Supporting Text

In Vitro Binding Assay. GST and GST ankyrin-G membrane-binding domain (MBD) were generated by using Sindbis pseudovirions in baby hamster kidney (BHK) cells as described (1). Cells were washed and incubated on ice with lysis buffer (50 mM Tris·Cl/150 mM NaCl/1% Triton X-100/0.05% Tween-20/1 mM Na2EDTA, pH 8.0) in the presence of protease inhibitors. Lysates were centrifuged, and the supernatant containing GST-MBD or GST was incubated with glutathione-Sepharose 4B. For pull-down experiments with GST-MBD or GST alone as a control, BHK cells were transfected with NF/Nav1.5 chimera constructs. Cell lysates were added to either GST-MBD beads or GST beads overnight at 4°C. The beads were washed three times with ice-cold lysis buffer, and bound proteins were eluted twice with one bed volume of 10 mM reduced glutathione in 50 mM Tris·Cl, pH 8.0. Cell lysates and proteins associated with GST-MBD and GST were probed by immunoblotting using antibodies to hemagglutinin (HA). Similarly, cell lysates were analyzed to ensure expression of the tagged protein, and batches of beads used in the assays were analyzed by using antibodies to GST to ensure the presence of the GST-MBD and GST proteins.

Imaging. After washes, cardiomyocytes were mounted with Vectashield (Vector) and no. 1 coverslips. Images were collected on Zeiss 510 Meta confocal microscope 40 power water 1.2 numerical aperture (NA) (Zeiss), or 100 power oil 1.45 NA (Zeiss), pinhole equals 1.0 (Airy Disc) by using Carl Zeiss Imaging software. Both channels were collected on PMT3, and images were collected by using calibrations to confirm that fluorescence was collected in the linear range. All cardiomyocytes were prepared identically, and imaged by using identical protocols [using "Re-Use" function (computer automatically images each sample with exact same imaging protocol; i.e. magnification, brightness, contrast, pinhole, scan time, averaging, etc.)]. Intensity measurements were performed using Carl Zeiss 510 imaging software. Images were imported into PHOTOSHOP (Adobe Systems, Mountain View, CA) for cropping. Identical settings were utilized in PHOTOSHOP when direct comparisons were necessary (i.e. intensity of HA-Nav1.5 vs HA-Nav1.5 E1053K).

Yeast Two-Hybrid Assays. The ankyrin-G MVD was subcloned in frame with the Gal4 activation domain into LEU2-marked prey vector pGAD424. Nav1.5 loop 2 was subcloned in-frame with the LexA DNA-binding domain into TRP1-marked bait vector pBTM116. Yeast strain L40, harboring the HIS3 reporter gene under the control of LexA-binding sites, was cotransformed with 50 ng each of the bait and prey vector by using the lithium/acetate method and then streaked out onto agar plates lacking tryptophan and leucine. For each transformation, six independent clones were selected and replica-plated onto media lacking tryptophan, leucine, and histidine to assess expression of the HIS3 reporter gene.

Immunoprecipitations. Adult rat heart was removed, rinsed with ice cold 0.32 M sucrose/2 mM EDTA in PBS (pH 7.4), and flash frozen in liquid nitrogen. The heart was ground into a fine powder by using an ice-cold mortar and pestle. Powder (0.5 g) was resuspended in 50 mM Tris·HCl, 10 mM NaCl (pH 7.4) with 0.32 M sucrose, 5 mM EDTA, 2.5 mM EGTA plus protease inhibitors by using a Dounce homogenizer. The lysate was centrifuged for 10 min at 1,000 ´ g to pellet large membranes and nuclei. The supernatant was removed, Triton X-100 (final concentration 1.5%) and deoxycholate (1.0% final concentration) were added, and the lysate was incubated at 4°C for 1 h. After centrifugation for 1 h at 100,000 ´ g, the detergent-soluble fraction was removed for coimmunoprecipitation experiments. Affinity-purified ankyrin-G Ig was prebound to Protein G beads and incubated with the detergent-soluble lysates for 6 h at 4º C. The beads were washed three times by using ice-cold 50 mM Tris·HCl, 10 mM NaCl (pH 7.4) with 0.32 M sucrose, 5 mM EDTA, 2.5 mM EGTA, 1.0% Triton X-100 plus protease inhibitors. Bound protein was eluted with 5´ PAGE buffer, and Western blotting was performed by using affinity-purified Nav1.5 antibody. For detection of Nav1.5, blots were incubated with 125I Protein A for 2 h. Protein was detected by phosphorimaging.

BHK Cell Culture and Transfection. BHK cells were cultured in minimum essential medium (-MEM, Invitrogen) supplemented with 5% (vol/vol) fetal bovine serum (Invitrogen) at 37°C under 5% CO2.

1. Lemaillet, G., Walker, B. & Lambert, S. (2003) J. Biol. Chem. 278, 27333-27339.