Abbott GW (2017), β Subunits Functionally Differentiate Human Kv4...

XB-ART-53097

Front Physiol 2017 Jan 01;8:66. doi: 10.3389/fphys.2017.00066.

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β Subunits Functionally Differentiate Human Kv4.3 Potassium Channel Splice Variants.

Abbott GW.

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The human ventricular cardiomyocyte transient outward K+ current (Ito) mediates the initial phase of myocyte repolarization and its disruption is implicated in Brugada Syndrome and heart failure (HF). Human cardiac Ito is generated primarily by two Kv4.3 splice variants (Kv4.3L and Kv4.3S, diverging only by a C-terminal, S6-proximal, 19-residue stretch unique to Kv4.3L), which are differentially remodeled in HF, but considered functionally alike at baseline. Kv4.3 is regulated in human heart by β subunits including KChIP2b and KCNEs, but their effects were previously assumed to be Kv4.3 isoform-independent. Here, this assumption was tested experimentally using two-electrode voltage-clamp analysis of human subunits co-expressed in Xenopus laevis oocytes. Unexpectedly, Kv4.3L-KChIP2b channels exhibited up to 8-fold lower current augmentation, 40% slower inactivation, and 5 mV-shifted steady-state inactivation compared to Kv4.3S-KChIP2b. A synthetic peptide mimicking the 19-residue stretch diminished these differences, reinforcing the importance of this segment in mediating Kv4.3 regulation by KChIP2b. KCNE subunits induced further functional divergence, including a 7-fold increase in Kv4.3S-KCNE4-KChIP2b current compared to Kv4.3L-KCNE4-KChIP2b. The discovery of β-subunit-dependent functional divergence in human Kv4.3 splice variants suggests a C-terminal signaling hub is crucial to governing β-subunit effects upon Kv4.3, and demonstrates the potential significance of differential Kv4.3 gene-splicing and β subunit expression in myocyte physiology and pathobiology.

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Species referenced: Xenopus laevis
Genes referenced: elavl2 gnl3 kcnd3 kcne4 kcnip2

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	Figure 1. KChIP2b differentially augments Kv4.3L and Kv4.3S current magnitude. (A) Exemplar current traces recorded at +40 mV (voltage protocol inset) from Xenopus oocytes 36–40 h after injection of 0.2 ng cRNA encoding Kv4.3L or Kv4.3S, with or without 1 ng KChIP2b (n = 14–18). Insets: left, scale bars; right, Voltage clamp protocol. Zero current level indicated by dashed line. (B) Mean ± SEM peak current magnitude at +40 mV for currents recorded as in (A) but with 0.04–5 ng Kv4.3L or S cRNA, with or without 1 or 5 ng KChIP2b cRNA, injected per oocytes, as indicated (n = 14–37). P < 0.01 vs. all other groups with similar quantity of cRNA injected, by ANOVA followed by Tukey's HSD test. (C) Sequence alignment (Clustal TCoffee) of human Kv4.2, Kv4.3L and Kv4.3S protein sequences in the S6 to C-terminal region, including the segment missing in Kv4.3s (underlined red). (D) Exemplar current traces recorded at +40 mV (voltage protocol as in panel A) from Xenopus oocytes 36–40 h after injection of 1 ng cRNA encoding hKv4.2 with or without 5 ng hKChIP2b cRNA (n = 16–18). Scale bar inset. Zero current level indicated by dashed line. (E) Mean ± SEM peak current magnitude at +40 mV for currents recorded as in (E) (n = 16–18). *P < 0.0001 between groups. (F) Comparison of Kv4.x current augmentation by hKChIP2b, analyzed from data in panels B and F (1 ng Kv4.x, 5 ng hKChP2b cRNA; n = 16–37).
