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

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 up-regulation of IKs 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 IKs 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 IKs, reconstituting physiological regulation of the channel complex. By contrast, nano-mediated tethering of RIIα or Cα to Q1-YFP constitutively inhibited IKs 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 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.

The Phosphodiesterase Inhibitor IBMX Blocks the Potassium Channel THIK-1 from the Extracellular Side.

The two-pore domain potassium channel (K2P-channel) THIK-1 has several predicted protein kinase A (PKA) phosphorylation sites. In trying to elucidate whether THIK-1 is regulated via PKA, we expressed THIK-1 channels in a mammalian cell line (CHO cells) and used the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) as a pharmacological tool to induce activation of PKA. Using the whole-cell patch-clamp recording, we found that THIK-1 currents were inhibited by application of IBMX with an IC50 of 120 µM. Surprisingly, intracellular application of IBMX or of the second messenger cAMP via the patch pipette had no effect on THIK-1 currents. In contrast, extracellular application of IBMX produced a rapid and reversible inhibition of THIK-1. In patch-clamp experiments with outside-out patches, THIK-1 currents were also inhibited by extracellular application of IBMX. Expression of THIK-1 channels in Xenopus oocytes was used to compare wild-type channels with mutated channels. Mutation of the putative PKA phosphorylation sites did not change the inhibitory effect of IBMX on THIK-1 currents. Mutational analysis of all residues of the (extracellular) helical cap of THIK-1 showed that mutation of the arginine residue at position 92, which is in the linker between cap helix 2 and pore helix 1, markedly reduced the inhibitory effect of IBMX. This flexible linker region, which is unique for each K2P-channel subtype, may be a possible target of channel-specific blockers. SIGNIFICANCE STATEMENT: The potassium channel THIK-1 is strongly expressed in the central nervous system. We studied the effect of 3-isobutyl-1-methyl-xanthine (IBMX) on THIK-1 currents. IBMX inhibits breakdown of cAMP and thus activates protein kinase A (PKA). Surprisingly, THIK-1 current was inhibited when IBMX was applied from the extracellular side of the membrane, but not from the intracellular side. Our results suggest that IBMX binds directly to the channel and that the inhibition of THIK-1 current was not related to activation of PKA.