Super-Resolution Imaging Using a Novel High-Fidelity Antibody Reveals Close Association of the Neuronal Sodium Channel Na(V)1.6 with Ryanodine Receptors in Cardiac Muscle

摘要

The voltage-gated sodium channel [pore-forming subunit of the neuronal voltage-gated sodium channel (Na(V)1.6)] has recently been found in cardiac myocytes. Emerging studies indicate a role for Na(V)1.6 in ionic homeostasis as well as arrhythmogenesis. Little is known about the spatial organization of these channels in cardiac muscle, mainly due to the lack of high-fidelity antibodies. Therefore, we developed and rigorously validated a novel rabbit polyclonal Na(V)1.6 antibody and undertook super-resolution microscopy studies of Na(V)1.6 localization in cardiac muscle. We developed and validated a novel rabbit polyclonal antibody against a C-terminal epitope on the neuronal sodium channel 1.6 (Na(V)1.6). Raw sera showed high affinity in immuno-fluorescence studies, which was improved with affinity purification. The antibody was rigorously validated for specificity via multiple approaches. Lastly, we used this antibody in proximity ligation assay (PLA) and super-resolution STochastic Optical Reconstruction Microscopy (STORM) studies, which revealed enrichment of Na(V)1.6 in close proximity to ryanodine receptor (RyR2), a key calcium (Ca2+) cycling protein, in cardiac myocytes. In summary, our novel Na(V)1.6 antibody demonstrates high degrees of specificity and fidelity in multiple preparations. It enabled multimodal microscopic studies and revealed that over half of the Na(V)1.6 channels in cardiac myocytes are located within 100 nm of ryanodine receptor Ca2+ release channels.