	Figure 2. KChIP2b differentially affects Kv4.3L and Kv4.3S inactivation rate and voltage dependence. (A) Exemplar current traces recorded from Xenopus oocytes 36–48 h after injection of 1 ng cRNA (as indicated) encoding Kv4.3L or Kv4.3S alone (black, n = 13–14) or in addition to 5 ng cRNA encoding KChIP2 (red, n = 13–31). Insets: right, Voltage clamp protocol; left, scale bars. Zero current level indicated by dashed line. (B) Mean ± SEM peak current/voltage relationship for currents as in (A); n-values as in (A). (C) Box plots showing individual and mean ± SEM values for fast and slow τ of inactivation, and relative amplitude of the fast component (double exponential fit), of Kv4.3L vs. Kv4.3S, recorded as in (A). n = 13–14. NS, P > 0.05. (D) Box plots showing individual and mean ± SEM values for τ of inactivation (single exponential fit) of Kv4.3L vs. Kv4.3S, with KChIP2 co-expression. Currents recorded as in (A); n = 13–14. ***P < 1 × 10−15. (E) Upper, exemplar current trace recorded using a steady-state inactivation protocol, from Xenopus oocytes 36–48 h after injection of cRNA encoding Kv4.3L (1 ng) and KChIP2 (5 ng). Insets: left, Voltage clamp protocol; right, scale bars. Zero current level indicated by dashed line. Lower, mean ± SEM fraction of available channels/voltage relationship for Kv4.3L vs. Kv4.3S, with/without KChIP2b; currents recorded as in (E); n = 6–8. (F) Upper, exemplar current trace recorded using an inactivation recovery protocol, from Xenopus oocytes 48 h after injection of cRNA encoding Kv4.3L (1 ng) and KChIP2 (5 ng). Insets: left and lower right, scale bars; center, voltage clamp protocol. Zero current level indicated by dashed line. Lower, mean ± SEM fractional recovery from inactivation/recovery time for Kv4.3L vs. Kv4.3S, with/without KChIP2b; recorded as in protocol above; n = 4–6.
	Figure 3. KCNE1 differentially affects Kv4.3L and Kv4.3S function with/without KChIP2b. (A) Mean ± SEM peak current/voltage relationship for current traces recorded from Xenopus oocytes 48 h after injection of cRNA encoding Kv4.3L or Kv4.3S (1 ng) with KCNE1 (5 ng), with or without KChIP2b (5 ng; n = 9–17). Inset: Voltage clamp protocol. *P < 0.01 compared to other currents at +40 mV. (B) Box plots showing individual and mean ± SEM values for τ of fast and slow components, and relative amplitude, of fast inactivation (double exponential fit) of Kv4.3L-KCNE1 vs. Kv4.3S-KCNE1 currents recorded as in (A); n = 7–11. P < 0.05; P < 0.01; NS, P > 0.05. (C) Box plot showing individual and mean ± SEM values for τ of Kv4.3L-KCNE1-KChIP2b vs. Kv4.3S-KCNE1-KChIP2b inactivation (single exponential fit) for currents recorded as in (B); n = 8–17. P < 0.01. (D) Mean ± SEM fraction of available channels/voltage relationship for Kv4.3L-KCNE1-KChIP2b vs. Kv4.3S-KCNE1-KChIP2b currents; voltage protocol upper right inset; n = 6. (E) Mean ± SEM fractional recovery from inactivation/recovery time for Kv4.3L-KCNE1 vs. Kv4.3S-KCNE1 with (n = 6) or without (n = 3–4) KChIP2b; voltage protocol upper right inset.
	Figure 4. KCNE2 differentially affects Kv4.3L and Kv4.3S function with/without KChIP2b. (A) Mean ± SEM peak current/voltage relationship for current traces recorded from Xenopus oocytes 36–48 h after injection of cRNA encoding Kv4.3L or Kv4.3S (1 ng) with KCNE2 (5 ng), with or without KChIP2 (5 ng; n = 14–18). Inset: Voltage clamp protocol. *P < 0.001 compared to Kv4.3L-KChIP2-KCNE2 at +40 mV. (B) Box plot showing individual and mean ± SEM values for τ of inactivation (single exponential fit) for currents recorded as in (A); n = 14–18. NS, P > 0.05; *P < 5 × 10−14. (C) Mean ± SEM fraction of available channels/voltage relationship for Kv4.3L-KCNE2 vs. Kv4.3S-KCNE2 (upper) and Kv4.3L-KCNE2-KChIP2 vs. Kv4.3S-KCNE2-KChIP2 (lower) currents; voltage protocol upper right inset; n = 6. (D) Mean ± SEM fractional recovery from inactivation/recovery time for Kv4.3L-KCNE2 vs. Kv4.3S-KCNE2 (upper) and Kv4.3L-KCNE2-KChIP2 vs. Kv4.3S-KCNE2-KChIP2 (lower) currents; voltage protocol upper inset; n = 5–7.
