PKA phosphorylation underlies functional recruitment of sarcolemmal SK2 channels in ventricular myocytes from hypertrophic hearts

Shanna Hamilton, Iuliia Polina, Radmila Terentyeva, Peter Bronk, Tae Yun Kim, Karim Roder, Richard T. Clements, Gideon Koren, Bum Rak Choi, Dmitry Terentyev

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

Key points: Small-conductance Ca2+-activated K+ (SK) channels expressed in ventricular myocytes are dormant in health, yet become functional in cardiac disease. SK channels are voltage independent and their gating is controlled by intracellular [Ca2+] in a biphasic manner. Submicromolar [Ca2+] activates the channel via constitutively-bound calmodulin, whereas higher [Ca2+] exerts inhibitory effect during depolarization. Using a rat model of cardiac hypertrophy induced by thoracic aortic banding, we found that functional upregulation of SK2 channels in hypertrophic rat ventricular cardiomyocytes is driven by protein kinase A (PKA) phosphorylation. Using site-directed mutagenesis, we identified serine-465 as the site conferring PKA-dependent effects on SK2 channel function. PKA phosphorylation attenuates ISK rectification by reducing the Ca2+/voltage-dependent inhibition of SK channels without changing their sensitivity to activating submicromolar [Ca2+]i. This mechanism underlies the functional recruitment of SK channels not only in cardiac disease, but also in normal physiology, contributing to repolarization under conditions of enhanced adrenergic drive. Abstract: Small-conductance Ca2+-activated K+ (SK) channels expressed in ventricular myocytes (VMs) are dormant in health, yet become functional in cardiac disease. We aimed to test the hypothesis that post-translational modification of SK channels under conditions accompanied by enhanced adrenergic drive plays a central role in disease-related activation of the channels. We investigated this phenomenon using a rat model of hypertrophy induced by thoracic aortic banding (TAB). Western blot analysis using anti-pan-serine/threonine antibodies demonstrated enhanced phosphorylation of immunoprecipitated SK2 channels in VMs from TAB rats vs. Shams, which was reversible by incubation of the VMs with PKA inhibitor H89 (1 μmol L–1). Patch clamped VMs under basal conditions from TABs but not Shams exhibited outward current sensitive to the specific SK inhibitor apamin (100 nmol L–1), which was eliminated by inhibition of PKA (1 μmol L–1). Beta-adrenergic stimulation (isoproterenol, 100 nmol L–1) evoked ISK in VMs from Shams, resulting in shortening of action potentials in VMs and ex vivo optically mapped Sham hearts. Using adenoviral gene transfer, wild-type and mutant SK2 channels were overexpressed in adult rat VMs, revealing serine-465 as the site that elicits PKA-dependent phosphorylation effects on SK2 channel function. Concurrent confocal Ca2+ imaging experiments established that PKA phosphorylation lessens rectification of ISK via reduction Ca2+/voltage-dependent inhibition of the channels at high [Ca2+] without affecting their sensitivity to activation by Ca2+ in the submicromolar range. In conclusion, upregulation of SK channels in diseased VMs is mediated by hyperadrenergic drive in cardiac hypertrophy, with functional effects on the channel conferred by PKA-dependent phosphorylation at serine-465.

Original languageEnglish (US)
Pages (from-to)2847-2873
Number of pages27
JournalJournal of Physiology
Volume598
Issue number14
DOIs
StatePublished - Jul 1 2020
Externally publishedYes

Bibliographical note

Funding Information:
This work was supported by American Heart Association Grant #18POST33960456 (SH); National Heart, Lung, and Blood Institute at the National Institutes of Health (NIH) NIH RO1HL135236 (RTC); NIH R01HL110791 (GK); NIH R01HL096669 (BRC); and American Heart Association Grant in Aid 15GRNT25650002 and NIH R01HL121796 (DT).

Publisher Copyright:
© 2019 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society

Keywords

  • Calcium transients
  • Cardiac electrophysiology
  • PKA
  • Small-conductance Ca-activated K channels
  • Ventricular arrhythmia

Fingerprint Dive into the research topics of 'PKA phosphorylation underlies functional recruitment of sarcolemmal SK2 channels in ventricular myocytes from hypertrophic hearts'. Together they form a unique fingerprint.

Cite this