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Results: 7

1.
Figure 4

Figure 4. From: Functional Rescue of Kv4.3 Channel Tetramerization Mutants by KChIP4a.

The proximal N-terminus of Kv4.3 is necessary for interaction of the C110A mutant and KChIP4a. (A and B) Current traces of the deletion mutant Kv4.3Δ24-C110A with and without WT KChIP4a were recorded in oocytes held at −80 mV in response to a family of depolarizing potentials from −60 mV to +40 mV in 10 mV increments for 1 s. (C) The scaled and superimposed currents at +40 mV (from panels A and B) are shown to compare the development of inactivation kinetics among Kv4.3Δ24-C110A alone and coexpression of Kv4.3Δ24-C110A and WT KChIP4a. (D) Summary of peak current amplitudes measured at +40 mV from C110A alone, coexpression of C110A and WT KChIP4a, Kv4.3Δ24-C110A alone, and coexpression of Kv4.3Δ24-C110A and WT KChIP4a. All data are shown as mean ± SE for 20–25 oocytes. Statistical significance (∗∗∗P < 0.001) was compared between groups as indicated; ns: no statistical significance between Kv4.3Δ24-C110A alone and coexpression of Kv4.3Δ24-C110A with WT KChIP4a.

Ping Liang, et al. Biophys J. 2010 June 16;98(12):2867-2876.
2.
Figure 1

Figure 1. From: Functional Rescue of Kv4.3 Channel Tetramerization Mutants by KChIP4a.

Functional rescue of Kv4.3 zinc mutants by coexpression of KChIP4a. (A) Current traces of WT Kv4.3 or zinc mutant channels with and without coexpression with WT KChIP4a were recorded in oocytes held at −80 mV in response to a family of depolarizing potentials from −60 mV to +40 mV in 10 mV increments for 1 s. Upper panel: Current traces of WT Kv4.3 or zinc mutant channels alone. Bottom panel: Current traces of coexpression of WT Kv4.3 or zinc mutant channels with WT KChIP4a. (B) Summary of peak currents recorded at +40 mV from panel A. Data are shown as the mean ± SE for 8–24 oocytes, and statistical significance (Student's t-test) is indicated as ∗∗∗P < 0.001. (C) Scaled and superimposed currents recorded at +40 mV from panel A are displayed to compare the development of inactivation kinetics among three zinc mutants with coexpression with WT KChIP4a. (D) Summary of normalized recovery from inactivation fitted with a single exponential function for varying lengths of time in steps from –80 to +40 mV, measured at +40 mV from zinc mutants with coexpression with WT KChIP4a.

Ping Liang, et al. Biophys J. 2010 June 16;98(12):2867-2876.
3.
Figure 6

Figure 6. From: Functional Rescue of Kv4.3 Channel Tetramerization Mutants by KChIP4a.

KIS suppresses surface expression of Kv4.3 channels and causes slow inactivation. (A) Kv4.3Δ24 current traces were recorded in oocytes held at −80 mV in response to a family of depolarizing potentials from −60 mV to +40 mV with a 10 mV increments for 1 s. The inset compares the inactivation-scaled Kv4.3Δ24 and WT Kv4.3 currents at +40 mV. (B) Chimera (KIS-Kv4.3Δ24) current traces were recorded with the same protocol as in panel A. (C) The scaled and superimposed currents at +40 mV (from panels A and B) are shown to compare the development of inactivation kinetics among Kv4.3Δ24 alone, WT Kv4.3/WT KChIP4a, and chimera (KIS-Kv4.3Δ24). (D) Summary of normalized peak current amplitudes measured at +40 mV from WT Kv4.3 alone, Kv4.3/WT KChIP4a, Kv4.3Δ24 alone, and chimera (KIS-Kv4.3Δ24). All data are shown as the mean ± SE for 20–25 oocytes. Statistical significance (Student's t-test) was compared between groups as indicated; P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Ping Liang, et al. Biophys J. 2010 June 16;98(12):2867-2876.
4.
Figure 2

Figure 2. From: Functional Rescue of Kv4.3 Channel Tetramerization Mutants by KChIP4a.

