Identification of a novel protein kinase A phosphorylation site, S449, on the delayed rectifier potassium channel, Kv1.2

Our laboratory has provided evidence of the contribution of vascular smooth muscle (VSM) delayed rectifier potassium channels (K DR ), composed of Kv1.2, Kv1.5, Kv1.6 and Kvβ subunits, to the control of myogenic constriction of arterial resistance vessels. We have also shown that K DR channels in the vasculature are regulated by vasodilatory agonists acting through protein kinase A (PKA). However, the molecular basis for this regulation is uncertain. The current study used electrophysiological and biochemical analyses to identify PKA phosphorylation sites on Kv1.2. Heterologous expression of Kv1 channel subunits revealed that Kv1.2 currents were enhanced following stimulation of PKA, while Kv1.5 currents were unaffected. Previous studies suggested that Kv1.2 is phosphorylated by PKA at threonine‐46 (T46). However, several lines of evidence are inconsistent with this view. Mutation of T46 did not reduce phosphorylation of Kv1.2 by PKA in vitro . Phosphoamino acid analysis demonstrated that phosphorylation occurred exclusively at serine. Two‐dimensional (2D) phosphopeptide mapping revealed one major phosphopeptide, which was detected in both wild‐type and mutant Kv1.2. Mutation of serine‐449 (S449A) significantly reduced 32 P‐incorporation into Kv1.2. In vitro phosphorylation assays indicated that this site is stoichiometrically phosphorylated by PKA. Sequencing by mass spectrometry confirmed that Kv1.2 is phosphorylated by PKA at S449. This study suggests that S449 is a PKA phosphorylation site on Kv1.2, and provides insight into the mechanism underlying the modulation of VSM K DR by PKA.