Regulation of Kv2.1 biogenesis and gating by candidate disease-linked Kv6.1 variants

Figures

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  Figure 1. Kv6.1 variant effects on Kv2.1 current magnitude and activation properties.A, AlphaFold structure of Kv6.1 depicting the position of the [W416C] mutation in the pore region and [L284P] mutation in the voltage sensing domain. B, current density at +30 mV was measured from indicated subunit combinations (1:1 cDNA ratio) after transient transfection in LM cells. One-way ANOVA followed by Tukey post hoc test was used to compare experimental groups, with ∗∗∗ indicating p < 0.001. C, conductance-voltage relationships for Kv2.1 coexpressed with each of the indicated Kv6.1 channels. Cells were stepped between −100 mV and +100 mV (200 ms in 10 mV steps, −100 mV holding potential), followed by a pulse to −30 mV for tail current measurement. Normalized tail current amplitudes were fit with Equation (1), (Kv2.1 V1/2 = −5.9 ± 2.5 mV, k = 11.5 ± 0.5 mV, n = 13; Kv2.1 + Kv6.1, V1/2 = 7.0 ± 2.1 mV, k = 18.5 ± 0.7 mV, n = 14; Kv2.1 + Kv6.1(L284P) V1/2 = 12.3 ± 2.0 mV, k = 19.0 ± 1.1 mV, n = 6; Kv2.1 + Kv6.1(W416C) V1/2 = 2.1 ± 2.4 mV, k = 12.3 ± 0.5 mV, n = 6). D–G, representative patch clamp records for Kv2.1 and indicated Kv6.1 variants. Voltage steps are shown in 20 mV intervals, and the…

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  Figure 2. Modulation of Kv2.1 inactivation properties by Kv6.1.A–D, exemplar recordings of indicated combinations of Kv2.1 and Kv6.1. To assess inactivation, cells were pulsed to 60 mV for 200 ms (I1) repolarized briefly to −100 mV, then stepped between −100 mV to +80 mV in 20 mV increments (4 s conditioning pulse) to induce channel inactivation, followed by another 200 ms test pulse to +60 mV (I2). The ratio of I2/I1 reflects the extent of inactivation during the conditioning pulse. Sweeps leading to maximal inactivation are highlighted in black each panel (+60 mV for Kv2.1, −20 mV Kv2.1 + Kv6.1 and variants). The Kv2.1 control group was cotransfected with soluble GFP to ensure a constant amount of plasmid DNA in the transfection. E, single sweeps from the inactivation protocol, with a conditioning step to −40 mV to illustrate inactivation at intermediate voltages in each group. F, mean voltage dependence of inactivation, determined by the ratio of I2/I1 in each sweep (n = 4–8 per group). G, fractional inactivation (measured using I2/I1 ratio) at −40 mV was measured from indicated subunit combinations (1:1 cDNA ratio) after transient transfection in LM cells. One-way ANOVA followed by…

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  Figure 3. Kv6.1 and variants influence total Kv2.1 expression.A, Kv2.1 was coexpressed with Kv6.1, Kv6.1[L284P], and Kv6.1[W416C] in 1:1 and 1:3 ratios as indicated, for 72 h in HEK293 cells. The Kv2.1 control group was cotransfected with soluble GFP to ensure a constant amount of plasmid DNA in the transfection. Whole-cell lysates were separated using SDS-PAGE and probed with anti-Kv2.1 and β-actin (loading control) antibodies. All the lanes in the representative blot are from the same blot. B, total expression of Kv2.1 in each group was first normalized to their respective β-actin signal and plotted as normalized to the Kv2.1 control expression in each experiment (n = 5). A repeated measures ANOVA test (on raw density normalized to β-actin) followed by a post hoc paired t test was used to compare experimental groups where appropriate (∗ indicates p < 0.05 relative to Kv2.1 alone and # indicates p < 0.05 relative to the Kv2.1:Kv6.1 condition).