Gating properties of rescued Kv4.3 C110A zinc mutant by KChIP4a. (A) Left panel: Scaled and superimposed currents at +40 mV to compare the development of macroscopic activation (τact) and inactivation kinetics between coexpression of WT Kv4.3/KChIP4a and C110A zinc mutant/KChIP4a. Middle and right panels: Comparison of activation and fast components of inactivation time constants (τinact, fast) at +40 mV between WT Kv4.3/KChIP4a and C110A/KChIP4a (in the panels, P4a denotes KChIP4a). The activation was fitted to curve with a single exponential equation, and the inactivation was fitted to curve with a double exponential equation. (B) Left panel: Recovery from inactivation of WT or C110A mutant channels coexpressed with KChIP4a. Right panel: Comparison of time constants for recovery from inactivation between WT Kv4.3/KChIP4a and C110A/KChIP4a. (C) Left panel: Tail current activation and prepulse inactivation plots of WT and C110A mutant channels. Middle and right panels: Comparison of V1/2 values for tail current activation and prepulse inactivation. A significant difference was found between C110A and WT Kv4.3 in the V1/2 of prepulse inactivation, but not in the tail current activation. Note that there is a strong (25 mV) rightward shift in prepulse inactivation for C110A mutant channels compared to WT Kv4.3. All data are shown as the mean ± SE for 5–13 oocytes, and statistical significance is indicated as ∗∗P < 0.005 and ∗∗∗P < 0.001.

Ping Liang, et al. Biophys J. 2010 June 16;98(12):2867-2876.
5.
Figure 5

Figure 5. From: Functional Rescue of Kv4.3 Channel Tetramerization Mutants by KChIP4a.

Enhanced surface expression of the myc-tagged C110A mutant and tetrameric assembly by KChIP4a. (A) In oocyte labeling, normalized protein expression by surface labeling in oocytes expressing the myc-Kv4.3 channel alone and in the presence of WT KChIP4a, compared with uninjected oocytes as a negative control or WT KChIP1 coinjected with Kv4.3 as a positive control. Right panel: Normalized protein expression by surface labeling in oocytes expressing myc-Kv4.3 C110A mutant alone and in the presence of either WT KChIP4a or KChIP4a double mutant (Y70A-K74A), which attenuated the surface expression increased by WT KChIP4a. For statistical comparison, WT Kv4.3 or C110A alone was compared with that of coexpressed KChIPs, P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 (Student's t-test); #P < 0.05 is only denoted for comparison between Kv4.3 C110A/WT KChIP4a and Kv4.3 C110A/KChIP4a Y70A-K74A double mutant. (B) Top panels: SEC-FPLC profiles of Kv4 channel proteins expressed with or without KChIPs and Western-blotted to identify fractions containing Kv4.3 subunits. WT Kv4.3 channels ran in the tetrameric fractions; however, Kv4.3 C110A channels ran as monomers. Bottom panels: Channels expressed with KChIPs Western-blotted for Kv4.3 C110A protein. Kv4.3 C110A channels showed a significant shift to tetrameric fractions with KChIP1 or KChIP4a proteins, but ran mainly as monomers with KChIP4a Y70A-K74A mutant. (C) Semiquantitative analysis of the ratio of tetramer/total protein based on panels in B.

Ping Liang, et al. Biophys J. 2010 June 16;98(12):2867-2876.
6.
Figure 3

Figure 3. From: Functional Rescue of Kv4.3 Channel Tetramerization Mutants by KChIP4a.

Double mutant Y70A-K74A of KChIP4a attenuates the functional rescue of the C110A zinc mutant. (A) Sequence alignment of the KChIP4a peptide (64-83) with the corresponding sequence of KChIP1 (residues 51–70). Residues shadowed in yellow denote amino acids that are conserved in KChIP1 and KChIP4a. Asterisks denote the two residues that when mutated in KChIP1 disrupt the second interface interaction with Kv4.3, as reported in our previous study. (B) Coexpression of Kv4.3 C110A and KChIP4a (Y70A-K74A); current traces were recorded with the same protocol as in Fig. 1. (C) Representative current traces of Kv4.3 C110A alone (black line), C110A/WT KChIP4a (red), and C110A/KChIP4a Y70A-K74A (blue) recorded at +40 mV from Xenopus oocytes. (D) Summary of normalized peak current amplitudes measured at +40 mV from Kv4.3 C110A alone and coexpression with WT or double mutant KChIP4a. Data are shown as mean ± SE for 8–25 oocytes. ∗∗∗P < 0.001 (Student's t-test), comparison between C110A as control and C110A/WT KChIP4a or C110A/KChIP4a Y70A-K74A double mutant; ##P < 0.001 (Student's t-test), comparison between C110A/WT KChIP4a and C110A/KChIP4a Y70A-K74A double mutant. (E) Fast component of the time constant of inactivation (τinact, fast) measured at +40 mV from C110A/WT KChIP4a and C110A/KChIP4a Y70A-K74A. Data are shown as mean ± SE for 8–13 oocytes. (F) Summary of time constants of recovery (τrecovery) from inactivation for varying lengths of time in steps from −80 to +40 mV, measured at +40 mV for coexpression of Kv4.3-C110A and different KChIPs. Data are shown as mean ± SE for 8–10 oocytes. ∗∗∗P < 0.001, comparison between controls (either C110A/WT KChIP1 or C110A/WT KChIP4a) and C110A/ KChIPs mutants.

