No abstract

The slow cardiac delayed rectifier current (IKs) is formed by KCNQ1 and KCNE1 subunits and is one of the major repolarizing currents in the heart. Decrease of IKs currents either due to inherited mutations or pathological remodeling is associated with increased risk for cardiac arrhythmias and sudden death. Ca 2+-dependent PKC isoforms (cPKC) are chronically activated in heart disease and diabetes. Recently, we found that sustained stimulation of the calcium-dependent PKCβII isoform leads to decrease in KCNQ1 subunit membrane localization and KCNQ1/KCNE1 channel activity, although the role of KCNE1 in this regulation was not explored. Here, we show that the auxiliary KCNE1 subunit expression is necessary for channel internalization. A mutation in a KCNE1 phosphorylation site (KCNE1 (S102A)) abolished channel internalization in both heterologous expression systems and cardiomyocytes. Altogether, our results suggest that KCNE1(S102) phosphorylation by PKCβII leads to KCNQ1/KCNE1 channel internalization in response to sustained PKC stimulus, while leaving KCNQ1 homomeric channels in the membrane. This preferential internalization is expected to have strong impact on cardiac repolarization. Our results suggest that KCNE1(S102) is an important anti-arrhythmic drug target to prevent IKs pathological remodeling leading to cardiac arrhythmias.

KCNQ (M-type) K þ channels and TRPC cation channels are regulated via G q/11 -protein-mediated signals in brain and peripheral ganglia. Stimulation of G q/11 -coupled receptors both consumes PIP 2 via PLC hydrolysis and stimulates PIP 2 synthesis via rises in Ca 2þ i and other signals. Using brain-slice electrophysiology and Ca 2þ imaging, we characterized threshold K þ currents in mouse dentate gyrus granule cells (DGGCs) and CA1 pyramidal cells, the effects of M 1 mAChR (M1R) stimulation on M current and on neuronal discharge properties, and elucidated the underlying signaling mechanisms. We found disparate signaling between DGGCs and CA1 neurons. DGGCs displayed M1R-induced enhancement of M current, rather than its suppression, due to profound stimulation of PIP 2 synthesis, which was paralleled by increased PIP 2 -regulated GIRK currents provoked by stimulation of endogenous GABA B receptors. Nonetheless, stimulation of M1Rs increased excitability under current-clamp, as previously reported. Using Cre-lox breeding, deficiency of KCNQ2-containing M channels in DGGCs ablated M1Rinduced enhancement of the M-type current. Simultaneously, M1R stimulation in DGGCs induced robust increases in [Ca 2þ ] i , largely via TRPC currents, which increased excitability. With TRPC channels blocked, M1R stimulation induced no increases in excitability, and with blockade of PI(4) P 5-kinase, M1R stimulation suppressed M current. On the other hand, M current in CA1 pyramidal neurons was suppressed by M1R stimulation, similar to widely-described M channel inhibition involving PIP 2 depletion in peripheral ganglia. Use of expressed hM3q DREADD receptors gave similar results. Both types of neurons displayed a much more slowly deactivating Kþ current that was, at most, only weakly affected by M1R agonists. Therefore, there are pleotropic mechanisms of cholinergic signals that direct cell-type specific, precise control of hippocampal function, with strong implications for hyperexcitability and epilepsy.

Plasma membrane phosphatidylinositol 4-phosphate (PI4P) is a precursor of PI(4,5)P2, an important regulator of a large number of ion channels. Although the role of the phospholipid PI(4,5)P2 in stabilizing ion channel function is well established, little is known about the role of phospholipids in channel membrane localization and specifically the role of PI4P in channel function and localization. The phosphatidylinositol 4-kinases (PI4Ks) synthesize PI4P. Our data show that inhibition of PI4K and prolonged decrease of levels of plasma membrane PI4P lead to a decrease in the KCNQ1/KCNE1 channel membrane localization and function. In addition, we show that mutations linked to Long QT syndrome that affect channel interactions with phospholipids lead to a decrease in membrane expression. We show that expression of a LQT1-associated C-terminal deletion mutant abolishes PI4Kinase-mediated decrease in membrane expression and rescues membrane expression for phospholipid-targeting mutations. Our results indicate a novel role for PI4P on ion channel regulation. Our data suggest that decreased membrane PI4P availability to the channel, either due to inhibition of PI4K or as consequence of mutations, dramatically inhibits KCNQ1/KCNE1 channel membrane localization and current. Our results may have implications to regulation of other PI4P binding channels.