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  Figure 4. Kv6.1 expression is diminished by coexpression with Kv2.1.A, Kv6.1 and variants were coexpressed with or without Kv2.1 in a 1:1 ratio as indicated, for 72 h in HEK293 cells. The Kv6.1 and variant control groups were cotransfected with GFP to ensure a constant amount of plasmid DNA in the transfection. Whole-cell lysates were separated by SDS-PAGE and probed using an anti-GFP antibody (Kv6.1 constructs are EGFP-tagged), using β-actin as a loading control. Protein ladder molecular weights are in kDa. B, total expression of Kv6.1 in each group was normalized to the β-actin loading control and is plotted as normalized to the WT Kv6.1 control (expressed alone) expression in the corresponding experiment (n = 4–6). A paired t test was used to compare the expression between experimental groups for Kv6.1 or each variant (∗ and ∗∗∗ indicate p values of p < 0.05 and p < 0.001, respectively).

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  Figure 5. Kv2.1 association with Kv6.1 and variants.A, coimmunoprecipitation of Kv2.1 by EGFP-Kv6.1 variants. Cell lysates were incubated with either anti-GFP or anti-GAPDH (negative control) as pull-down antibodies, and immunoprecipitates were probed with anti-Kv2.1. B, conversely, pulldowns were done with anti-Kv2.1 or anti-GAPDH (negative control), and immunoprecipitates were probed with anti-GFP (n = 3). For both panels A and B, the “+ Kv6.1 or + variants” groups indicate “Kv2.1+ Kv6.1 or variants.” C, cells were transfected with Nanoluc-Kv2.1 and EGFP-Kv6.1 (WT + variants), as indicated, and emission spectra were collected during incubation with Nano-Glo (Promega) substrate. D, spectra for each Nanoluc-Kv2.1 + EGFP-Kv6.1 variant were subtracted from the Nanoluc emission spectrum, to illustrate the appearance of the EGFP BRET acceptor signal (n = 3). The inset bar graph summarizes the area under the curve (AUC) of the EGFP signal for each condition. The control group (Nanoluc-Kv2.1 alone) was co-transfected with soluble GFP to ensure a constant amount of plasmid DNA in the transfection mixture for all experiments. BRET, bioluminescence resonance energy transfer.

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  Figure 6. HALO-label detection of cell surface Kv2.1 expression.A, schematic of a HALO-Kv2.1: Kv6.1 assembly. Membrane-impermeant Halo dye JF635i (shown in red) reacts with the HaloTag in the extracellular S1-S2 linker of Kv2.1 at the cell surface. B, cumulative probability of JF635i integrated intensity from individual cells in each transfection condition (five independent experiments). C, log of integrated intensity of the HaloJF635i normalized to the Kv2.1 alone group (max value, 1) and the dsRed alone group (min value, 0), for indicated combinations of Kv2.1 and Kv6.1 variants. A repeated measures ANOVA (on the raw median log JF635i integrated intensity in each experiment) followed by a Tukey’s post hoc test was used to compare experimental groups where appropriate (∗ indicates p < 0.05 relative to HALO-Kv2.1 alone and # indicates p < 0.05 relative to the HALO-Kv2.1:Kv6.1 WT condition). D, HEK293 cells were transfected with combinations of HALO-Kv2.1 and Kv6.1 (WT or variants) as indicated, labeled with membrane-impermeant Halo-JF635i, and cell-by-cell fluorescence intensity was quantified using high content microscopy. The scale bar (in white) represents 100 μm. E, insets from panel D…

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  Figure 7. Stoichiometric interplay affects expression of Kv6.1 and Kv2.1.A, HEK293 cells were transfected with a constant (200 ng) amount of Kv6.1 (left) or Kv6.1[W416C] (right), and increasing amounts of Kv2.1 (0, 100 ng, 200 ng, 400 ng, 600 ng, 800 ng) for 72 h, and probed for Kv2.1, Kv6.1, or β-actin, as indicated (n = 3). The groups were cotransfected with varying amounts of soluble GFP to ensure constant plasmid DNA in the transfection mixture. B, Kv2.1 bands were normalized to the Kv2.1 signal from the 1:0.5 (WT Kv6.1:Kv2.1) ratio condition. C, Kv6.1 bands were normalized to the Kv6.1 signal from the 1:0 (Kv6.1:Kv2.1) ratio condition. A paired t test was used to compare Kv6.1 versus Kv6.1[W416C] expression at each ratio (∗ indicates p < 0.05, panel C). Data in (B, C) are expressed as mean ± SEM from three independent experiments.