	Figure 5. KCNE3 differentially affects Kv4.3L and Kv4.3S current magnitude with KChIP2b. (A) Mean ± SEM peak current/voltage relationship for current traces recorded from Xenopus oocytes 36–48 h after injection of cRNA encoding Kv4.3L or Kv4.3S (1 ng) with KCNE3 (5 ng) with or without KChIP2 (5 ng; n = 11–20). Inset: Voltage clamp protocol. ***P < 0.001 compared to other currents at +40 mV. (B) Box plots showing individual and mean ± SEM values for τ of fast and slow components, and relative amplitude, of fast inactivation (double exponential fit) of Kv4.3L-KCNE3 vs. Kv4.3S-KCNE3 currents recorded as in (A); n = 10–13. NS, P > 0.05. (C) Box plot showing individual and mean ± SEM values for τ of Kv4.3L-KCNE3-KChIP2 vs. Kv4.3S-KCNE3-KChIP2 inactivation (single exponential fit) for currents recorded as in (B); n = 11–15. NS, P > 0.05. (D) Mean ± SEM fraction of available channels/voltage relationship for Kv4.3L-KCNE3 vs. Kv4.3S-KCNE3 with/without KChIP2; voltage protocol upper right inset; n = 5–7. (E) Mean ± SEM fractional recovery from inactivation/recovery time for Kv4.3L-KCNE3-KChIP2 vs. Kv4.3S-KCNE3-KChIP2; voltage protocol upper inset; n = 4–7.
	Figure 6. KCNE4 differentially affects Kv4.3L and Kv4.3S function with KChIP2b. (A) Mean ± SEM peak current/voltage relationship for current traces recorded from Xenopus oocytes 36–48 h after injection of cRNA encoding Kv4.3L (filled squares) and (circles)/or (open squares) Kv4.3S (1 ng), with KCNE4 (5 ng), with/without KChIP2 (5 ng; n = 10–17). Lower right inset: Voltage clamp protocol. P < 0.01 compared to all other currents at +40 mV, by one-way ANOVA with post-hoc Tukey HSD-test. Upper right inset: no effect on current density of individually increasing Kv4.3L (red columns) or Kv4.3S (open columns) cRNA injected, from 1 to 5 ng per oocyte, when co-injected with KCNE4S and KChIP2b cRNA (n = 11–17). NS = P > 0.05 by one-way ANOVA with post-hoc Tukey HSD-test; all other group comparisons P < 0.01. (B) Box plot showing individual and mean ± SEM values for τ of inactivation (single exponential fit) for Kv4.3L and / or Kv4.3S co-expressed with KCNE4 and KChIP2; n = 7–15. *P < 5 × 10−6 vs. other groups; other comparisons P > 0.05. (C) Mean ± SEM fraction of available channels/voltage relationship for Kv4.3L and/or Kv4.3S co-expressed with KCNE4 and KChIP2; voltage protocol upper right inset; n = 5–6. (D) Mean ± SEM fractional recovery from inactivation/recovery time for Kv4.3L and/or Kv4.3S co-expressed with KCNE4 and KChIP2; voltage protocol upper inset; n = 5–6. Arrow, overshoot following recovery from inactivation.
