Closed-state inactivation in Kv4.3 isoforms is differentially modulated by protein kinase C

Abstract

Kv4.3, with its complex open- and closed-state inactivation (CSI) characteristics, is a primary contributor to early cardiac repolarization. The two alternatively spliced forms, Kv4.3-short (Kv4.3-S) and Kv4.3-long (Kv4.3-L), differ by the presence of a 19-amino acid insert downstream from the sixth transmembrane segment. The isoforms are similar kinetically; however, the longer form has a unique PKC phosphorylation site. To test the possibility that inactivation is differentially regulated by phosphorylation, we expressed the Kv4.3 isoforms in Xenopus oocytes and examined changes in their inactivation properties after stimulation of PKC activity. Whereas there was no difference in open-state inactivation, there were profound differences in CSI. In Kv4.3-S, PMA reduced the magnitude of CSI by 24% after 14.4 s at -50 mV. In contrast, the magnitude of CSI in Kv4.3-L increased by 25% under the same conditions. Mutation of a putatively phosphorylated threonine (T504) to aspartic acid within a PKC consensus recognition sequence unique to Kv4.3-L eliminated the PMA response. The change in CSI was independent of the intervention used to increase PKC activity; identical results were obtained with either PMA or injected purified PKC. Our previously published 11-state model closely simulated our experimental data. Our data demonstrate isoform-specific regulation of CSI by PKC in Kv4.3 and show that the carboxy terminus of Kv4.3 plays an important role in regulation of CSI.

Cardiac Potassium Channel Subtypes: New Roles in Repolarization and Arrhythmia

“…Accordingly, several isoforms with different functional phenotypes exist (22,200,223,327,402,404,460,469,499). The alternative splicing increases the redundancy of the channels, but also helps to diversify the function of the multimeric proteins.…”

Overexpression of a non-muscle RBFOX2 isoform triggers cardiac conduction defects in myotonic dystrophy

“…We further propose that compared to the muscle isoform, RBFOX240 stimulates the higher-order assembly of LASR complex 46,47 , which boosts its splicing activity, resulting in altered isoform expression of proteins that can disrupt the normal cardiac rhythm and function. Indeed, we demonstrated that RBFOX240 promotes the generation of pathogenic splice variants of voltage-gated sodium and potassium channels that are known to exhibit slower conduction velocity and increased susceptibility to arrhythmias 52,54,68,69 . Our MD simulations showed that a RBFOX240-mediated splicing switch in Nav1.5 transcript changes the amino acid composition of the voltage sensing element within domain I that promotes persistent lipid-interactions and stabilizes the 310-helical conformation of S4 helix, which reduces the energy barrier and allows for rapid deactivation of the channel.…”

“…Amongst these amino acids is a conserved threonine residue, which is phosphorylated by protein kinase C to facilitate regulation of transient outward current Ito 68 . The KV4.3 isoform lacking the 57-nt exon has been shown to promote faster inactivation of the channel, which slows down the ion flux and lengthens the duration of action potential-particularly affecting the early recovery and plateau phases of repolarization 69 .…”

β Subunits Control the Effects of Human Kv4.3 Potassium Channel Phosphorylation

“…The two known human Kv4.3 splice variants, Kv4.3L and Kv4.3S, were previously thought to function similarly at baseline, but differently with respect to their regulation by PKC, because of PKC phosphorylation of T504 in the C-terminal 19-residues that are found only in Kv4.3L (Po et al, 2001 ; Xie et al, 2009 ). Yet, the author recently found that co-expression with KChIP2b or KCNEs co-expression reveals important functional differences between Kv4.3L and Kv4.3S, namely that KChIP2 favorably augments Kv4.3S vs. Kv4.3L activity (four- vs. two-fold), and that Kv4.3S-KChIP2b channels are faster inactivating, and exhibit negative-shifted steady-state inactivation, compared to Kv4.3L-KChIP2b (Abbott, 2017 ).…”

“…Protein kinase C (PKC) phosphorylation of T504 within this consensus site mediates inhibition of human Kv4.3L, facilitating physiologically important α-adrenergic regulation of cardiac I to (Po et al, 2001 ). In contrast, others found that for rat Kv4.3, PKC stimulation (10 nM PMA for 30 min) inhibited both long and short forms, T504A-Kv4.3L-indepedently, with the only isoform-dependent difference being differentially altered closed-state inactivation (decreasing it in Kv4.3S, increasing it in Kv4.3L; Xie et al, 2009 ).…”