Coupling of functionality to trafficking of KCNQ2/3 potassium channels at the axon initial segment

Figures

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  Fig. 1.. Halo-TAC-KCNQ3 retains channel functionality and enables live-cell imaging of heteromeric KCNQ2/3 complexes. (A) Representative current traces of KCNQ3T and Halo-TAC-KCNQ3T obtained by whole-cell patch-clamp recordings in HEK293T cells. The dotted line indicates the zero-current level. (B) Current densities at 80 mV for KCNQ3T (Q3T) and Halo-TAC-KCNQ3T (Halo-TAC-Q3T). P = 0.090. (C) G–V curves of KCNQ3T and Halo-TAC-KCNQ3T. V1/2 for KCNQ3T: −35.11 ± 1.67 mV and for Halo-TAC-KCNQ3T: −35.38 ± 1.87 mV. P = 0.927. (B and C) n = 4 cells (Q3T) and 5 cells (Halo-TAC-Q3T). P-values were obtained by Welch’s t test. (D) Representative confocal microscopic images of primary cultured hippocampal neurons expressing EGFP-Nav1.2 II-III linker and Halo-TAC-KCNQ3 with KCNQ2 or an empty vector. (E) Representative confocal microscopic images of primary cultured hippocampal neurons expressing EGFP-Nav1.2 II-III linker, Halo-TAC-KCNQ3-ETD/AAA, and KCNQ2. (D and E) HaloTag was labeled with AF660 and TMR. The arrow indicates the position of AIS. (Scale bar, 20 µm.) (F) EGFP and AF660 intensity profiles along the axon in neurons expressing EGFP-Nav1.2 II-III linker with following constructs: KCNQ2 and…

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  Fig. 2.. KCNQ3 functionality determines AIS targeting of KCNQ2/3. (A) Schematic representation of the KCNQ3 activation process. (B) Representative current traces of Halo-TAC-KCNQ3T and its mutants obtained by whole-cell patch-clamp recordings in HEK293T cells. The dotted line indicates the zero-current level. (C) Representative confocal microscopic images of primary cultured hippocampal neurons expressing EGFP-Nav1.2 II-III linker and KCNQ2 with Halo-TAC-KCNQ3 or its mutants. HaloTag was labeled with AF660 and TMR. The arrow indicates the position of AIS. (Scale bar, 20 µm.) (D–F) AF660 intensities at the AIS and dendrite, ratios of AF660 intensity between AIS and dendrite, and TMR intensities at the AIS of neurons expressing EGFP-Nav1.2 II-III linker and KCNQ2 with Halo-TAC-KCNQ3 or its mutants (n = 5 to 11). *P < 0.05, **P < 0.01, and ***P < 0.001 (Dunnett’s test, comparisons were made with the “WT” column for the same group). (G) Positive correlation between the current densities of the Halo-TAC-KCNQ3T constructs measured in HEK293T cells (SI Appendix, Fig. S3A) and the AIS localization of the corresponding Halo-TAC-KCNQ3 constructs (E). Spearman’s rank correlation coefficient (R) =…

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  Fig. 3.. Coupling of KCNQ2/3 functionality to trafficking is not attributable to plastic AIS remodeling dependent on neuronal excitability. (A) Whole-cell patch-clamp recordings from primary cultured hippocampal neurons expressing KCNQ2 with Halo-TAC-KCNQ3 (WT or H258N). Representative action potentials evoked by 200 pA current injection for 800 ms. The dotted line indicates the −65 mV level. (B and C) Firing rates evoked by current injections with increments of 50 pA (50 pA, P = 0.363; 100 pA, P = 0.466; 150 pA, P = 0.707; 200 pA, P = 0.599; 250 pA, P = 0.677; 300 pA, P = 0.706) and resting membrane potentials (RMP) (P = 0.151) in neurons expressing KCNQ2 with Halo-TAC-KCNQ3 (WT, n = 7; H258N, n = 6). P-values were obtained by Welch’s t test. (D–G) Waveforms, phase plots, maximum amplitudes (P = 0.249), and half-widths (P = 0.114) of spontaneous action potentials at resting membrane potential in neurons expressing KCNQ2 with Halo-TAC-KCNQ3 (WT, n = 5; H258N, n = 5). P-values were obtained by Welch’s t test. (H) Representative confocal microscopic images of primary cultured hippocampal neurons expressing EGFP-Nav1.2 II-III linker and KCNQ2 with Halo-TAC-KCNQ3 or its mutants. The arrow…

