Characterization of the cardiac sodium channel SCN5A mutation, N1325S, in single murine ventricular myocytes

Sodium current amplitude, kinetics and regulation depend on the properties of the pore-forming protein (mostly NaV1.5 in adult heart) and on the specific molecular partners with which the channel protein associates. The composition of the voltage-gated sodium channel macromolecular complex is location-specific; yet, the exact position of NaV1.5 in the subcellular landscape of the intercalated disc (ID), remains unclear. We implemented diffraction unlimited microscopy (direct stochastic optical reconstruction microscopy, or “dSTORM”) to localize the pore-forming subunit of the cardiac sodium channel NaV1.5 with a resolution of 20nm on the XY plane. In isolated adult ventricular myocytes, NaV1.5 was found in distinct semi-circular clusters. When the entire population of clusters within a 500 nm window from the ID was considered (more than 350 individual clusters analyzed), 75% of them localized to N-cadherin rich sites. NaV1.5-distal clusters were found at an average 313±15 nm from the cell end. Introducing an astigmatic lens in the light path allowed us to solve cluster location in three dimensions, at resolutions of 20 nm in XY and 40 nm in the z plane. Three-dimensional images confirmed the preferential localization at or near N-cadherin plaques, and further suggested that NaV1.5 arrives to the membrane via N-cadherin-anchored paths, most likely microtubules. In additional experiments, we developed a novel approach to correlate the image of NaV1.5 clusters by dSTORM with the cellular ultrastructure as resolved by electron microscopy on the same sample. This “correlative light-electron microscopy” method confirmed the preference of NaV1.5 clusters at sites of mechanical coupling. Overall, we provide the first ultrastructural description of NaV1.5 at the cardiac ID and its relation with the major electron-dense domains of the adult heart. Our data support a model by which microtubule-mediated delivery of NaV1.5 anchors at N-cadherin-rich sites, likely “mixed junctions” also containing desmosomal molecules (such as plakophilin-2; see Cerrone et al; Circulation 129:1092-1103, 2014) and connexin43. These findings have major implications to the understanding of sodium current disruption in diseases affecting the integrity of the ID.

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Abstract 1503: Knockdown of Mog1 Expression Reduces Sodium Currents by Reducing the Trafficking of Cardiac Sodium Channel Na V 1.5 to the Cell Membrane

Circulation ◽

10.1161/circ.118.suppl_18.s_338-c ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Susmita Chakrabarti ◽

Sandro Yong ◽

Shin Yoo ◽

Ling Wu ◽

Qing Kenneth Wang

Keyword(s):

Sodium Channel ◽

Ventricular Myocytes ◽

Sodium Current ◽

Expression System ◽

Heart Association ◽

Hek293 Cells ◽

Similar Reduction ◽

Mammalian Expression ◽

Ventricular Cells ◽

Cardiac Sodium Channel

The cardiac sodium channel (Na v 1.5) plays a significant role in cardiac physiology and leads to cardiac arrhythmias and sudden death when mutated. Modulation of Na v 1.5 activity can also arise from changes to accessory subunits or proteins. Our laboratory has recently reported that MOG1, a small protein that is highly conserved from yeast to humans, is a co-factor of Na v 1.5. Increased MOG1 expression has been shown to increase Na v 1.5 current density. In adult mouse ventricular myocytes, these two proteins were found to be co-localized at the intercalated discs. Here, we further characterize the regulatory role of MOG1 using the RNA interference technique. Sodium current was recorded in voltage-clamp mode from a holding potential of −100 mV and activated to −20 mV. In 3-day old mouse neonatal ventricular cells transfected with siRNA against mouse MOG1 decreased sodium current densities (pA/pF) compared to control or scramble siRNA treated cells (−10.2±3.3, n=11 vs. −165±16, n=20 or −117.9±11.7, n=11). A similar reduction in sodium current was observed in mammalian expression system consisting of HEK293 cells stably expressing human Na v 1.5, by transfecting siRNAs against either human or mouse MOG1 (−41.7±8.3, n=7 or, −82.6±9.6, n=7 vs. −130.6±11.5, n=7; −111.5±8.5, n=7, respectively). Immunocytochemistry revealed that the expression of MOG1 and Na v 1.5 were decreased in both HEK and neonatal cells when compared to scramble siRNAs or control groups. These results show that MOG1 is an essential co-factor for Na v 1.5 by way of a channel trafficking. Such interactions between MOG1 and Na v 1.5 suggest that early localization of MOG1 on the membrane of neonatal cardiomyocytes may be necessary for proper localization and the distribution of Na v 1.5 during cardiac development. This research has received full or partial funding support from the American Heart Association, AHA National Center.

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