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

Acute cardiac hypoxia produces life-threatening elevations in late sodium current (ILATE) in the human heart. Here, we show the underlying mechanism: hypoxia induces rapid SUMOylation of NaV1.5 channels so they reopen when normally inactive, late in the action potential. NaV1.5 is SUMOylated only on lysine 442, and the mutation of that residue, or application of a deSUMOylating enzyme, prevents hypoxic reopenings. The time course of SUMOylation of single channels in response to hypoxia coincides with the increase in ILATE, a reaction that is complete in under 100 s. In human cardiac myocytes derived from pluripotent stem cells, hypoxia-induced ILATE is confirmed to be SUMO-dependent and to produce action potential prolongation, the pro-arrhythmic change observed in patients.

SUMOylation determines the voltage required to activate cardiac IKs channels.

IKs 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 IKs channels depends on their covalent modification by small ubiquitin-like modifier (SUMO) proteins. IKs channels are comprised of four KCNQ1 pore-forming subunits, two KCNE1 accessory subunits, and up to four SUMOs, one on Lys424 of each KCNQ1 subunit. Each SUMO shifts the half-maximal activation voltage (V1/2) of IKs ∼ +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-Lys424 sites. SUMOylation of KCNQ1 is KCNE1 dependent and determines the native attributes of cardiac IKs in vivo.

Individual IKs channels at the surface of mammalian cells contain two KCNE1 accessory subunits.

KCNE1 (E1) ß-subunits assemble with KCNQ1 (Q1) voltage-gated K(+) channel α-subunits to form IKslow (IKs) channels in the heart and ear. The number of E1 subunits in IKs channels has been an issue of ongoing debate. Here, we use single-molecule spectroscopy to demonstrate that surface IKs channels with human subunits contain two E1 and four Q1 subunits. This stoichiometry does not vary. Thus, IKs channels in cells with elevated levels of E1 carry no more than two E1 subunits. Cells with low levels of E1 produce IKs channels with two E1 subunits and Q1 channels with no E1 subunits--channels with one E1 do not appear to form or are restricted from surface expression. The plethora of models of cardiac function, transgenic animals, and drug screens based on variable E1 stoichiometry do not reflect physiology.

Structure and functional interaction of the extracellular domain of human GABA(B) receptor GBR2.

Inhibitory neurotransmission is mediated primarily by GABA. The metabotropic GABA(B) receptor is a G protein-coupled receptor central to mammalian brain function. Malfunction of GABA(B) receptor has been implicated in several neurological disorders. GABA(B) receptor functions as a heterodimeric assembly of GBR1 and GBR2 subunits, where GBR1 is responsible for ligand-binding and GBR2 is responsible for G protein coupling. Here we demonstrate that the GBR2 ectodomain directly interacts with the GBR1 ectodomain to increase agonist affinity by selectively stabilizing the agonist-bound conformation of GBR1. We present the crystal structure of the GBR2 ectodomain, which reveals a polar heterodimeric interface. We also identify specific heterodimer contacts from both subunits, and GBR1 residues involved in ligand recognition. Lastly, our structural and functional data indicate that the GBR2 ectodomain adopts a constitutively open conformation, suggesting a structural asymmetry in the active state of GABA(B) receptor that is unique to the GABAergic system.

Characterization of KCNQ1 atrial fibrillation mutations reveals distinct dependence on KCNE1.

The I(Ks) potassium channel, critical to control of heart electrical activity, requires assembly of α (KCNQ1) and ß (KCNE1) subunits. Inherited mutations in either I(Ks) channel subunit are associated with cardiac arrhythmia syndromes. Two mutations (S140G and V141M) that cause familial atrial fibrillation (AF) are located on adjacent residues in the first membrane-spanning domain of KCNQ1, S1. These mutations impair the deactivation process, causing channels to appear constitutively open. Previous studies suggest that both mutant phenotypes require the presence of KCNE1. Here we found that despite the proximity of these two mutations in the primary protein structure, they display different functional dependence in the presence of KCNE1. In the absence of KCNE1, the S140G mutation, but not V141M, confers a pronounced slowing of channel deactivation and a hyperpolarizing shift in voltage-dependent activation. When coexpressed with KCNE1, both mutants deactivate significantly slower than wild-type KCNQ1/KCNE1 channels. The differential dependence on KCNE1 can be correlated with the physical proximity between these positions and KCNE1 as shown by disulfide cross-linking studies: V141C forms disulfide bonds with cysteine-substituted KCNE1 residues, whereas S140C does not. These results further our understanding of the structural relationship between KCNE1 and KCNQ1 subunits in the I(Ks) channel, and provide mechanisms for understanding the effects on channel deactivation underlying these two atrial fibrillation mutations.