	Figure 7. KCNE5 differentially affects Kv4.3L and Kv4.3S function. (A) Mean ± SEM peak current/voltage relationship for current traces recorded from Xenopus oocytes 36–48 h after injection of cRNA encoding Kv4.3L or Kv4.3S (1 ng) with KCNE5 (1–5 ng) with/without KChIP2b (5 ng; n = 6–12). P < 0.05 between Kv4.3L and Kv4.3S with similar β subunits. Inset: Voltage clamp protocol. (B) Box plots showing individual and mean ± SEM values for τ of fast and slow components, and relative amplitude, of fast inactivation (double exponential fit) of Kv4.3L-KCNE5 vs. Kv4.3S-KCNE5 currents recorded as in (A); n = 5–7. NS, P > 0.05. (C) Box plot showing individual and mean ± SEM values for τ of inactivation (single exponential fit) for Kv4.3L vs. Kv4.3S co-expressed with KCNE5 and KChIP2; n = 9–12. **P < 5 × 10−8. (D) Mean ± SEM fraction of available channels/voltage relationship for Kv4.3L-KCNE5 vs. Kv4.3S-KCNE5 channels with (n = 5–12) or without (n = 5–9) KChIP2; voltage protocol inset. (E) Mean ± SEM fractional recovery from inactivation/recovery time for Kv4.3L-KCNE5 vs. Kv4.3S-KCNE5 channels with (n = 7–8) or without (n = 5–6) KChIP2; voltage protocol inset.
	Figure 8. Kv4.3S-KChIP2 function is modified by a peptide corresponding to Kv4.3L residues 488–506. (A) Mean ± SEM peak current at 40 mV for current traces recorded from Xenopus oocytes 48–72 h after injection of 1.5 ng cRNA each encoding the following: Kv4.2 with (n = 10) or without (n = 7–8) KChIP2; Kv4.3L with (n = 13–20) or without (n = 8–12) KChIP2; Kv4.3S with (n = 13–17) or without (n = 8) KChIP2, 30–90 min following injection of L peptide (red columns) or control peptide (open columns; final concentration 25 μM). P < 0.05; all other same-subunit combination comparisons P > 0.05. (B) Mean ± SEM inactivation τ at 40 mV for current traces recorded from Xenopus oocytes 48–72 h after injection of cRNAs as in (A). P < 0.05; all other same-subunit combination comparisons P > 0.05.
	Figure 9. Subunit cRNA ratios mimicking those in human HF Kv4.3S- and KChIP2-dependently reduce current. (A) Mean ± SEM peak current/Voltage relationship for current traces recorded from Xenopus oocytes 48–72 h after injection of cRNAs as indicated (n = 12–15; *P < 0.001, *P < 1 × 10−5 vs. current magnitude for other groups at +40 mV). Lower right inset: Voltage clamp protocol. (B) Box plot showing individual and mean ± SEM values for τ of inactivation (single exponential fit) for currents generated as in (A), symbols as in (A) (n = 12–15; P < 0.05, ****P < 1 × 10−5). (C) Averaged current traces elicited by ventricular action potential-like Voltage protocol (center) recorded in oocytes expressing channel subunit ratios as in (A), line colors as in (A), scaled to mean current magnitudes as in (A) (left) or normalized for easier comparison of gating kinetics (right). Each trace is an average of 120–300 raw traces recorded from 4 to 10 cells.

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	Figure 1. KChIP2b differentially augments Kv4.3L and Kv4.3S current magnitude. (A) Exemplar current traces recorded at +40 mV (voltage protocol inset) from Xenopus oocytes 36–40 h after injection of 0.2 ng cRNA encoding Kv4.3L or Kv4.3S, with or without 1 ng KChIP2b (n = 14–18). Insets: left, scale bars; right, Voltage clamp protocol. Zero current level indicated by dashed line. (B) Mean ± SEM peak current magnitude at +40 mV for currents recorded as in (A) but with 0.04–5 ng Kv4.3L or S cRNA, with or without 1 or 5 ng KChIP2b cRNA, injected per oocytes, as indicated (n = 14–37). P < 0.01 vs. all other groups with similar quantity of cRNA injected, by ANOVA followed by Tukey's HSD test. (C) Sequence alignment (Clustal TCoffee) of human Kv4.2, Kv4.3L and Kv4.3S protein sequences in the S6 to C-terminal region, including the segment missing in Kv4.3s (underlined red). (D) Exemplar current traces recorded at +40 mV (voltage protocol as in panel A) from Xenopus oocytes 36–40 h after injection of 1 ng cRNA encoding hKv4.2 with or without 5 ng hKChIP2b cRNA (n = 16–18). Scale bar inset. Zero current level indicated by dashed line. (E) Mean ± SEM peak current magnitude at +40 mV for currents recorded as in (E) (n = 16–18). *P < 0.0001 between groups. (F) Comparison of Kv4.x current augmentation by hKChIP2b, analyzed from data in panels B and F (1 ng Kv4.x, 5 ng hKChP2b cRNA; n = 16–37).
