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Super-Resolution Imaging Using a Novel High-Fidelity Antibody Reveals Close Association of the Neuronal Sodium Channel NaV1.6 with Ryanodine Receptors in Cardiac Muscle

Published online by Cambridge University Press:  14 January 2020

Heather L. Struckman ,
Stephen Baine Open the ORCID record for Stephen Baine [Opens in a new window] ,
Justin Thomas ,
Louisa Mezache ,
Kirk Mykytyn ,
Sándor Györke ,
Przemysław B. Radwański  and
Rengasayee Veeraraghavan Open the ORCID record for Rengasayee Veeraraghavan [Opens in a new window]
Heather L. Struckman
Affiliation:
Department of Biomedical Engineering, College of Engineering, The Ohio State University, Columbus, OH, USA
Stephen Baine
Affiliation:
Dorothy M. Davis Heart and Lung Research Institute, College of Medicine, The Ohio State University Wexner Medical Center, Columbus, OH, USA Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, Columbus, OH, USA
Justin Thomas
Affiliation:
Division of Pharmacy Practice and Sciences, College of Pharmacy, The Ohio State University, Columbus, OH, USA
Louisa Mezache
Affiliation:
Department of Biomedical Engineering, College of Engineering, The Ohio State University, Columbus, OH, USA
Kirk Mykytyn
Affiliation:
Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, OH, USA
Sándor Györke
Affiliation:
Dorothy M. Davis Heart and Lung Research Institute, College of Medicine, The Ohio State University Wexner Medical Center, Columbus, OH, USA Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, Columbus, OH, USA
Przemysław B. Radwański*
Affiliation:
Dorothy M. Davis Heart and Lung Research Institute, College of Medicine, The Ohio State University Wexner Medical Center, Columbus, OH, USA Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, Columbus, OH, USA Division of Pharmacy Practice and Sciences, College of Pharmacy, The Ohio State University, Columbus, OH, USA
Rengasayee Veeraraghavan*
Affiliation:
Department of Biomedical Engineering, College of Engineering, The Ohio State University, Columbus, OH, USA Dorothy M. Davis Heart and Lung Research Institute, College of Medicine, The Ohio State University Wexner Medical Center, Columbus, OH, USA Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, Columbus, OH, USA
*
*Authors for correspondence: Przemysław B. Radwański, E-mail: Przemyslaw.Radwanski@osumc.edu; Rengasayee Veeraraghavan, E-mail: veeraraghavan.12@osu.edu
*Authors for correspondence: Przemysław B. Radwański, E-mail: Przemyslaw.Radwanski@osumc.edu; Rengasayee Veeraraghavan, E-mail: veeraraghavan.12@osu.edu
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Abstract

The voltage-gated sodium channel [pore-forming subunit of the neuronal voltage-gated sodium channel (NaV1.6)] has recently been found in cardiac myocytes. Emerging studies indicate a role for NaV1.6 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 NaV1.6 antibody and undertook super-resolution microscopy studies of NaV1.6 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 (NaV1.6). 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 NaV1.6 in close proximity to ryanodine receptor (RyR2), a key calcium (Ca2+) cycling protein, in cardiac myocytes. In summary, our novel NaV1.6 antibody demonstrates high degrees of specificity and fidelity in multiple preparations. It enabled multimodal microscopic studies and revealed that over half of the NaV1.6 channels in cardiac myocytes are located within 100 nm of ryanodine receptor Ca2+ release channels.

Keywords

antibodycardiac physiologyexcitation–contraction couplingsodium channelssuper-resolution microscopy
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 26 , Issue 1 , February 2020 , pp. 157 - 165
Copyright
Copyright © Microscopy Society of America 2020

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