Kv11.1 (ERG1) K+ channels localize in cholesterol and sphingolipid enriched membranes and are modulated by membrane cholesterol

The localization of ion channels to specific membrane microdomains can impact the functional properties of channels and their role in cellular physiology. We determined the membrane localization of human Kv11.1 (hERG1) alpha-subunit protein, which underlies the rapidly activating, delayed rectifier K(+) current (I(Kr)) in the heart. Immunocytochemistry and membrane fractionation using discontinuous sucrose density gradients of adult canine ventricular tissue showed that Kv11.1 channel protein localized to both the cell surface and T-tubular sarcolemma. Furthermore, density gradient membrane fractionation using detergent (Triton X-100) and non-detergent (OptiPrep) methods from canine ventricular myocytes or HEK293 cells demonstrated that Kv11.1 protein, along with MiRP1 and Kv7.1 (KCNQ1) proteins, localize in cholesterol and sphingolipid enriched membrane fractions. In HEK293 cells, Kv11.1 channels, but not long QT-associated mutant G601S-Kv11.1 channels, also localized to cholesterol and sphingolipid enriched membrane fractions. Depletion of membrane cholesterol from HEK293 cells expressing Kv11.1 channels using methyl-beta-cyclodextrin (MbetaCD) caused a positive shift of the voltage dependence of activation and an acceleration of deactivation kinetics of Kv11.1 current (I(Kv11.1)). Cholesterol loading of HEK293 cells reduced the steep voltage dependence of I(Kv11.1) activation and accelerated the inactivation kinetics of I(Kv11.1). Incubation of neonatal mouse myocytes in MbetaCD also accelerated the deactivation kinetics of I(Kr). We conclude that Kv11.1 protein localizes in cholesterol and sphingolipid enriched membranes and that membrane cholesterol can modulate I(Kv11.1) and I(Kr).     

Activation of hypothalamic RIP‐Cre neurons promotes beiging of WAT via sympathetic nervous system

Activation of brown adipose tissue (BAT) and beige fat by cold increases energy expenditure. Although their activation is known to be differentially regulated in part by hypothalamus, the underlying neural pathways and populations remain poorly characterized. Here, we show that activation of rat‐insulin‐promoter‐Cre (RIP‐Cre) neurons in ventromedial hypothalamus (VMH) preferentially promotes recruitment of beige fat via a selective control of sympathetic nervous system (SNS) outflow to subcutaneous white adipose tissue (sWAT), but has no effect on BAT. Genetic ablation of APPL2 in RIP‐Cre neurons diminishes beiging in sWAT without affecting BAT, leading to cold intolerance and obesity in mice. Such defects are reversed by activation of RIP‐Cre neurons, inactivation of VMH AMPK, or treatment with a β3‐adrenergic receptor agonist. Hypothalamic APPL2 enhances neuronal activation in VMH RIP‐Cre neurons and raphe pallidus, thereby eliciting SNS outflow to sWAT and subsequent beiging. These data suggest that beige fat can be selectively activated by VMH RIP‐Cre neurons, in which the APPL2–AMPK signaling axis is crucial for this defending mechanism to cold and obesity.     

Small GTPase Determinants for the Golgi Processing and Plasmalemmal Expression of Human Ether-a-go-go Related (hERG) K+ Channels

