VAPB determines surface expression and dendritic localization of HCN2. A) Live cell imaging of HeLa cells transfected with an N-terminally EGFP-tagged HCN2 carrying an extracellular HA-epitope (^EGFPHCN2^HA_Ex) alone or cotransfected with VAPB or the TM segment of VAPB (TM^VAPB). B) Chemiluminescence assays of fixed non-permeabilized HeLa cells, analyzing the surface expression as relative light units (RLUs) for ^EGFPHCN2^HA_Ex alone and after cotransfection with VAPB (1.6 ± 0.1). Upper inset illustrates a representative control Western blot showing an unaltered HCN2 protein expression. C) Chemiluminescence surface expression assay as in B, but using TM^VAPB (1.6 ± 0.1). D) Immunocytochemistry of ^HAVAPB transfected cortical neurons. Endogenous HCN2 (green) is colocalizing (white) with ^HAVAPB (magenta) in the soma and dendrites. Anti–MAP2-staining illustrating an intact neuronal network and dendrites (blue). E) Immunocytochemistry experiment as in D, but transfecting the ALS8 mutation ^HAVAPB^P56S (magenta), leading to an aggregation of VAPB^P56S in the soma of the neurons. Also, HCN2 fluorescence (green) was focused in the soma and dendritic localization was lost, despite an intact neuronal network (α-MAP2, blue). Scale bars, 20 µm (A, D, E). All data are presented as means ± sem. The number of experiments (n) is indicated in the respective bar graphs. **P < 0.01 (unpaired Student’s t test).

Codistribution of VAPs with HCN2 and contribution to thalamic I_h. A–E), Distribution of HCN2, VAPB, and VAPA mRNA in mouse brain and spinal cord. ISH analysis of HCN2, VAPB, and VAPA using DIG-labeled riboprobes, revealing mRNA expression of VAPB in cortical areas (A), hippocampus (B), thalamus (C), cerebellum (D) (arrows point to interneurons in the granular layer), and spinal cord (E). Note the overlapping distribution of VAPB with HCN2 and VAPA mRNA. Am, amygdala; CA, cornu ammonis; DG, dentate gyrus; DH, dorsal horn; gcl, granule cell layer; Hb, habenulae; ic, internal capsule; LG, lateral geniculate ncl.; m, molecular cell layer; pcl, Purkinje cell layer; RTh, reticular thalamic ncl.; Sth, subthalamic ncl.; VB, ventrobasal thalamus; Th, thalamus; VH, ventral horn. F) Representative current traces elicited in slice patch-clamp experiments of the ventrobasal thalamus (VB) of wild-type animals (control) and VAPB^−/− mice. G) The I_h current was significantly reduced in VAPB^−/− mice (15.4 ± 1.1 pA/pF) compared with control animals (22.2 ± 2.3 pA/pF). H) Average activation curves of the VB I_h current for control and VAPB^−/− mice. V_1/2 of activation for control (−91.6 ± 1.3 mV, n = 8) and VAPB^−/− (−87.5 ± 1.2 mV, n = 7). Scale bars: 500 µm (A–C, E), 100 µm (D). All data are presented as means ± sem. The number of experiments (n) is indicated in the respective bar graphs. *P < 0.05 (unpaired Student’s t test).

Bradycardia in knock-down zebrafish embryos and VAPB^−/− mice. A) Zebrafish embryos at 72 hpf. Control-injected and MO-injected embryos against VAPA (^MOVAPA) or VAPB (^MOVAPB) exhibit no significant abnormalities (left), particularly no structural heart defects (right). Note a light cardiac edema in ^MOVAPB and a strong edema in ^MOVAPA. B) Heart rate in beats per minute (bpm) of control and ^MOVAPA, ^MOVAPB, or ^MOVAPA/B injected zebrafish embryos at 72 hpf. C) Representative examples of calcium transients (relative fluorescence intensity) in the cardiac atrium (black) and cardiac ventricle (gray) of control-injected zebrafish at 72 hpf, displaying a regular atrio-ventricular propagation of excitation from atrium to ventricle in a 1:1-ratio. D, E), Representative calcium transients recorded in ^MOVAPA/B double knock-down morphants, illustrating strongly reduced heart rates with variable frequency. F) Representative example of atrial and ventricular calcium measurements from a ^MOVAPA/B morphant with a 2:1 atrio-ventricular block, in which only every second atrial excitation leads to a ventricular excitation. Data were obtained from 3 independent batches of injections (A–F). G) Heart rate in beats per minute (bpm) of VAPB^−/− mice compared with wild-type littermates (control), analyzed by using tail-cuff measurements. H) Representative surface ECG recordings of VAPB^−/− mice and their wild-type littermates (control). ECGs of VAPB^−/− mice show bradycardia and an increased T-wave amplitude. I–N) Analyses of the ECG parameters of VAPB^−/− mice. I) Heart rate in beats per minute (bpm). J) PQ interval (PQ) duration. K) QRS complex (QRS) duration. L) Frequency corrected QT interval (QTc). M) Frequency-corrected T_peak to T_end duration (T_p-T_ec). N) T-wave amplitude (JT_p). Scale bars: 500 µm (A); 500 ms (C–F), and 100 ms (H). All data are presented as means ± sem. The number of animals (n) is indicated in the respective bar graphs. N.s., not significant. *P < 0.05, **P < 0.01, ***P < 0.001 [Student’s t test (G, H, J–N) or Welch’s t test (B, I)].

VAPB modulates I_f of spontaneously beating cardiac HL-1 cells. A) Immunocytochemistry of VAPB in HL-1 cells. Scale bar, 20 µm B) Western blot illustrating the knock-down of VAPB expression in HL-1 cells by shRNA transfection. Control, HL-1 cells transfected with scrambled shRNA. C) Representative I_f currents of HL-1 cells under control conditions and after VAPB transfection. D) Percentage of HL-1 cells containing I_f under control conditions (38%) and after VAPB transfection (58%). E) Beating frequency under control conditions (158 ± 4) and after VAPB transfection (179 ± 4), analyzed by optical counting of contractions in original Claycomb medium containing norepinephrine (60). F) Accelerated activation kinetics of VAPB-transfected HL-1 cells (n = 9–10). G) Activation curves of HL-1 cells under control conditions (n = 10) and after VAPB transfection (n = 11). H) Positive shift in the V_1/2 of activation of I_f recorded in VAPB transfected HL-1 cells. Control (scrambled shRNA), −90.3 ± 3.4 mV (n = 10); VAPB transfected, −79.6 ± 2.7 mV (n = 11). I) Representative action potential measurements of wild-type HL-1 and shRNA transfected cells (shVAPB). J) Analysis of the diastolic depolarization (DD duration). K) Beating frequency of HL-1 cells under control conditions and after VAPB knock-down. L) VAPB knock-down slows the activation kinetics (n = 5) of endogenous I_f currents. M) Transfection of shRNA (n = 5) shifts the voltage-dependence of activation (V_1/2) of I_f to more negative potentials (n = 6). N) V_1/2 values for control (scrambled shRNA) were −88.2 ± 3.1 mV (n = 6) and for shRNA-transfection (shVAPB), −96.2 ± 2.3 mV (n = 5), respectively. (I, J), Scrambled shRNA was used as control. All data are presented as means ± sem. The number of experiments (n) is indicated in the bar graphs. N.s., not significant. *P < 0.05, **P < 0.01, ***P < 0.001 [unpaired Student’s t test (D, G, H, M) or Mann-Whitney U test (E, F, J–L, N)

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