	Figure 2. KChIP2b differentially affects Kv4.3L and Kv4.3S inactivation rate and voltage dependence. (A) Exemplar current traces recorded from Xenopus oocytes 36–48 h after injection of 1 ng cRNA (as indicated) encoding Kv4.3L or Kv4.3S alone (black, n = 13–14) or in addition to 5 ng cRNA encoding KChIP2 (red, n = 13–31). Insets: right, Voltage clamp protocol; left, scale bars. Zero current level indicated by dashed line. (B) Mean ± SEM peak current/voltage relationship for currents as in (A); n-values as in (A). (C) Box plots showing individual and mean ± SEM values for fast and slow τ of inactivation, and relative amplitude of the fast component (double exponential fit), of Kv4.3L vs. Kv4.3S, recorded as in (A). n = 13–14. NS, P > 0.05. (D) Box plots showing individual and mean ± SEM values for τ of inactivation (single exponential fit) of Kv4.3L vs. Kv4.3S, with KChIP2 co-expression. Currents recorded as in (A); n = 13–14. ***P < 1 × 10−15. (E) Upper, exemplar current trace recorded using a steady-state inactivation protocol, from Xenopus oocytes 36–48 h after injection of cRNA encoding Kv4.3L (1 ng) and KChIP2 (5 ng). Insets: left, Voltage clamp protocol; right, scale bars. Zero current level indicated by dashed line. Lower, mean ± SEM fraction of available channels/voltage relationship for Kv4.3L vs. Kv4.3S, with/without KChIP2b; currents recorded as in (E); n = 6–8. (F) Upper, exemplar current trace recorded using an inactivation recovery protocol, from Xenopus oocytes 48 h after injection of cRNA encoding Kv4.3L (1 ng) and KChIP2 (5 ng). Insets: left and lower right, scale bars; center, voltage clamp protocol. Zero current level indicated by dashed line. Lower, mean ± SEM fractional recovery from inactivation/recovery time for Kv4.3L vs. Kv4.3S, with/without KChIP2b; recorded as in protocol above; n = 4–6.
	Figure 3. KCNE1 differentially affects Kv4.3L and Kv4.3S function with/without KChIP2b. (A) Mean ± SEM peak current/voltage relationship for current traces recorded from Xenopus oocytes 48 h after injection of cRNA encoding Kv4.3L or Kv4.3S (1 ng) with KCNE1 (5 ng), with or without KChIP2b (5 ng; n = 9–17). Inset: Voltage clamp protocol. *P < 0.01 compared to other currents at +40 mV. (B) Box plots showing individual and mean ± SEM values for τ of fast and slow components, and relative amplitude, of fast inactivation (double exponential fit) of Kv4.3L-KCNE1 vs. Kv4.3S-KCNE1 currents recorded as in (A); n = 7–11. P < 0.05; P < 0.01; NS, P > 0.05. (C) Box plot showing individual and mean ± SEM values for τ of Kv4.3L-KCNE1-KChIP2b vs. Kv4.3S-KCNE1-KChIP2b inactivation (single exponential fit) for currents recorded as in (B); n = 8–17. P < 0.01. (D) Mean ± SEM fraction of available channels/voltage relationship for Kv4.3L-KCNE1-KChIP2b vs. Kv4.3S-KCNE1-KChIP2b currents; voltage protocol upper right inset; n = 6. (E) Mean ± SEM fractional recovery from inactivation/recovery time for Kv4.3L-KCNE1 vs. Kv4.3S-KCNE1 with (n = 6) or without (n = 3–4) KChIP2b; voltage protocol upper right inset.
