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 depicted. Statistical analysis: one-way ANOVA (P < 0.02) with Tukey’s multiple comparisons test. Specific P values were as follows: *P < 0.0292; **P < 0.0025. (E) Representative currents from the conditions in panel D using the same protocols as those in panel A.