Brugada syndrome trafficking–defective Nav1.5 channels can trap cardiac Kir2.1/2.2 channels

Abstract

Cardiac Nav1.5 and Kir2.1-2.3 channels generate Na (INa) and inward rectifier K (IK1) currents, respectively. The functional INa and IK1 interplay is reinforced by the positive and reciprocal modulation between Nav15 and Kir2.1/2.2 channels to strengthen the control of ventricular excitability. Loss-of-function mutations in the SCN5A gene, which encodes Nav1.5 channels, underlie several inherited arrhythmogenic syndromes, including Brugada syndrome (BrS). We investigated whether the presence of BrS-associated mutations alters IK1 density concomitantly with INa density. Results obtained using mouse models of SCN5A haploinsufficiency, and the overexpression of native and mutated Nav1.5 channels in expression systems - rat ventricular cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) - demonstrated that endoplasmic reticulum (ER) trafficking-defective Nav1.5 channels significantly decreased IK1, since they did not positively modulate Kir2.1/2.2 channels. Moreover, Golgi trafficking-defective Nav1.5 mutants produced a dominant negative effect on Kir2.1/2.2 and thus an additional IK1 reduction. Moreover, ER trafficking-defective Nav1.5 channels can be partially rescued by Kir2.1/2.2 channels through an unconventional secretory route that involves Golgi reassembly stacking proteins (GRASPs). Therefore, cardiac excitability would be greatly affected in subjects harboring Nav1.5 mutations with Golgi trafficking defects, since these mutants can concomitantly trap Kir2.1/2.2 channels, thus unexpectedly decreasing IK1 in addition to INa.

The p.P888L SAP97 polymorphism increases the transient outward current (Ito,f) and abbreviates the action potential duration and the QT interval

“…Mouse ventricular myocyte isolation. Single mouse cardiomyocytes were isolated by enzymatic dissociation with collagenase type II (Worthington Biochemical Corporation Lakewood, NJ, USA) and protease (type XIV, Sigma Chemical Co. London, UK) from the three groups of mice following previously described methods 26 .…”

“…Dr. Hugues Abriel (University of Bern, Switzerland) and Dr. Stéphane Hatem (Sorbonne University, France) kindly provided the cDNA encoding the human cardiac SAP97 isoform that contains the I3 but not the I1A domain (I3-I1A) tagged with ds-Red. We introduced the p.P888L variant using the QuikChange Site-Directed Mutagenesis kit (Stratagene, USA) as previously described 6,26 .…”

“…Chinese hamster ovary (CHO) cells were cultured as previously described 6,26,27 and co-transfected with the cDNA encoding Kv4.3 channels and WT or p.P888L SAP97 using FUGENE XtremeGENE (Roche Diagnostics, Switzerland) according to manufacturer´s instructions.…”

“…Further, Kv4.3, SAP97, and CaMKII form a tripartite complex that allows the regulation of Kv4.3 channels by CaMKII 12 . Therefore, we decided to specifically analyze whether the differential effects of p.P888L SAP97 on Kv4.3 channels can be attributed to CaMKII by silencing its expression using siRNAs 6,26 .Transfection of CHO cells with a combination of siRNAs against all CaMKII isoforms reduced their expression by ~55% ( Supplementary Fig. III).…”

“…Currents were recorded at 21-23 °C using the whole-cell patch-clamp technique and filtered at half the sampling frequency 6,21,26,27 . Series resistance was compensated manually and ≥80% compensation was achieved.…”