	Figure 4. KCNE2 differentially affects Kv4.3L and Kv4.3S function with/without KChIP2b. (A) Mean ± SEM peak current/voltage relationship for current traces recorded from Xenopus oocytes 36–48 h after injection of cRNA encoding Kv4.3L or Kv4.3S (1 ng) with KCNE2 (5 ng), with or without KChIP2 (5 ng; n = 14–18). Inset: Voltage clamp protocol. *P < 0.001 compared to Kv4.3L-KChIP2-KCNE2 at +40 mV. (B) Box plot showing individual and mean ± SEM values for τ of inactivation (single exponential fit) for currents recorded as in (A); n = 14–18. NS, P > 0.05; *P < 5 × 10−14. (C) Mean ± SEM fraction of available channels/voltage relationship for Kv4.3L-KCNE2 vs. Kv4.3S-KCNE2 (upper) and Kv4.3L-KCNE2-KChIP2 vs. Kv4.3S-KCNE2-KChIP2 (lower) currents; voltage protocol upper right inset; n = 6. (D) Mean ± SEM fractional recovery from inactivation/recovery time for Kv4.3L-KCNE2 vs. Kv4.3S-KCNE2 (upper) and Kv4.3L-KCNE2-KChIP2 vs. Kv4.3S-KCNE2-KChIP2 (lower) currents; voltage protocol upper inset; n = 5–7.
	Figure 5. KCNE3 differentially affects Kv4.3L and Kv4.3S current magnitude with KChIP2b. (A) Mean ± SEM peak current/voltage relationship for current traces recorded from Xenopus oocytes 36–48 h after injection of cRNA encoding Kv4.3L or Kv4.3S (1 ng) with KCNE3 (5 ng) with or without KChIP2 (5 ng; n = 11–20). Inset: Voltage clamp protocol. ***P < 0.001 compared to other currents at +40 mV. (B) Box plots showing individual and mean ± SEM values for τ of fast and slow components, and relative amplitude, of fast inactivation (double exponential fit) of Kv4.3L-KCNE3 vs. Kv4.3S-KCNE3 currents recorded as in (A); n = 10–13. NS, P > 0.05. (C) Box plot showing individual and mean ± SEM values for τ of Kv4.3L-KCNE3-KChIP2 vs. Kv4.3S-KCNE3-KChIP2 inactivation (single exponential fit) for currents recorded as in (B); n = 11–15. NS, P > 0.05. (D) Mean ± SEM fraction of available channels/voltage relationship for Kv4.3L-KCNE3 vs. Kv4.3S-KCNE3 with/without KChIP2; voltage protocol upper right inset; n = 5–7. (E) Mean ± SEM fractional recovery from inactivation/recovery time for Kv4.3L-KCNE3-KChIP2 vs. Kv4.3S-KCNE3-KChIP2; voltage protocol upper inset; n = 4–7.
	Figure 6. KCNE4 differentially affects Kv4.3L and Kv4.3S function with KChIP2b. (A) Mean ± SEM peak current/voltage relationship for current traces recorded from Xenopus oocytes 36–48 h after injection of cRNA encoding Kv4.3L (filled squares) and (circles)/or (open squares) Kv4.3S (1 ng), with KCNE4 (5 ng), with/without KChIP2 (5 ng; n = 10–17). Lower right inset: Voltage clamp protocol. P < 0.01 compared to all other currents at +40 mV, by one-way ANOVA with post-hoc Tukey HSD-test. Upper right inset: no effect on current density of individually increasing Kv4.3L (red columns) or Kv4.3S (open columns) cRNA injected, from 1 to 5 ng per oocyte, when co-injected with KCNE4S and KChIP2b cRNA (n = 11–17). NS = P > 0.05 by one-way ANOVA with post-hoc Tukey HSD-test; all other group comparisons P < 0.01. (B) Box plot showing individual and mean ± SEM values for τ of inactivation (single exponential fit) for Kv4.3L and / or Kv4.3S co-expressed with KCNE4 and KChIP2; n = 7–15. *P < 5 × 10−6 vs. other groups; other comparisons P > 0.05. (C) Mean ± SEM fraction of available channels/voltage relationship for Kv4.3L and/or Kv4.3S co-expressed with KCNE4 and KChIP2; voltage protocol upper right inset; n = 5–6. (D) Mean ± SEM fractional recovery from inactivation/recovery time for Kv4.3L and/or Kv4.3S co-expressed with KCNE4 and KChIP2; voltage protocol upper inset; n = 5–6. Arrow, overshoot following recovery from inactivation.
