The C-terminal HRET sequence of Kv1.3 regulates gating rather than targeting of Kv1.3 to the plasma membrane

Figures

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  Figure 1. Point- and C-terminal deletion mutants of Kv1.3 channels. (A) Schematic illustration of wild type (WT) Kv1.3 pore-forming alpha subunit with N-terminal EGFP/mCherry, FLAG epitope, point mutations (H399K, A413V, W384F), the HRET sequence and the last four amino acids of C-terminus (FTDV). (B) Amino acid sequences of WT Kv1.3 and mutant Kv1.3 s starting at amino acid 382 (“……” represents rest of the C-terminus not relevant to this study). Amino acids are listed by their one letter designations, FL: full-length C-terminus, ΔC: C-terminal truncated at 439, NOHRET: truncation after postion 435. XHRETE: deletion mutant removing HRETE sequence. polyA: replacement HRETE with AAAAA, Atail: HRETE changed for AAAAA, and rest of C-terminus is removed. Starting amino acid of C-terminus 427 is indicated. (C) Current traces of WT (mCherry tagged, left), A413V-FL (center) and H399K-FL (right) channels expressed in HEK cells. WT (outside-out patch configuration): cells were depolarized to + 50 mV from a holding potential of −120 mV for 2 seconds. A413V-FL (whole-cell configuration): cells were kept at −120 mV then depolarized to + 50 mV for 30 ms. H399K (whole-cell configuration): cells were…

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  Figure 2. Current of A413V truncated mutants in HEK and CHO cells. (A) Representative current traces recorded in HEK transfected with the EGFP-tagged A413V-ΔC or the A413V-NOHRET construct (arrows). Currents were recorded upon 30-ms-long depolarizations to + 50 mV from a holding potential of −120 mV. (B) Representative current traces recorded in CHO transfected with the EGFP-tagged A413V-ΔC or the A413V-NOHRET construct. Recording conditions were the same as in panel A. (C,D) Box plot of the current density measured in HEK (C) or CHO (D) transfected with the full length (A413V-FL), A413V-ΔC or A413V-NOHRET constructs. The data are reported as mean (dashed line), median (solid line), first (top box) and third quartiles (bottom box), and maximum and minimum. Current density was determined as detailed in the materials and methods.

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  Figure 3. TEA inhibition of full length and C-terminal removed H399K channel. (A) Whole-cell currents of EGFP- and FLAG-tagged H399K-FL channels in CHO cells were recorded during 200-ms-long pulses to 0 mV (not to + 50 mV to reduce whole-cell current) from a holding potential of −120 mV. Traces are shown before (black) and after addition of 100 mM TEA (purple). (B) Currents of the EGFP- and FLAG-tagged H399K-ΔC construct in the presence (purple) and absence (black) of 100 mM TEA. The same voltage-clamp protocol was used as on panel A was used to evoke the currents.

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  Figure 4. Targeting of deletion mutants of Kv1.3 to the plasma membrane. Representative images of EGFP- and FLAG-tagged Kv1.3 mutant channels expressed in CHO. (A) CHO cells expressing the A413V-FL/H399K-FL, (B) A413V/H399K C-terminal truncated Kv1.3 (A413V-ΔC/H399K-ΔC) or C) A413V-NOHRET/H399K-NOHRET plasmid is displayed. The 1st column shows the EGFP signal of Kv1.3 channel in green. Cells were labeled with anti-FLAG primary and Alexa Fluor 647 GAMIG secondary antibodies to verify cell membrane localization of the channel protein (2nd column, red). The 3rd column shows the merge of red and green channels of the EGFP and anti-FLAG fluorescence signal whereas brightfield images of the cells are shown in the 4th column. (For details of fluorescent labeling see Materials and Methods).

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  Figure 5. WT Kv1.3 fails to rescue the A413V phenotype when co-transfected with A413V-NOHRET subunits. (A) Whole-cell current recorded in a CHO cell co-transfected with EGFP-tagged A413V-NOHRET and mCherry tagged WT Kv1.3 plasmids (1:1 molar ratio). The cell was depolarized to + 50 mV from a holding potential of −120 mV. (B) Confocal images of a CHO cell expressing both FLAG and EGFP-tagged A413V-NOHRET and mCherry-tagged WT Kv1.3. Cells were stained with anti-FLAG and Alexa-647 GAMIG antibodies. a: A413V-NOHRET (green), b: mCherry-WT Kv1.3 (red), c: FLAG (blue), d: overlay of red, green blue channels, e: brightfield image of the cells. Scale bar is 5 µm.

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  Figure 6. The gating current of WT-NOHRET channels is missing. (A) Gating currents in a WT-W384F channel expressing CHO cell. Cell was held at −120 mV then depolarized to different test potentials (from −100 mV to 20/100 mV in 10 mV steps) for 50 ms. (B) The test potential dependence of the integrated gating current (Qon, on-gating) for the cells recorded in panel A (W384F) and C (WT-NOHRET). Qon was evaluated as described in the Material and Methods section. (C) Gating current recordings in a CHO cell transfected with WT-NOHRET construct. An identical pulse protocol was applied as in A.

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  Figure 7. Elimination of the HRETE motif does not affect Kv1.3 operation. (A) Current traces of WT-FL, WT-XHRETE, WT-polyA and WT-Atail channels were recorded upon 15-ms-long + 50 mV depolarization to study activation kinetics of the four constructs (red line: WT-FL, blue: WT-polyA, black: WT-XHRETE, WT-Atail: green). For fitting and evaluation of activation kinetics see Materials and Methods. Holding potential was set to −120 mV, measurements were performed in outside-out configuration and currents were normalized to the peak value for the panel A and B. (B) Typical current recordings showing the inactivation kinetics of the WT-FL, WT-polyA, WT-XHRETE and WT-Atail channels. Cells were depolarized to + 40 mV for 2 s from −120 mV holding potential, inactivation kinetics was evaluated as described in Materials and Methods. (C) Voltage-dependence of steady-state activation of four Kv1.3 channel phenotypes: the normalized conductance-test potential relationships were obtained and analyzed as described in the Materials and Methods. Normalized conductance as a function of the test potential is shown for a WT-FL (filled circle, red line), a WT-polyA (empty triangles, blue line), a WT-XHRETE…