An interaction between the III-IV linker and CTD in NaV1.5 confers regulation of inactivation by CaM and FHF

Abstract

Voltage gated sodium channel (VGSC) activation drives the action potential upstroke in cardiac myocytes, skeletal muscles, and neurons. After opening, VGSCs rapidly enter a non-conducting, inactivated state. Impaired inactivation causes persistent inward current and underlies cardiac arrhythmias. VGSC auxiliary proteins calmodulin (CaM) and fibroblast growth factor homologous factors (FHFs) bind to the channel’s C-terminal domain (CTD) and limit pathogenic persistent currents. The structural details and mechanisms mediating these effects are not clear. Building on recently published cryo-EM structures, we show that CaM and FHF limit persistent currents in the cardiac NaV1.5 VGSC by stabilizing an interaction between the channel’s CTD and III-IV linker region. Perturbation of this intramolecular interaction increases persistent current and shifts the voltage dependence of steady-state inactivation. Interestingly, the NaV1.5 residues involved in the interaction are sites mutated in the arrhythmogenic long QT3 syndrome (LQT3). Along with electrophysiological investigations of this interaction, we present structural models that suggest how CaM and FHF stabilize the interaction and thereby limit the persistent current. The critical residues at the interaction site are conserved among VGSC isoforms, and subtle substitutions provide an explanation for differences in inactivation among the isoforms.

E1784K, the most common Brugada syndrome and long-QT syndrome type 3 mutant, disrupts sodium channel inactivation through two separate mechanisms

“…We have shown that E1784K-induced increases in noninactivating current are dependent on a positive charge at position 1784, and that mutants at position 1784 can alter fast inactivation voltage dependence and rates without affecting the fraction of noninactivating current. These data, in conjunction with other recent studies on this region, confirm that the C terminus forms multiple interactions with the sodium channel inactivation machinery (Clairfeuille et al, 2019;Gade et al, 2020). We confirm that an interaction occurs between residue E1784E of the C terminus and the DIII-DIV linker at sites K1492 and K1493.…”

“…1 B). Similar to previous data Deschênes et al, 2000;Gade et al, 2020;Abdelsayed et al, 2015), E1784K shows a trend toward depolarization compared with E1784E (P = 0.1554; Fig. 1 B).…”

“…Similar to the previous study on K1493E (Gade et al, 2020), our homology model of Nav1.5 built on the American cockroach structure in the closed, noninactivated state (Shen et al, 2017) shows that residue E1784E comes within 5Å of residue K1493 in the DIII-DIV linker (Fig. 6, A and C).…”

“…We constructed a homology model of the Nav1.5 sequence on the NavPaS structure using the MODELLER program run at the University of California San Francisco RBVI (Sali and Blundell, 1993). As shown previously, this model places residue E1784E within 5Å of residue K1493 in the DIII-DIV linker (Gade et al, 2020). A model constructed on the SWISS-MODEL server (not depicted) showed a similar result (Bordoli et al, 2009;Arnold et al, 2006;Biasini et al, 2014).…”

Fibroblast growth factor homologous factors tune arrhythmogenic late NaV1.5 current in calmodulin binding–deficient channels

“…By comparison with cardiomyocytes, the Na V 1.5 IQ/AA channels exhibit strong late Na + current when expressed heterologously. To determine regulatory factors that prevent late Na + current for IQ/AA mutant ch, we considered fibroblast growth factor homologous factors (FHFs), which are within the Na V 1.5 proteomic subdomain in the heart, can bind to the C-terminal domain of Na V 1.5, and have been implicated in the regulation of late Na + current ( 11 , 12 , 19 – 21 ). We found that FGF13, the predominant cardiac isoform in rodents ( 22 ), diminished late current of the IQ/AA mutant, suggesting that endogenous FHFs may serve to prevent late Na + current in cardiomyocytes.…”