Characterization of SEMA3A-Encoded Semaphorin as a Naturally Occurring Kv4.3 Protein Inhibitor and its Contribution to Brugada Syndrome

Rationale: Semaphorin 3A (SEMA3A)-encoded semaphorin is a chemorepellent that disrupts neural patterning in the nervous and cardiac systems. In addition, SEMA3A has an amino acid motif that is analogous to hanatoxin, an inhibitor of voltage-gated K+ channels. SEMA3A-knockout mice exhibit an abnormal ECG pattern and are prone to ventricular arrhythmias and sudden cardiac death. Objective: Our aim was to determine whether SEMA3A is a naturally occurring protein inhibitor of K(v)4.3 (I-to) channels and its potential contribution to Brugada syndrome. Methods and Results: K(v)4.3, Na(v)1.5, Ca(v)1.2, or K(v)4.2 were coexpressed or perfused with SEMA3A in HEK293 cells, and electrophysiological properties were examined via whole-cell patch clamp technique. SEMA3A selectively altered K(v)4.3 by significantly reducing peak current density without perturbing K(v)4.3 cell surface protein expression. SEMA3A also reduced I-to current density in cardiomyocytes derived from human-induced pluripotent stem cells. Disruption of a putative toxin binding domain on K(v)4.3 was used to assess physical interactions between SEMA3A and K(v)4.3. These findings in combination with coimmunoprecipitations of SEMA3A and K(v)4.3 revealed a potential direct binding interaction between these proteins. Comprehensive mutational analysis of SEMA3A was performed on 198 unrelated SCN5A genotype-negative patients with Brugada syndrome, and 2 rare SEMA3A missense mutations were identified. The SEMA3A mutations disrupted SEMA3A's ability to inhibit K(v)4.3 channels, resulting in a significant gain of K(v)4.3 current compared with wild-type SEMA3A. Conclusions: This study is the first to demonstrate SEMA3A as a naturally occurring protein that selectively inhibits K(v)4.3 and SEMA3A as a possible Brugada syndrome susceptibility gene through a K(v)4.3 gain-of-function mechanism.