Cardiac-specific, inducible ClC-3 gene deletion eliminates native volume-sensitive chloride channels and produces myocardial hypertrophy in adult mice.

Native volume-sensitive outwardly rectifying anion channels (VSOACs) play a significant role in cell volume homeostasis in mammalian cells. However, the molecular correlate of VSOACs has been elusive to identify. The short isoform of ClC-3 (sClC-3) is a member of the mammalian ClC gene family and has been proposed to be a molecular candidate for VSOACs in cardiac myocytes and vascular smooth muscle cells. To directly test this hypothesis, and assess the physiological role of ClC-3 in cardiac function, we generated a novel line of cardiac-specific inducible ClC-3 knock-out mice. These transgenic mice were maintained on a doxycycline diet to preserve ClC-3 expression; removal of doxycycline activates Cre recombinase to inactivate the Clcn3 gene. Echocardiography revealed dramatically reduced ejection fraction and fractional shortening, and severe signs of myocardial hypertrophy and heart failure in the knock-out mice at both 1.5 and 3 weeks off doxycycline. In mice off doxycycline, time-dependent inactivation of ClC-3 gene expression was confirmed in atrial and ventricular cells by qRT-PCR and Western blot analysis. Electrophysiological examination of native VSOACs in isolated atrial and ventricular myocytes 3 weeks off doxycycline revealed a complete elimination of the currents, whereas at 1.5 weeks, VSOAC current densities were significantly reduced, compared to age-matched control mice maintained on doxycycline. These results indicate that ClC-3 is a key component of native VSOACs in mammalian heart and plays a significant cardioprotective role against cardiac hypertrophy and failure.

Cardiac-specific overexpression of the human short CLC-3 chloride channel isoform in mice.

1. ClC-3 has been proposed as a molecular candidate responsible for volume-sensitive outwardly rectifying anion channels (VSOAC) in cardiac and smooth muscle cells. To further test this hypothesis, we produced a novel line of transgenic mice with cardiac-specific overexpression of the human short ClC-3 isoform (hsClC-3). 2. Northern and western blot analyses demonstrated that mRNA and protein levels of the short isoform (sClC-3) in the heart were significantly increased in hsClC-3-overexpressing (OE) mice compared with wild-type (WT) mice. Heart weight : bodyweight ratios for OE mice were significantly smaller compared with age-matched WT mice. 3. Electrocardiogram recordings indicated no difference at rest, whereas echocardiographic recordings revealed consistent reductions in left ventricular diastolic diameter, left ventricular posterior wall thickness at end of diastole and interventricular septum thickness in diastole in OE mice. 4. The VSOAC current densities in atrial cardiomyocytes were significantly increased by ClC-3 overexpression compared with WT cells. No differences in VSOAC current properties in OE and WT atrial myocytes were observed in terms of outward rectification, anion permeability (I(-) > Cl(-) > Asp(-)) and inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid and glibenclamide. The VSOAC in atrial myocytes from both groups were totally abolished by phorbol-12,13-dibutyrate (a protein kinase C activator) and by intracellular dialysis of an N-terminal anti-ClC-3 antibody. 5. Cardiac cell volume measurements revealed a significant acceleration of the rate of regulatory volume decrease (RVD) in OE myocytes compared with WT. 6. In conclusion, enhanced VSOAC currents and acceleration of the time-course of RVD in atrial myocytes of OE mice is strong evidence supporting an essential role of sClC-3 in native VSOAC function in mouse atrial myocytes.