Ping Liang, et al. Biophys J. 2010 June 16;98(12):2867-2876.
7.
Figure 7

Figure 7. From: Functional Rescue of Kv4.3 Channel Tetramerization Mutants by KChIP4a.

Core KChIP4a-dependent rescue of Kv4.3 channel tetramerization mutants. (A) Upper panel: WT Kv4.3 current traces in the absence or presence of WT KChIP1, WT KChIP4a, and KChIP4a Δ34 were recorded in oocytes held at −80 mV in response to a family of depolarizing potentials from −60 mV to +40 mV with a 10 mV increments for 1 s, respectively. The normalized peak current amplitude at +40 mV is onefold (n = 21) for WT Kv4.3 alone, compared with 3.5 ± 0.2-fold (n = 19) for WT Kv4.3/WT KChIP1, 0.7 ± 0.1-fold (n = 13) for WT Kv4.3/WT KChIP4a, and 5.2 ± 0.3-fold (n = 8) for WT Kv4.3/ KChIP4a Δ34, respectively. Lower panel: Kv4.3-C110A current traces in the absence or presence of WT KChIP1, WT KChIP4a, and KChIP4a Δ34 were recorded using the same protocol as in the upper panel. The C110A mutant shows nonfunctional current. (B) The scaled and superimposed currents at +40 mV (from upper panel A) are shown to compare the development of the inactivation kinetics of WT Kv4.3 alone (black line) or WT Kv4.3/WT KChIP1 (red line) as controls with WT Kv4.3/WT KChIP4a (green line) and WT Kv4.3/KChIP4a Δ34 (blue line). The fast component of time constant of inactivation (τinact, fast) is 32.6 ± 1.7 (n = 12) for WT Kv4.3 alone, compared with 74.9 ± 1.1 (n = 8) for WT Kv4.3/WT KChIP1, 413.4 ± 42.1 (n = 12) for WT Kv4.3/WT KChIP4a, and 75.5 ± 3.3 (n = 5) for WT Kv4.3/ KChIP4a Δ34, respectively. Note the crossover of the inactivation kinetics of Kv4.3 current and coexpression of WT KChIP1 or KChIP4a Δ34 at +40 mV. (C) The scaled and superimposed currents at +40 mV (from lower side panel A) are shown to compare the development of inactivation kinetics among coexpression of C110A and WT KChIP1 (red line), coexpression of C110A and WT KChIP4a (green line), and coexpression of C110A and KChIP4a Δ34 (blue line). The fast component of the time constant of inactivation (τinact, fast) is 40.3 ± 1.5 (n = 6) for C110A/KChIP4a Δ34, as compared with 29.2 ± 2.2 (n = 6) for C110A/WT KChIP1 and 149.5 ± 9.1 (n = 13) for C110A/WT KChIP4a, respectively. (D) The normalized peak current amplitude at +40 mV is 3.9 ± 0.2-fold (n = 21) for C110A/WT KChIP1, 3.7 ± 0.3-fold (n = 13) for C110A/WT KChIP4a and 5.9 ± 1.0-fold (n = 10) for C110A/KChIP4a Δ34, as compared with WT Kv4.3 alone. (E) Summary of the time constants of recovery (τrecovery) from inactivation for varying lengths of time at steps from −80 to +40 mV, measured at +40 mV from WT Kv4.3 or Kv4.3-C110A and different KChIPs. All data are shown as mean ± SE; the statistical comparison (Student's t-test) for P < 0.05, P < 0.01, and P < 0.001 was made with regard to WT Kv4.3 alone; and the groups denoted with #P < 0.05, ##P < 0.01, and ###P < 0.001 were compared with either WT Kv4.3/WT KChIP4a or Kv4.3 C110A/ WT KChIP4a complexes.

Ping Liang, et al. Biophys J. 2010 June 16;98(12):2867-2876.

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