PROJECT SUMMARY Changes in ion channel biophysics and function due to mutation or disease-associated remodeling are frequently assessed in isolated cells; yet translation of these mechanistic changes for therapeutic target development is difficult and often fails due to the inherent lack of tissue-scale regulation that is missing in isolated cell experiments. Sodium channel gain-of-function is a critical example of this translational challenge, with well-identified ion channel biophysical dysfunction (i.e., gating); yet an estimated 16- 64% of congenital sodium channel gain-of-function patients present without an electrocardiographic phenotype. Our prior work demonstrated a proof-of-concept that modulation of ion concentrations in extracellular nanodomains can conceal or unmask this gain-of-function, and these dynamics are an inherently tissue-scale phenomenon, as the sodium channel enriched nanodomains adjacent to gap junctions at the intercalated disc. The current proposal seeks to demonstrate that this concept has significant potential for therapeutic target development and pre-clinical predictive assessment. Preliminary data demonstrate that extracellular sodium and potassium (through co-regulation by intercalated disc- localized potassium channels) can modulate the presentation of sodium channel gain-of-function in a manner that depends on intercalated disc structure. We propose computational simulations (novel structurally detailed tissue models) and experiments (isolated myocytes, ex vivo hearts, and in vivo genetically-modified mice and peptide treated guinea pigs) to test the hypothesis that sodium and potassium concentrations can serve as critical biomarkers that are co-factors for risk of electrophysiological dysfunction in the setting of sodium channel gain-of-function and concomitant perinexus expansion. While ion concentrations are already established biomarkers in clinical care, our proposal will test the hypothesis that so called ‘normal’ ranges may indeed be pathological in patients with sodium channel gain-of-function. Indeed, the standard collection of these biomarkers is an asset for future work that will seek to identify patients at greater risk for electrophysiological dysfunction. Upon successful completion of these aims, we will produce new mechanistic understanding of the manifestation of sodium channel gain-of-function, which will demonstrate a mechanistic equivalency between detection and therapy.