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bioRxiv · 10.1101/2022.09.12.507626

Divergent regulation of KCNQ1/E1 by targeted recruitment of protein kinase A to distinct sites on the channel complex

Zou, X.·Shanmugam, S. K.·Kanner, S. A.·Sampson, K. J.·Kass, R. S.·Colecraft, H. M.
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Abstract

The slow delayed rectifier potassium current, IKs, conducted through pore-forming Q1 and auxiliary E1 ion channel complexes is important for human cardiac action potential repolarization. During exercise or fright, IKs is up-regulated by protein kinase A (PKA)-mediated Q1 phosphorylation to maintain heart rhythm and optimum cardiac performance. Sympathetic upregulation of IKs requires recruitment of PKA holoenzyme (two regulatory- RI or RII- and two catalytic C 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 IKs in the absence of AKAP9. Targeted recruitment of endogenous C to E1-YFP using a GFP/YFP nanobody (nano) fused to RII enabled acute cAMP-mediated enhancement of IKs, reconstituting physiological regulation of the channel complex. By contrast, nano-mediated tethering of RII or C to Q1-YFP constitutively inhibited IKs by retaining the channel intracellularly in the endoplasmic reticulum and Golgi. Proteomic analysis revealed distinct phosphorylation sites are modified by C targeted to Q1-YFP compared to free C. Thus, functional outcomes of synthetically recruited PKA on IKs regulation is critically dependent on the site of recruitment within the channel complex. The results reveal insights into divergent regulation of IKs 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, X., Shanmugam, S. K., Kanner, S. A., Sampson, K. J., Kass, R. S., Colecraft, H. M.. 2022-09-14. Divergent regulation of KCNQ1/E1 by targeted recruitment of protein kinase A to distinct sites on the channel complex. https://doi.org/10.1101/2022.09.12.507626

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