	Figure 7. KCNE5 differentially affects Kv4.3L and Kv4.3S function. (A) Mean ± SEM peak current/voltage relationship for current traces recorded from Xenopus oocytes 36–48 h after injection of cRNA encoding Kv4.3L or Kv4.3S (1 ng) with KCNE5 (1–5 ng) with/without KChIP2b (5 ng; n = 6–12). P < 0.05 between Kv4.3L and Kv4.3S with similar β subunits. Inset: Voltage clamp protocol. (B) Box plots showing individual and mean ± SEM values for τ of fast and slow components, and relative amplitude, of fast inactivation (double exponential fit) of Kv4.3L-KCNE5 vs. Kv4.3S-KCNE5 currents recorded as in (A); n = 5–7. NS, P > 0.05. (C) Box plot showing individual and mean ± SEM values for τ of inactivation (single exponential fit) for Kv4.3L vs. Kv4.3S co-expressed with KCNE5 and KChIP2; n = 9–12. **P < 5 × 10−8. (D) Mean ± SEM fraction of available channels/voltage relationship for Kv4.3L-KCNE5 vs. Kv4.3S-KCNE5 channels with (n = 5–12) or without (n = 5–9) KChIP2; voltage protocol inset. (E) Mean ± SEM fractional recovery from inactivation/recovery time for Kv4.3L-KCNE5 vs. Kv4.3S-KCNE5 channels with (n = 7–8) or without (n = 5–6) KChIP2; voltage protocol inset.
	Figure 8. Kv4.3S-KChIP2 function is modified by a peptide corresponding to Kv4.3L residues 488–506. (A) Mean ± SEM peak current at 40 mV for current traces recorded from Xenopus oocytes 48–72 h after injection of 1.5 ng cRNA each encoding the following: Kv4.2 with (n = 10) or without (n = 7–8) KChIP2; Kv4.3L with (n = 13–20) or without (n = 8–12) KChIP2; Kv4.3S with (n = 13–17) or without (n = 8) KChIP2, 30–90 min following injection of L peptide (red columns) or control peptide (open columns; final concentration 25 μM). P < 0.05; all other same-subunit combination comparisons P > 0.05. (B) Mean ± SEM inactivation τ at 40 mV for current traces recorded from Xenopus oocytes 48–72 h after injection of cRNAs as in (A). P < 0.05; all other same-subunit combination comparisons P > 0.05.
	Figure 9. Subunit cRNA ratios mimicking those in human HF Kv4.3S- and KChIP2-dependently reduce current. (A) Mean ± SEM peak current/Voltage relationship for current traces recorded from Xenopus oocytes 48–72 h after injection of cRNAs as indicated (n = 12–15; *P < 0.001, *P < 1 × 10−5 vs. current magnitude for other groups at +40 mV). Lower right inset: Voltage clamp protocol. (B) Box plot showing individual and mean ± SEM values for τ of inactivation (single exponential fit) for currents generated as in (A), symbols as in (A) (n = 12–15; P < 0.05, ****P < 1 × 10−5). (C) Averaged current traces elicited by ventricular action potential-like Voltage protocol (center) recorded in oocytes expressing channel subunit ratios as in (A), line colors as in (A), scaled to mean current magnitudes as in (A) (left) or normalized for easier comparison of gating kinetics (right). Each trace is an average of 120–300 raw traces recorded from 4 to 10 cells.