BIOLOGICAL ANTIARRHYTHMICS-SODIUM CHANNEL INTERACTING PROTEINS

Abstract

Voltage gated Na channels (Na_V) are essential for excitation of tissues. Mutations in Na_Vs cause a spectrum of human disease from autism and epilepsy to cardiac arrhythmias to skeletal myotonias. The carboxyl termini (CT) of Na_V channels are hotspots for disease-causing mutations and are richly invested with protein interaction sites. We have focused on the regulation of Na_V by two proteins that bind in this region: calmodulin (CaM) and non-secreted fibroblast growth factors (iFGF or FHF). CaM regulates Na_V gating, mediating Ca²⁺-dependent inactivation (CDI) in a channel isoform-specific manner, while Ca²⁺-free CaM (apo-CaM) binding broadly regulates Na_V opening and suppresses the arrhythmogenic late Na current (I_Na-L). FHFs inhibit CDI, in Na_V isoforms that exhibit this property, and potently suppress I_Na-L, the latter requiring the amino terminus of the FHF. A peptide comprised of the first 39 amino acids of FHF1_A is sufficient to inhibit I_Na-L, constituting a credible specific antiarrhythmic.

Fig. 1.

Schematic of the voltage-gated sodium channel (Na_V). Transmembrane glycoprotein with four internally homologous domains (I–IV), each comprised of six membrane-spanning alpha helices (cylinders), the fourth (orange) being positively charged and serving as a voltage sensor. The loops between the fifth and sixth membrane-spanning helices in each domain form the ion selective pore. There are two classes of long and short variants that differ in the length of the loop connecting the first and second homologous domains. There are a number of ancillary subunits, represented by the single membrane spanning β subunit (red cylinder). The channel is phosphorylated at a number of sites by several kinases, shown here as blue (PKA), pink (CaMKII), and red (PKC) circles. The carboxy terminus is structurally ordered in the region adjacent to the membrane and is comprised of six alpha helices (purple); the sixth contains an IQ motif that binds CaM and FHF binds in the pre-IQ region. Abbreviations: Syn, syntrophin; EFL, EF hand-like motif

Fig. 2.

Schematic of CaM and isoform-specific dynamic Ca²⁺ regulation of Na_V channels. The skeletal muscle isoform, Na_V1.4, exhibits Ca²⁺-dependent inactivation (CDI). At low levels of [Ca²⁺], the N-lobe of CaM is tethered to the proximal CT near the EFL, and calcification of the N-lobe promotes unbinding from the EFL, with the N-lobe serving as an inactivation particle. Differences in CaM binding in Na_V1.5 produce tethering of the calcified N-lobe to the distal CT; thus, NaV1.5 does not exhibit CDI. Modified from Yoder, et al. 2014.

Fig. 3.

CaM binding tunes the late current. Panels A-C show WT channels expressed in HEK293 cells under control conditions and in the presence of a CaM chelator and CaM. Time-compressed currents are shown as well as single channel currents recorded between 50 and 300 msec after the depolarization. In panel A, the open probability plot, and an amplified inset of the first 100 msec is shown below the currents. Panels D-F and G-I are currents through IQ/AA and S1904L mutants, respectively, in the same format as shown for WT. From Kang, et al. 2021.

Fig. 4.

Summary plot of the effect of siRNA knock down of FHF2 on persistent Na current in mouse ventricular myocytes. In the presence of the targeted siRNA, persistent current was significantly increased in IQ/AA mice but not WT and non-transgenic controls. From Chakouri, et al. 2022.

Fig. 5.

Summary plot of the effect of isoforms of FHF on persistent Na current through mutant NaV1.5 channels. Type A FHFs with an intact amino terminus (NT) inhibit I_Na-L; type B variants do not. FHF1_S, which retains part of the NT, suppresses the late current. From Chakouri, et al. 2022.

Fig. 6.

Na currents from human iPSC cardiomyocytes reveal FHF inhibition of late current. Cardiomyocytes were generated from a control subject and patient with LQTS due to a ΔKPQ mutation. Knock down of FHF2 had no impact currents in either cell. FHF1_A dramatically reduced late channel openings in the mutant cardiomyocytes. Consistent with isoform-specific FHF expression and the importance of A-type FHFs in tuning I_Na-L. From Chakouri, et al. 2022.

Fig. 7.

Panel A: Summary plot of the inhibition of persistent current by FHF1_A amino terminal fragments. The fragment comprising amino acids 1-39 is as potent an inhibitor as full length FHF1_A or the complete amino terminal fragment and significantly more potent than ranolazine. Panel B: Adenovirus encoded FixR inhibits late openings of Na_V1.5 ΔKPQ mutant channels in hiPSC-CMs. Panel C: FixR inhibits I_Na-L in several contexts, mutations that cause LQTS, and channel phosphorylation by PKA and CaMKII known to be associated with structural heart disease. Modified from Chakouri, et al. 2022.