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  Figure 8. Kv2.1 coassembly underlies suppression of Kv6.1[W416C].A, HEK293 cells were transfected with a constant (200 ng) amount of Kv2.1 and increasing amounts of WT Kv6.1 (left) or Kv6.1[W416C] (right), (Kv6.1 cDNA amounts: 0, 100 ng, 200 ng, 400 ng, 600 ng, 800 ng) for 72 h, and probed for Kv2.1, Kv6.1, or β-actin, as indicated (n = 3). The groups were cotransfected with soluble GFP wherever applicable to ensure a constant amount of plasmid DNA in the transfection mixture. Protein ladder molecular weights are in kDa. B, Kv2.1 signal density was normalized to Kv2.1 signal from the 1:0 (Kv2.1:Kv6.1) ratio condition. A paired t test was used to compare Kv2.1 coexpressed with either Kv6.1 or Kv6.1[W416C] expression at each ratio (∗ indicates p < 0.05, panel B). Data are expressed as mean ± SEM from three independent experiments. C, Kv6.1 signal density was normalized to WT Kv6.1 signal from the 1:0.5 (Kv2.1:Kv6.1) ratio condition. Data are expressed as mean ± SEM from three independent experiments. D, schematic diagram indicating stability of Kv2.1 and Kv6.1[W416C] heteromers or homomers of each channel under either excess Kv2.1 or excess Kv6.1[W416C] compared to the other one.

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  Figure 9. Coassembly with Kv2.1 underlies Kv6.1 phosphorylation.A, Kv2.1 was coexpressed with Kv6.1 variants in HEK293 cells for 72 h. Lysates were treated with Lambda phosphatase (LP) as indicated, separated by SDS-PAGE and probed with an anti-Kv2.1 antibody (n = 3). B, Kv2.1 and Kv6.1 variants were cotransfected in HEK293 cells in 1:1 ratio. Lysates were treated with LP as indicated, separated by SDS-PAGE, and probed with an anti-GFP antibody (n = 3). Arrows denote shifted mobility of Kv6.1 and Kv6.1[L284P] when coexpressed with Kv2.1. Mobility shifts are not seen for Kv6.1 (or variants) when expressed alone. C, Kv2.1 and Kv6.1 variants were cotransfected in HEK293 cells, in 3:1 (Kv2.1:Kv6.1) ratio. Lysates were treated with LP as indicated, separated by a 100 μM phos-tag SDS-PAGE gel, and probed with an anti-GFP antibody (n = 2 of 3:1 ratio and n = 4 for Kv2.1: Kv6.1 in 1:1 ratio, not shown here). Phosphorylated Kv6.1 and Kv6.1[L284P] bands are highlighted with arrows. A higher exposure is shown in the dotted box for clearer depiction of phosphorylated bands. Anti–β-actin was used as a loading control in panels A and B. The control groups (individual subunits expressed alone) were…

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  Figure 10. Common features of Kv2.2 and Kv2.1 modulation by Kv6.1.A, current density at +30 mV was measured from indicated subunit combinations (1:1 cDNA ratio of Kv2.2 and Kv6.1 WT or Kv6.1[W416C]) after transient transfection in LM cells. Control groups were cotransfected with soluble GFP to ensure a constant amount of plasmid DNA in the transfection mixture in all experiments. One-way ANOVA followed by Tukey post hoc test was used to compare groups (∗ indicates p < 0.05; ∗∗ indicates p < 0.001), n = 6. B, mOrange-Kv2.2 was coexpressed with Kv6.1, Kv6.1[L284P], and Kv6.1[W416C] in 1:1 ratio for 72 h in HEK293 cells. Whole-cell lysates were separated on SDS-PAGE and probed with anti-Kv2.2 and Na+/K+-ATPase (loading control) antibodies. C, Kv2.2 signal in each group from panel B is plotted as normalized to the Kv2.2 control (channel expressed alone) (n = 3). A repeated measures ANOVA test (on raw Kv2.2 density normalized to β-actin) followed by a post hoc paired t test was used to compare experimental groups (∗ indicates p < 0.05 relative to Kv2.2 alone). D, Kv6.1 or variants were expressed alone or with Kv2.2 (1:1 ratio), for 72 h in HEK293 cells. Whole-cell lysates were separated by…