SUMOylation determines the voltage required to activate cardiac I _Ks channels

Abstract

I Ks channels open in response to depolarization of the membrane voltage during the cardiac action potential, passing potassium ions outward to repolarize ventricular myocytes and end each beat. Here, we show that the voltage required to activate I Ks channels depends on their covalent modification by small ubiquitin-like modifier (SUMO) proteins. I Ks channels are comprised of four KCNQ1 poreforming subunits, two KCNE1 accessory subunits, and up to four SUMOs, one on Lys 424 of each KCNQ1 subunit. Each SUMO shifts the half-maximal activation voltage (V 1/2 ) of I Ks ∼ +8 mV, producing a maximal +34-mV shift in neonatal mouse cardiac myocytes or Chinese hamster ovary (CHO) cells expressing the mouse or human subunits. Unexpectedly, channels formed without KCNE1 carry at most two SUMOs despite having four available KCNQ1-Lys 424 sites. SUMOylation of KCNQ1 is KCNE1 dependent and determines the native attributes of cardiac I Ks in vivo.C ardiac I Ks channels pass the slow component of the delayed rectifier potassium current that is necessary to produce normal heart rate and rhythm. Thus, I Ks varies in magnitude and timing subject to neuronal and hormonal influences (1). Abnormal changes in I Ks function, due to inherited mutations in KCNQ1 or KCNE1, or suppression of the current by medications, can produce long QT syndrome (LQTS) and life-threatening cardiac arrhythmias (2). KCNQ1 (also called K V 7.1 and previously K V LQT) is a classical voltage-gated potassium α-subunit with six transmembrane segments and a single pore-forming P loop. Human KCNQ1 has variants up to 676 residues in length, whereas KCNE1 (minK) has just 129 residues and a single transmembrane span (Fig. 1A). Despite its small size, KCNE1 is essential to the operation of I Ks . Compared with channels formed by KCNQ1 subunits alone, KCNE1 produces a right shift in the half-maximal voltage required to activate the channel (V 1/2 ), slows the kinetics of activation and deactivation, increases the channel unitary conductance, alters the ion selectivity of the conduction pore and, modifies its pharmacology (3-8). KCNE1 also endows I Ks channel with a high affinity for PIP 2 (9) and allows responsiveness to PKA-mediated phosphorylation following β-adrenergic stimulation (10). Here, we demonstrate that the V 1/2 inherent to native cardiac I Ks channels results from, and is modified by, KCNE1-dependent SUMOylation of KCNQ1.SUMOylation is the enzyme-mediated linkage of one of three SUMO isoforms to the e-amino group of specific Lys residues on a target protein (11). Present in all eukaryotic cells, the pathway was recognized to regulate the activity of nuclear transcription factors when we discovered it resident at the plasma membrane (12, 13) and to regulate the excitability of cerebellar granule neurons via SUMOylation of K2P1 and Na V 1.2 (14, 15), and of hippocampal neurons via modification of K V 2.1 (16).This study was inspired by the work of Qi et al. (17), who describe partial deficiency of the deSUMOylating enzyme SENP2 in mice, showing it to pro...

Hypoxia Produces Pro-arrhythmic Late Sodium Current in Cardiac Myocytes by SUMOylation of NaV1.5 Channels

“…We delivered 1 nM purified SUMO1 peptide into iPS-CMs by the patch pipette; a concentration that evoked maximal effects on Na V 1.2 channels in cerebellar granule neurons (CGNs) and three different K + channels in CGNs, hippocampal neurons, and rat cardiac myocytes (Xiong et al, 2017;Plant et al, 2011Plant et al, , 2012Plant et al, , 2016. SUMO1 induced changes in cardiac I Na like those produced by hypoxia: I LATE increased to $4.2% of peak I Na without a change in the peak magnitude ( Figures 1B and 1C; Table 1).…”

“…SUMOylation is the enzyme-mediated linkage of one of three SUMO isoforms to the ε amino group of specific lysine residues in a target protein (Henley et al, 2014). Present in all eukaryotic cells, the SUMO pathway was known to regulate the trafficking and activity of nuclear transcription factors when we described it to operate as well at the plasma membrane by direct SUMOylation of Na + and K + channels to regulate excitability Plant et al, 2010Plant et al, , 2011Plant et al, , 2012Plant et al, , 2016Xiong et al, 2017). Given the important role of hypoxia-induced increases in I LATE in heart disease, we tested the hypothesis that SUMOylation of Na V 1.5 channels was the underlying mechanism.…”

Expression of SUMO enzymes is fiber type dependent in skeletal muscles and is dysregulated in muscle disuse

“…38,39 Since when SUMO has been discovered, 40 the importance related to this PTM has been investigated in eukaryotic cell lines, but only recently, it has been associated to control functions in differentiated organs like muscles and brain. 26,[41][42][43] .…”

Doxorubicin induces caspase-mediated proteolysis of KV7.1

“…Briefly, to initiate differentiation, medium was changed to CDM3, consisting of RPMI 1640 (Corning), 500 μg ml -1 Oryza sativa-derived recombinant human albumin (Oryzogen, Wuhan, China), and 213 μg ml -1 L-ascorbic acid 2-phosphate (Wako, Tokyo, Japan). For days 0-1, media was supplemented with 3 μM of the glycogen synthase kinase 3- inhibitor CHIR99021 (Biorbyt, San Francisco, USA) 21,22 and 10 ng ml -1 BMP4 (Peprotech, Hamburg, Germany). On day 1, media was changed to CDM3 and on d2 media was changed to CDM3 supplemented with 2 μM of the Wnt inhibitor Wnt-C59 (Biorbyt).…”

“…For K v 7.1, it has also been shown that the A-kinase anchoring protein yotiao binds to helix D, leading to the recruitment of protein kinase A (PKA) and protein phosphatase 1, thereby forming a macromolecular complex important for regulating I Ks activity [20]. In addition to PKA-mediated phospho-rylation, ubiquitination and sumoylation have been reported to be important for regulation of K v 7.1 activity at the posttranslational level [21,22].…”