Background: Na v 1.5 cardiac Na + channel mutations can cause arrhythmogenic syndromes. Some of these mutations exert a dominant negative effect on wild-type channels. Recent studies showed that Na + channels can dimerize, allowing coupled gating. This leads to the hypothesis that allosteric interactions between Na + channels modulate their function and that these interactions may contribute to the negative dominance of certain mutations. Methods: To investigate how allosteric interactions affect microscopic and macroscopic channel function, we developed a modeling paradigm in which Markovian models of two channels are combined. Allosteric interactions are incorporated by modifying the free energies of the composite states and/or barriers between states. Results: Simulations using two generic 2-state models (C-O, closed-open) revealed that increasing the free energy of the composite states CO/OC leads to coupled gating. Simulations using two 3-state models (closed-open-inactivated) revealed that coupled closings must also involve interactions between further composite states. Using two 6-state cardiac Na + channel models, we replicated previous experimental results mainly by increasing the energies of the CO/OC states and lowering the energy barriers between the CO/OC and the CO/OO states. The channel model was then modified to simulate a negative dominant mutation (Na v 1.5 p.L325R). Simulations of homodimers and heterodimers in the presence and absence of interactions showed that the interactions with the variant channel impair the opening of the wild-type channel and thus contribute to negative dominance.

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Cardiac voltage‐gated sodium (Na+) channels (Nav1.5) are crucial for myocardial electrical excitation. Recent studies based on single‐channel recordings have suggested that Na+ channels interact functionally and exhibit coupled gating. However, the analysis of such recordings frequently relies on manual interventions, which can lead to bias. Here, we developed an automated pipeline to de‐trend and idealize single‐channel currents, and assessed possible functional interactions in cell‐attached patch clamp experiments in HEK293 cells expressing human Nav1.5 channels as well as in adult mouse and rabbit ventricular cardiomyocytes. Our pipeline involved de‐trending individual sweeps by linear optimization using a library of predefined functions, followed by digital filtering and baseline offset. Subsequently, the processed sweeps were idealized based on the idea that the ensemble average of the idealized current identified by thresholds between current levels reconstructs at best the ensemble average current from the de‐trended sweeps. This reconstruction was achieved by non‐linear optimization. To ascertain functional interactions, we examined the distribution of the numbers of open channels at every time point during the activation protocol and compared it to the distribution expected for independent channels. We also examined whether the channels tended to synchronize their openings and closings. However, we did not uncover any solid evidence of such interactions in our recordings. Rather, our results indicate that wild‐type Nav1.5 channels are independent entities or exhibit only very weak functional interactions that are probably irrelevant under physiological conditions. Nevertheless, our unbiased analysis will be important for further studies examining whether auxiliary proteins potentiate functional Na+ channel interactions. imageKey points Nav1.5 channels are critical for cardiac excitation. They are part of macromolecular interacting complexes, and it was previously suggested that two neighbouring channels may functionally interact and exhibit coupled gating. Manual interventions when processing single‐channel recordings can lead to bias and inaccurate data interpretation. We developed an automated pipeline to de‐trend and idealize single‐channel currents and assessed possible functional interactions between Nav1.5 channels in HEK293 cells and cardiomyocytes during activation protocols using the cell‐attached patch clamp technique. In recordings consisting of up to 1000 sweeps from the same patch, our analysis did not reveal any evidence of functional interactions or coupled gating between wild‐type Nav1.5 channels. Our unbiased analysis may be useful in further studies examining how Na+ channel interactions are affected by mutations and auxiliary proteins.