Silbernagel N , Walecki M , Schäfer MK , Kessler M , Zobeiri M , Rinné S , Kiper AK , Komadowski MA , Vowinkel KS , Wemhöner K , Fortmüller L , Schewe M , Dolga AM , Scekic-Zahirovic J , Matschke LA , Culmsee C , Baukrowitz T , Monassier L , Ullrich ND , Dupuis L , Just S , Budde T , Fabritz L , Decher N .

FS/13/43/30324 British Heart Foundation

	Figure 1. VAPB selectively increases HCN1 and HCN2 currents. A) Y2H direct interaction assay. Transformation control (-LW), leucine, and tryptophan dropout. Interaction read-out (-LWHA), additional dropout of histidine and adenine. pAL-Alg5, positive control. pPR3-N, negative control. B) Topology of VAPB. C) GSTVAPB pull-down of HCN2EGFP using transfected HeLa cells. D) GSTVAPA, GSTVAMP1, or GSTVAMP2 pull-down of HCN2EGFP using transfected HeLa cells. E) GSTVAPA pull-down of HCN2 and endogenous VAPB, using HCN2EGFP transfected HeLa cells. F) Pull-down of in vitro translated HCN2 (untagged). G) Pull-down of HCN2 from rat brain lysates. H, I) Representative currents (H) of HCN2 expressed in oocytes alone or with VAPB and the relative current amplitudes (I) analyzed over 3 d. J) Relative currents of HCN1, HCN2, and HCN4 alone or coexpressed with VAPB. K, L) Relative currents of different potassium channels (K) coexpressed with VAPB and of HCN2 (L) coexpressed with VAPA, VAPB, or VAPC. M) Relative currents of HCN2 coexpressed with a mixture of VAPA/B (1:1). N) Representative macropatch recordings in different configurations: on cell (o.c.), inside-out after patch excision (i.o.), and after application of 100 µM cAMP (i.o.+100 µM cAMP). O, P) Activation curves for HCN2 alone (n = 6) (O), recorded as in N, or after coexpression with VAPB (n = 8) (P). Q) V1/2 values for HCN2 expressed alone or with VAPB in different patch modes. R) Relative currents of HCN2HAEx alone or with VAPB. S) Relative surface expression of HCN2HAEx expressed alone or with VAPB, analyzed as relative light units (RLUs). T) Relative currents of HCN2 expressed alone or with VAPB or VAPBP56S. All data are presented as means ± sem. The number of cells (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 (H, K, L, O–S) or Mann-Whitney U test (I, J, M, N, T)].
	Figure 2. Domains mediating the interaction of VAPB with HCN2. A) Schematic illustration of a HCN subunit. The CNBD and some of the truncation constructs studied are indicated. B) All truncation constructs exhibited a positive interaction, evident from growth on -LWHA dropout medium. C) Representative current traces and the relative currents for different C-terminal deletions expressed alone or with VAPB. D) Representative current traces and the relative current amplitudes for the N-terminal truncated NTKHCN2 expressed alone or with VAPB. E) Relative current amplitudes of NTKHCN2HAEx (extracellular HA-tag) expressed alone or with VAPB. F) Relative surface expression of NTKHCN2HAEx expressed alone or with VAPB analyzed as relative light units (RLUs). G) Schematic illustration, representative traces, and currents of a HCN2 channel chimera with the N terminus of HCN4 (HCN4-NHCN2) expressed alone or with VAPB. H) Relative currents of HCN2 expressed alone or coexpressed with VAPB (1.7 ± 0.1), TMVAPB (1.6 ± 0.2), the MSP domain (MSPVAPB), the MSP with half of the CC domain (MSP-CC0.5VAPB), or with the complete CC domain (MSP-CCVAPB). I, J) Relative current amplitudes of HCN2HAEx expressed alone or with TMVAPB (1.3 ± 0.1) (I) and the respective changes in the relative surface membrane expression analyzed as RLUs, using a single cell chemiluminescence assay (TMVAPB 1.8 ± 0.2) (J). All data are presented as means ± sem. The number of experiments (n) is indicated in the respective bar graphs. N.s., not significant. *P < 0.05, **P < 0.01, ***P < 0.001 [unpaired Student’s t test (E), Mann-Whitney U test (C, D, F–H, J), or Welch’s t test (I)].
	Figure 3. 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 (EGFPHCN2HAEx) alone or cotransfected with VAPB or the TM segment of VAPB (TMVAPB). B) Chemiluminescence assays of fixed non-permeabilized HeLa cells, analyzing the surface expression as relative light units (RLUs) for EGFPHCN2HAEx 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 TMVAPB (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 HAVAPBP56S (magenta), leading to an aggregation of VAPBP56S 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).
	Figure 4. Codistribution of VAPs with HCN2 and contribution to thalamic Ih. 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 Ih 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 Ih current for control and VAPB−/− mice. V1/2 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).
	Figure 5. 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 Tpeak to Tend duration (Tp-Tec). N) T-wave amplitude (JTp). 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)].
	Figure 6. VAPB modulates If 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 If currents of HL-1 cells under control conditions and after VAPB transfection. D) Percentage of HL-1 cells containing If 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 V1/2 of activation of If 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 If currents. M) Transfection of shRNA (n = 5) shifts the voltage-dependence of activation (V1/2) of If to more negative potentials (n = 6). N) V1/2 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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