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

KCNE1 (E1) β-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

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

Molecular Identification and Functional Investigation of ClC-3 as a Key Component of Native Volume-Sensitive Outwardly Rectifying Anion Channels (VSOACs) in Mammalian Heart

Chloride is the most abundant extracellular anion of different organisms and chloride transport proteins in mammalian heart have been shown to play many important functional roles in a great variety of different processes in cardiac physiology and pathophysiology. Most mammalian cells, including cardiac myocytes, have the capability of adjusting cell volume when challenged with extracellular osmolarity changes. Regulatory volume decrease (RVD) is a common physiological process in most mammalian cells. It is also regarded as one of the most fundamental features and basic functions of mammalian cells. The mammalian ClC gene family is a widely distributed chloride transport protein family, which comprises at present nine members (ClC-1~ClC-7, and two ClC-K isoforms), including both secondary active chloride-proton transporters and gated chloride channels. The mammalian ClC chloride channel superfamily has aroused more and more interest in the chloride channel research field due to its important physiological and pathophysiological significance, which has been revealed from several different human genetic diseases and has been well verified from various knock-out mouse models. Specifically, two members of the ClC gene family have been proposed to encode for functionally distinct chloride currents in mammalian hearts: ClC-2 for the hyperpolarization and cell-swelling activated inwardly rectifying chloride current (ICl,ir), and ClC-3 for the volume-sensitive outwardly rectifying chloride current (ICl,vol). Although convincing evidence has accumulated over the years supporting an essential role of ClC-3 for native volume sensitive outwardly rectifying anion currents (VSOACs) in mammalian cardiomyocytes and vascular smooth muscle cells, the ClC-3 hypothesis has been controversial. Specifically, the presence of native VSOAC currents from ClC-3 global knock-out mice cast considerable doubt on the validity of ClC-3 being responsible for the native VSOAC channels. However, complex compensatory changes were revealed from the ClC-3 global knock-out mice as there are significant alterations in both the mRNA and protein expression profiles from knock-out mouse cardiac myocytes due to the conventional global ClC-3 gene inactivation method. Accordingly, several important altered properties of the native VSOAC currents wereidentified, including the lost of sensitivity to the activation of protein kinase C (PKC) and inhibition by a ClC-3 antibody. The purpose of my research project is to further test the ClC-3 hypothesis and unravel the elusive issue of molecular identification of native VSOACs and its relevant essential regulatory characteristics in mammalian heart. We developed new improved technical approaches that involves the generation of three novel lines of cardiac-specific ClC-3 transgenic mice, the heart-specific human short-isoform ClC-3 overexpressing mice, and the heart-specific conditional and inducible ClC-3 knock-out mice. Echocardiography and electrocardiography techniques were utilized to assess mouse heart function and further evaluate the possible physiological role and pathophysiological significance of ClC-3 in mammalian heart with cardiac-specific ClC-3 transgenic manipulations. Furthermore, whole-cell patch clamp techniques were used to investigate and characterize the basic properties of native VSOAC currents in freshly isolated mouse cardiac myocytes from these three heart-specific ClC-3 transgenic mouse lines and their relevant age-matched control animals. The results indicate that heart-specific overexpression of human short-isoform ClC-3 in mice significantly increased the current densities of native VSOACs in the transgenic mice compared to the control ones, with no changes in basic biophysical and pharmacological properties. Phenotypic characterization of these transgenic mice revealed significantly decreased heart weight: body weight ratios and significantly accelerated RVD process in the freshly dispersed atrial myocytes from the human short-isoform ClC-3 overexpressing mice, compared to age-matched control mice. Electrophysiological studies with the freshly isolated atrial myocytes from the cardiac specific conditional knock-out mice demonstrated two different groups of cells. In group one, native VSOAC currents were significantly decreased when challenged with extracellular hypotonic stimulation, compared to those recorded from age-matched control mice, which is consistent with ClC-3 being responsible for native VSOACs. However, in the other group of cells, native VSOAC currents were still present with similar current densities as observed from age-matched control mice, which is consistent with the extensive compensatory changes revealed with the conventional global ClC-3 knock-out mice. Phenotypic studies of these heart-specific conditional ClC-3 knock-out mice disclosed cardiac hypertrophy and significantly compromised heart function. Furthermore, for the cardiac-specific inducible ClC-3 knock-out mouse model, the results demonstrated a time-dependent elimination of native VSOAC currents in freshly isolated cardiac myocytes from knock-out mice. These novel cardiac specific ClC-3 inducible knock-out mice were regularly maintained on the special doxycycline food. Doxycycline removal from the diet activated the Cre-recombinase and led to the deletion of ClC-3 gene in the adult mouse heart. Interestingly, it turned out that 3 weeks off doxycycline was a key time point to significantly knock down both mRNA and protein expression levels of ClC-3 and eliminate the native VSOAC currents in the acutely isolated cardiomyocytes from the knock-out mice. This approach also eliminated possible confounding effects of compensatory changes in other proteins as we observed in the heart-specific conditional ClC-3 knock-out mouse model. Accordingly, echocardiography analysis at the same time point also revealed significantly increased heart weight: body weight ratios, potent cardiac hypertrophy and seriously compromised heart function in the inducible knock-out mice, indicated by the cardiac functional parameters of significantly decreased left ventricular ejection fraction (LVEF) and fractional shortening (FS%), when compared to the age-matched control animals. In summary, these experimental approaches using novel ClC-3 transgenic mice with various cardiac specific ClC-3 transgenic constructs provided valuable biological information and greater insight into the physiological role of ClC-3 in mouse heart, compared to the conventional global gene knock-out methodology. Our data collected from the comprehensive study of the three novel heart-specific ClC-3 transgenic mouse lines strongly suggests an essential role of the ClC-3 chloride channel as a key component of native VSOACs in mammalian heart

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

KCNE1 (E1) β-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

Fifth-Order Mapped Semi-Lagrangian Weighted Essentially Nonoscillatory Methods Near Certain Smooth Extrema

Fifth-order mapped semi-Lagrangian weighted essentially nonoscillatory (WENO) methods at certain smooth extrema are developed in this study. The schemes contain the mapped semi-Lagrangian finite volume (M-SL-FV) WENO 5 method and the mapped compact semi-Lagrangian finite difference (M-C-SL-FD) WENO 5 method. The weights in the more common scheme lose accuracy at certain smooth extrema. We introduce mapped weighting to handle the problem. In general, a cell average is applied to construct the M-SL-FV WENO 5 reconstruction, and the M-C-SL-FD WENO 5 interpolation scheme is proposed based on an interpolation approach. An accuracy test and numerical examples are used to demonstrate that the two schemes reduce the loss of accuracy and improve the ability to capture discontinuities