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  Fig. 4.. AnkG binding and functionality of KCNQ3 govern insertion, immobilization, and lifetime of KCNQ2/3 on the AIS surface. (A, Left) A representative TIRFM image of a primary cultured hippocampal neuron expressing EGFP-Nav1.2 II-III linker, KCNQ2, and Halo-TAC-KCNQ3 labeled with TMR. (Scale bar, 5 µm.) (Right) Magnified view of the area within the yellow square in the Left panel, showing the trajectories of Halo-TAC-KCNQ3 molecules for 40 s as yellow lines. (Scale bar, 2 µm.) (B) Representative diffusion trajectories for 40 s of Halo-TAC-KCNQ3 (WT, ETD/AAA, and H258N) labeled with TMR at the AIS. Red, yellow, and blue lines indicate trajectories of immobile, slow-diffusion, and fast-diffusion states of the molecules within an interval of 33 ms, respectively. (Scale bar, 2 µm.) (C) konapp of Halo-TAC-KCNQ3 and its mutants on the cell surface at the AIS (WT, n = 10; ETD/AAA, n = 9; H258N, n = 9). *P < 0.05 and **P < 0.01 (Steel–Dwass test, only comparisons with the “WT” column are depicted). (D) Probability density distributions of the displacements of Halo-TAC-KCNQ3 and its mutants during 33 ms at the AIS. (E and F) Diffusion coefficients and occupancies of immobile, slow-diffusion,…

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  Fig. 5.. Functional state of KCNQ3 determines its binding affinity for ankG. (A) Representative TIRFM images of HEK293T cells expressing Halo-TAC-KCNQ3 labeled with TMR and ankG-SNAP labeled with 647-SiR. (Scale bar, 5 µm.) (B) Colocalization probabilities of Halo-TAC-KCNQ3 and its mutants with ankG-SNAP. P = 0.038 for WT versus H258N; P = 0.034 for FDR/RNE versus FDQ/RNE. (C) ka of Halo-TAC-KCNQ3 and its mutants colocalized with ankG-SNAP. P = 0.924 for WT versus H258N; P = 0.026 for FDR/RNE versus FDQ/RNE. (D) Dissociation curves of Halo-TAC-KCNQ3 and its mutants from ankG-SNAP which were fitted with a double exponential function. (E) kd obtained from Halo-TAC-KCNQ3 and its mutants colocalized with ankG-SNAP. P = 0.016 for WT versus H258N; P = 0.007 for FDR/RNE versus FDQ/RNE. (F) Kd calculated as kd/ka for Halo-TAC-KCNQ3 and its mutants in (C and E). P = 0.043 for WT versus H258N; P = 0.003 for FDR/RNE versus FDQ/RNE. n = 8 cells (WT), 7 cells (H258N), 6 cells (FDR/RNE), and 7 cells (FDQ/RNE). P-values were obtained by Welch’s t test. Data are mean ± SE.

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  Fig. 6.. Dynamic equilibrium of the binding reaction between KCNQ3 and ankG. The three kinetic parameters (ka, kd, and Kd) for KCNQ3-FDQ/RNE (resting state) and KCNQ3-FDR/RNE (activated state) are shown. The equilibrium between KCNQ3 and ankG depends on the functional state of KCNQ3. KCNQ3 has higher ankG binding affinity in the activated state than in the resting state.