Key result
Targeted recruitment of PKA to KCNE1 reconstituted physiological regulation of IKs, whereas targeting PKA to KCNQ1 constitutively inhibited the channel by retaining it intracellularly.
Why the study?
Mutations abolishing Q1 and AKAP9 functional interactions cause long QT syndrome types 1 and 11, raising sudden cardiac death risk; whether targeted protein phosphorylation can reconstitute PKA regulation of I Ks without AKAP9 was investigated.
Does targeted recruitment of PKA to distinct sites on the KCNQ1/E1 channel complex differentially regulate IKs current and channel trafficking in heterologous cell models?
Does targeted recruitment of PKA to distinct sites on the KCNQ1/E1 channel complex differentially regulate IKs current and channel trafficking in heterologous cell models?
Targeted protein phosphorylation of the KCNQ1/E1 channel complex yields divergent functional outcomes depending on the site of PKA recruitment, suggesting a novel approach for developing therapeutics for long QT syndrome.
Clarifies PKA recruitment for sympathetic IKs upregulation; leaves open whether this axis can be targeted to modulate arrhythmia risk.
The slow delayed rectifier potassium current, I Ks , conducted through pore-forming Q1 and auxiliary E1 ion channel complexes is important for human cardiac action potential repolarization. During exercise or fright, I Ks is up-regulated by protein kinase A (PKA)-mediated Q1 phosphorylation to maintain heart rhythm and optimum cardiac performance. Sympathetic upregulation of I Ks requires recruitment of PKA holoenzyme (two regulatory- RI or RII- and two catalytic Cα subunits) to Q1 C-terminus by an A kinase anchoring protein (AKAP9). Mutations in Q1 or AKAP9 that abolish their functional interaction result in long QT syndrome type 1 and 11, respectively, which increases the risk of sudden cardiac death during exercise. Here, we investigated the utility of a targeted protein phosphorylation (TPP) approach to reconstitute PKA regulation of I Ks in the absence of AKAP9. Targeted recruitment of endogenous Cα to E1-YFP using a GFP/YFP nanobody (nano) fused to RIIα enabled acute cAMP-mediated enhancement of I Ks , reconstituting physiological regulation of the channel complex. By contrast, nano-mediated tethering of RIIα or Cα to Q1-YFP constitutively inhibited I Ks by retaining the channel intracellularly in the endoplasmic reticulum and Golgi. Proteomic analysis revealed distinct phosphorylation sites are modified by Cα targeted to Q1-YFP compared to free Cα. Thus, functional outcomes of synthetically recruited PKA on I Ks regulation is critically dependent on the site of recruitment within the channel complex. The results reveal insights into divergent regulation of I Ks by phosphorylation across different spatial and time scales, and suggest a TPP approach to develop new drugs to prevent exercise-induced sudden cardiac death.
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Zou et al. (2022) studied Long QT Syndrome (preclinical model). Targeted recruitment of PKA (nanoRIIα or nanoCα) vs. Naked nanobody (nano) or free Cα was evaluated on IKs current amplitude and channel surface density. Targeted recruitment of PKA to KCNE1 reconstituted physiological regulation of IKs, whereas targeting PKA to KCNQ1 constitutively inhibited the channel by retaining it intracellularly.
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