Divergent regulation of KCNQ1/E1 by targeted recruitment of protein kinase A to distinct sites on the channel complex
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eLife assessment
This important study provides a substantial advance with a method by which a protein target resistant to therapeutic approaches can be uniquely modulated by a cellular protein kinase ferried by nanobodies to a precise molecular site of recruitment. Evidence for this major claim is compelling, but evidence for some of the minor claims seems incomplete. The work will be of broad interest to cell biologists, cardiovascular researchers, and drug developers.
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Abstract
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 up-regulation 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 that 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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eLife assessment
This important study provides a substantial advance with a method by which a protein target resistant to therapeutic approaches can be uniquely modulated by a cellular protein kinase ferried by nanobodies to a precise molecular site of recruitment. Evidence for this major claim is compelling, but evidence for some of the minor claims seems incomplete. The work will be of broad interest to cell biologists, cardiovascular researchers, and drug developers.
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Reviewer #1 (Public Review):
This study aims to compare the impact on KCNQ1/ KCNE1 channel complexes of localizing PKA components in distinct ways: by targeting of PKA domains to the C-terminus of KCNQ1 or KCNE1 or overexpression of an untargeted catalytic domain. The evidence is compelling that targeting PKA domains to the C-terminus of KCNQ1 causes distinct phosphorylation as well as decreasing channel conductance and channel protein at the cell surface when compared to overexpression of PKA subunits with other constructs. The study effectively deploys a symbiotic combination of techniques to link electrophysiology, surface expression, and phosphorylation changes responding to targeted recruitment. Support seems incomplete for the minor claims of retention specifically to the ER/Golgi, and that targeted recruitment of PKA domains to …
Reviewer #1 (Public Review):
This study aims to compare the impact on KCNQ1/ KCNE1 channel complexes of localizing PKA components in distinct ways: by targeting of PKA domains to the C-terminus of KCNQ1 or KCNE1 or overexpression of an untargeted catalytic domain. The evidence is compelling that targeting PKA domains to the C-terminus of KCNQ1 causes distinct phosphorylation as well as decreasing channel conductance and channel protein at the cell surface when compared to overexpression of PKA subunits with other constructs. The study effectively deploys a symbiotic combination of techniques to link electrophysiology, surface expression, and phosphorylation changes responding to targeted recruitment. Support seems incomplete for the minor claims of retention specifically to the ER/Golgi, and that targeted recruitment of PKA domains to KCNE1 was successful and distinct from untargeted overexpression. This study demonstrates the potential for engineering submolecularly-targeted phosphorylation to post-translationally modify a single protein in multiple ways with the same kinase. That distinct intramolecular patterns of phosphorylation can be encoded by the recruitment point of a kinase is very interesting and expected to be of value to the studies of ion channel modulation, kinase activity, and the development of related biotechnology.
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Reviewer #2 (Public Review):
This is a well-conceived and interesting study that investigates how a targeted protein phosphorylation (TPP) approach could be implemented to reconstitute PKA regulation of the cardiac KCNQ1/KCNE1 (IKs) potassium channel in the absence of an A-kinase anchoring protein (AKAP9). Using a genetically encoded GFP/YFP nanobody-based system they showed distinctive modulation of cAMP-mediated IKs activity. To that aim, they used an anti-GFP nanobody to recruit either the PKA holoenzyme RIIα or Cα subunits to YFP-tagged Q1 or YFP-E1 of reconstituted IKs channel complexes in CHO and HEK cells. They showed that targeted recruitment of endogenous Cα to E1-YFP using nano-RIIα modestly enhanced PKA-mediated IKs activity, whereas tethering of either nano-RIIα or nano-Cα to Q1-YFP retained KCNQ1 in the ER and Golgi thereby …
Reviewer #2 (Public Review):
This is a well-conceived and interesting study that investigates how a targeted protein phosphorylation (TPP) approach could be implemented to reconstitute PKA regulation of the cardiac KCNQ1/KCNE1 (IKs) potassium channel in the absence of an A-kinase anchoring protein (AKAP9). Using a genetically encoded GFP/YFP nanobody-based system they showed distinctive modulation of cAMP-mediated IKs activity. To that aim, they used an anti-GFP nanobody to recruit either the PKA holoenzyme RIIα or Cα subunits to YFP-tagged Q1 or YFP-E1 of reconstituted IKs channel complexes in CHO and HEK cells. They showed that targeted recruitment of endogenous Cα to E1-YFP using nano-RIIα modestly enhanced PKA-mediated IKs activity, whereas tethering of either nano-RIIα or nano-Cα to Q1-YFP retained KCNQ1 in the ER and Golgi thereby reducing IKs function. Using (LC-MS/MS), they further demonstrated that compared to free Cα, Cα targeted to Q1-YFP phosphorylated KCNQ1 subunit in multiple sites. Overall, the experiments are nicely done and yield sound data. The contribution of the paper is significant because it provides knowledge about the distinctive regulation of IKs by PKA, which could be used in the future to develop potential new drugs to prevent exercise-induced sudden cardiac death.
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Reviewer #3 (Public Review):
In this latest installment of a growing body of work from Henry Colecraft's lab in which native enzymes, ion channels, and other machinery are hijacked for therapeutic potential, cells can be made to respond to beta-adrenergic signals even when lacking the critical adaptor protein AKAP9. Normally, the cardiac repolarizing current IKs is enhanced in the face of beta-adrenergic signaling when increased cAMP activates PKA anchored to the channel protein by AKAP9. PKA phosphorylates the channel, increasing function or density in the membrane, especially during exercise or fright. Under these circumstances, when AKAP9 is missing in patients, the action potential fails to repolarize in a timely manner and arrhythmias can result. In this study, targeting the PKA catalytic or regulatory subunit to the E1 auxiliary …
Reviewer #3 (Public Review):
In this latest installment of a growing body of work from Henry Colecraft's lab in which native enzymes, ion channels, and other machinery are hijacked for therapeutic potential, cells can be made to respond to beta-adrenergic signals even when lacking the critical adaptor protein AKAP9. Normally, the cardiac repolarizing current IKs is enhanced in the face of beta-adrenergic signaling when increased cAMP activates PKA anchored to the channel protein by AKAP9. PKA phosphorylates the channel, increasing function or density in the membrane, especially during exercise or fright. Under these circumstances, when AKAP9 is missing in patients, the action potential fails to repolarize in a timely manner and arrhythmias can result. In this study, targeting the PKA catalytic or regulatory subunit to the E1 auxiliary channel subunit via a targeting nanobody restores at least some of the normal modulation in the presence of cAMP. This primary finding demonstrates a potential therapeutic approach when mutations disrupt its interaction with the channel complex.
A secondary finding of the study is that, contrary to expectation, targeting the enzyme to the Q1 alpha subunit C-terminus does not restore modulation but rather inhibits current by tying up the protein in the ER. Retention apparently depends on phosphorylation because a kinase-dead PKA catalytic subunit exhibits normal current. These findings demonstrate that the efficacy of correction is critically dependent on the site of recruitment. The results represent a starting point whereby kinase-based signaling can be synthetically harnessed to restore normal function in a disease setting.
The strengths of the study are the therapeutic potential of its principal finding and the clever approach to redirecting cellular components. Controls for the constructs are carefully designed and executed. Most of the conclusions are supported by the data presented. The weaknesses are minor and include providing more than an exemplar to support findings of enhanced phosphorylation and an accounting of how the findings from immunofluorescent images were quantitatively established. The study represents a major contribution to an emerging field of study in which modulation is induced by the proximity of enzymes to otherwise undruggable targets.
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