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

KEY POINTS: Small‐conductance Ca(2+)‐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 [Ca(2+)] in a biphasic manner. Submicromolar [Ca(2+)] activates the channel via constitutively‐bound calmodulin, whereas higher [Ca(2+)] 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 I (SK) rectification by reducing the Ca(2+)/voltage‐dependent inhibition of SK channels without changing their sensitivity to activating submicromolar [Ca(2+)](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 Ca(2+)‐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 I (SK) 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 Ca(2+) imaging experiments established that PKA phosphorylation lessens rectification of I (SK) via reduction Ca(2+)/voltage‐dependent inhibition of the channels at high [Ca(2+)] without affecting their sensitivity to activation by Ca(2+) 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.