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

Nicole J Boczek, Dan Ye, Eric K Johnson, Wei Wang, Lia Crotti, David J Tester, Federica Dagradi, Yuka Mizusawa, Margherita Torchio, Marielle Alders, John R Giudicessi, Arthur A M Wilde, Peter J Schwartz, Jeanne M Nerbonne, Michael J Ackerman

Rationale: 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: To determine whether SEMA3A is a naturally occurring protein inhibitor of Kv4.3 (Ito) channels and its potential contribution to Brugada syndrome (BrS). Methods and Results: Kv4.3, Nav1.5, Cav1.2, or Kv4.2 were co-expressed or perfused with SEMA3A in HEK293 cells and electrophysiological properties were examined via whole-cell patch clamp technique. SEMA3A selectively altered Kv4.3 by significantly reducing peak current density without perturbing Kv4.3 cell-surface protein expression. SEMA3A also reduced Ito current density in cardiomyocytes derived from human induced pluripotent stem cells. Disruption of a putative toxin binding domain on Kv4.3 was used to assess physical interactions between SEMA3A and Kv4.3. These findings in combination with co-immunoprecipitations of SEMA3A and Kv4.3 revealed a potential direct binding interaction between these proteins. Comprehensive mutational analysis of SEMA3A was performed on 198 unrelated SCN5A-genotype negative patients with BrS and two rare SEMA3A missense mutations were identified. The SEMA3A mutations disrupted SEMA3A's ability to inhibit Kv4.3 channels, resulting in a significant gain of Kv4.3 current compared to WT-SEMA3A. Conclusions: This study is the first to demonstrate semaphorin3A as a naturally occurring protein that selectively inhibits Kv4.3 and SEMA3A as a possible BrS-susceptibility gene through a Kv4.3 gain-of-function mechanism.