 alone; blue squares, +145D; orange triangles, +145E; and green diamonds, +145A. The values represent the mean ± SE of 5–8 independent…

Figure 3.. Association and plasma membrane targeting of Kv1.3 in the presence of KCNE4 are not affected by the 145D/E polymorphism. HEK293 cells were transfected with Kv1.3 and different KCNE4 variants (145D, 145E, and 145A). (A) Representative coIP experiments. Total lysates were IP against CFP (KCNE4) and IB against Kv1.3 and KCNE4 (CFP). SM, starting material; IP-, absence of antibody. (B) Quantification of Kv1.3-KCNE4 coIP results from four independent experiments. The values represent the mean ± SE. Statistical analysis: RM one-way ANOVA. (C–E) HEK293 cells were transfected with Kv1.3-HA with or without KCNE4-CFP variants. Nonpermeabilized cells were incubated with an anti-HA antibody and a secondary antibody conjugated to Cy5. The Cy5 intensity was measured by flow cytometry. (C) Plot of HEK293 cells expressing Kv1.3-HA and CFP as a negative control (top), Kv1.3 + KCNE4 145D (center), and Kv1.3 + KCNE4 145E (bottom) with Cy5 (y axis) and CFP (x axis) fluorescence intensity for each combination (left). On the right, histogram for Cy5 intensity of the CFP-positive cells for each condition (circled in red in the left). (D) Schematic representation of cells expressing Kv1.3-HA (black)…

Figure S1.. Plasma membrane trafficking of the KCNE4 variants. (A) Representative confocal images of HEK293 cells transfected with KCNE4-CFP 145D, 145E, and 145A (blue) and a DsRed membrane marker (red). The panels on the right show the respective pixel-by-pixel profiles indicated by the white arrows in the merged panels. Blue, KCNE4 variants; red, membrane marker. (B) MOC for KCNE4 overlapping with the membrane marker (Mb). The individual values and the mean ± SE were plotted for KCNE4 145D (37 cells), 145E (55 cells), and 145A (25 cells). Statistical analysis: one-way ANOVA. P: 0.225, n.s., not significant.

Figure S2.. Both KCNE4 (145D and 145E) variants similarly impair the plasma membrane targeting of Kv1.3. Representative confocal microscopy images of a triple-colocalization experiment of Kv1.3, KCNE4, and DsRed membrane marker. (A) Representative images of HEK293 cells transfected with a membrane marker (red), Kv1.3-YFP (green), and KCNE4-CFP variants (blue). In the right panels, the plot profiles correspond to the sections indicated with white arrows in the merged panels. Scale bar: 10 µm. (B) MOC of Kv1.3 at the plasma membrane (Mb) in the absence (Kv1.3 alone, 45 cells) or in the presence of KCNE4 variants (+145D, 39 cells; +145E, 51 cells; or +145A, 27 cells). P: <0.0001. (C) MOC for KCNE4 present at the plasma membrane (Mb) in the presence of Kv1.3. P: 0.764. (D) MOC for Kv1.3 colocalization with KCNE4. P: 0.093. Individual values and mean ± SE were plotted. Statistical analysis: Kruskal–Wallis with Dunn’s multiple comparisons test. n.s., not significant. ***P < 0.001. Black circles, Kv1.3 alone; blue squares, +145D; orange triangles, +145E; and green diamonds, +145A.

Figure 4.. 145D/E polymorphism does not alter the KCNE4-dependent acceleration of slow inactivation of Kv1.3. (A) Representative 5-s-long traces at +60 mV of HEK293 cells transfected with Kv1.3 without or with KCNE4 (+145D, +145E, +145A). (B) Normalization of representative currents in A to visualize the slow inactivation of the channel. (C) 5-s traces were fitted with a monoexponential equation to calculate the τinactivation. The values represent the mean ± SE of 5–19 cells for each condition from four different transfections, as depicted. Statistical analysis: one-way ANOVA (P < 0.026) with Tukey’s multiple comparisons test. Specific P values were as follows: *P < 0.05 (0.0344 vs. +145D; 0.0371 vs. +145E; 0.0229 vs. +145A). (D) Representative currents of a train of 15 depolarizing pulses at a frequency of 2.5 Hz at +60 mV. (E) Relativized peak current for each of the 15 pulses from the protocol in D. (F) Current decay from E was fitted to a monoexponential decay to calculate the decay constant. The values represent the mean ± SE of 5–9 cells for each condition, as depicted. In each experiment, two different random conditions—always including Kv1.3 alone—from four different transfections,…

Figure S3.. Steady-state inactivation of Kv1.3 in the presence of KCNE4 variants. HEK293 cells were transfected with Kv1.3 in the absence or presence of different KCNE4 variants (+145D, +145E, +145A). (A) Representative two-step currents analyzing the steady-state inactivation of Kv1.3. The cells were held at −80 mV. Currents were elicited by a 2-s depolarizing prepulse from −60 mV to +40 mV followed by a 250-ms test pulse at +40 mV to measure the voltage dependence of inactivation. (B) Relativized peak currents of the test pulse were fitted to a Boltzmann equation. Black circles, Kv1.3 alone; blue squares, +145D; orange triangles, +145E; and green diamonds, +145A. The values represent the mean ± SE of 2–3 cells.

Figure 5.. Charge and position at residue 145 govern KCNE4-dependent Kv1.3 current reduction. HEK293 cells were transfected with Kv1.3 in the absence or presence of a series of KCNE4 variants. (A) Representative currents of Kv1.3 in the presence of KCNE4 WT (145D) and its mutants (+145K, +145N, and +145Q). The cells were held at −80 mV, and the currents were elicited by 250-ms-long pulses to +60 mV in 10-mV steps. Tail currents were measured at −40 mV. (B) Peak current density at +60 mV. The values represent the mean ± SE of 5–13 cells from four different transfections, as depicted. Statistical analysis: one-way ANOVA (P < 0.02) with Tukey’s multiple comparisons test. Specific P values were as follows: *P < 0.05 (0.0426 +145K vs. +145N); **P < 0.01 (0.0095 +145D vs. +145Q); ***P < 0.001 (0.0010 +145D vs. +145N). (C) Schematic representation of KCNE4 highlighting the anionic cluster containing the distal 145 polymorphic position (in bold). Residues just before and after 145 were mutated to either D or E. (D) Peak current density of Kv1.3 in the presence of KCNE4 WT (DDE), KCNE4 DED, and KCNE4 EDE. The values represent the mean ± SE of 7–8 cells from three different transfections, as…