A Versatile Chemical-Genetic Approach to Examine Sodium Channelopathies

The voltage-gated sodium channel NaV1.5 controls cardiac excitability and is an established therapeutic target. Mutations in the SCN5A gene, which encodes NaV1.5, are associated with inherited arrhythmia syndromes, including Brugada and Long-QT.  To advance the general understanding of NaV1.5-related conduction biology, we have developed a chemical-genetic model to achieve acute and reversible silencing of NaV1.5 in vitro and in vivo. To this end, a human NaV1.5 chimeric channel was engineered to contain a high-affinity, isoform-specific binding site for acylsulfonamide (GX) drugs. The GX drug binding site is comprised of an extracellular-facing pocket formed by the DIV voltage-sensor (VSD4) thus enabling a structure-based chimera design strategy.  The NaV1.5-GX channel has WT voltage-dependent gating and, unlike WT NaV1.5, is rapidly and reversibly inhibited by nanomolar GX compound.  Using CRISPR, the GX binding site has been engineered into the homologous region of the endogenous Scn5a locus, thus phenocopying the chimeric construct.  Inheritance of the NaV1.5-GX allele follows expected Mendelian ratios, allowing for the production of a NaV1.5GX/GX homozygous strain.  In the absence of GX compound, NaV1.5GX/GX hearts display normal cardiac phenotypes in vivo measured by EKG and echocardiography.  Patch-clamped NaV1.5GX/GX sodium channels  in isolated adult myocytes have WT gating but nanomolar GX compound ablates the NaV1.5 mediated current.