The pro-arrhythmic Long QT syndrome (LQT) is linked to 10 different genes (LQT1–10). Approximately 40% of genotype-positive LQT patients have LQT2, which is characterized by mutations in the human ether-a-go-go related gene (hERG). hERG encodes the voltage-gated K⁺ channel α-subunits that form the pore of the rapidly activating delayed rectifier K⁺ current in the heart. The purpose of this study was to elucidate the mechanisms that regulate the intracellular transport or trafficking of hERG, because trafficking is impaired for about 90% of LQT2 missense mutations. Protein trafficking is regulated by small GTPases. To identify the small GTPases that are critical for hERG trafficking, we coexpressed hERG and dominant negative (DN) GTPase mutations in HEK293 cells. The GTPases Sar1 and ARF1 regulate the endoplasmic reticulum (ER) export of proteins in COPII and COPI vesicles, respectively. Expression of DN Sar1 inhibited the Golgi processing of hERG, decreased hERG current (I_hERG) by 85% (n ≥ 8 cells per group, *, p < 0.01), and reduced the plasmalemmal staining of hERG. The coexpression of DN ARF1 had relatively small effects on hERG trafficking. Surprisingly, the coexpression of DN Rab11B, which regulates the endosomal recycling, inhibited the Golgi processing of hERG, decreased I_hERG by 79% (n ≥ 8 cells per group; *, p < 0.01), and reduced the plasmalemmal staining of hERG. These data suggest that hERG undergoes ER export in COPII vesicles and endosomal recycling prior to being processed in the Golgi. We conclude that hERG trafficking involves a pathway between the ER and endosomal compartments that influences expression in the plasmalemma.The human KCNH2 or ether-a-go-go related gene (hERG)³ encodes the voltage-gated K⁺ channel α-subunits that oligomerize to form the pore of the rapidly activating delayed rectifier K⁺ current (I_Kr) in cardiac myocytes (1–3). Hundreds of hERG mutations are linked to the congenital pro-arrhythmic Type 2 Long QT syndrome (LQT2) and functional studies suggest that these mutations result in a loss of normal hERG K⁺ channel (hERG) function (4, 5). In LQT2, missense mutations are the dominant abnormality and many LQT2 missense mutations reduce hERG K⁺ current (I_hERG) by decreasing the intracellular transport or trafficking of hERG to the Golgi apparatus (Golgi) and the cell surface membrane (plasmalemma) (6). Therefore, disruption of hERG K⁺ channel trafficking appears to be a principal mechanism for disease.Movement of proteins between membrane-bound intracellular compartments is mediated by small transport vesicles, which bud from a donor compartment to fuse with an appropriate acceptor compartment. The trafficking of many transmembrane and secretory proteins between the ER and Golgi compartments is dependent on the small GTPases ADP-ribosylation factor 1 (ARF1) and Sar1, which regulate the formation of coat-associated protein complex I (COPI) and II (COPII) vesicles, respectively (7–19). These small GTPases facilitate the polymerization of transport vesicle protein coats on the donor membrane. Vesicular cargo selection, docking, and fusion to the target membrane are regulated by adaptor proteins, SNARE proteins, and Rab GTPases. To rationally develop novel therapeutic targets that may increase the expression of trafficking-deficient LQT2 mutant channels, the molecular mechanisms that regulate the trafficking of hERG need to be explored. The purpose of this study is to identify transport proteins that regulate the trafficking of wild type (WT) hERG. We used a strategy of testing specific WT GTPases or ones containing dominant negative (DN) mutations to interfere with their function.     

Proteomic analysis during larval development and metamorphosis of the spionid polychaete Pseudopolydora vexillosa

Background  

While the larval-juvenile transition (metamorphosis) in the spionid polychaete Pseudopolydora vexillosa involves gradual morphological changes and does not require substantial development of juvenile organs, the opposite occurs in the barnacle Balanus amphitrite. We hypothesized that the proteome changes during metamorphosis in the spionids are less drastic than that in the barnacles. To test this, proteomes of pre-competent larvae, competent larvae (ready to metamorphose), and juveniles of P. vexillosa were compared using 2-dimensional gel electrophoresis (2-DE), and they were then compared to those of the barnacle.     

NHS-A isoform of the NHS gene is a novel interactor of ZO-1

Mutations in the NHS (Nance-Horan Syndrome) gene lead to severe congenital cataracts, dental defects and sometimes mental retardation. NHS encodes two protein isoforms, NHS-A and -1A that display cell-type dependent differential expression and localization. Here we demonstrate that of these two isoforms, the NHS-A isoform associates with the cell membrane in the presence of intercellular contacts and it immunoprecipitates with the tight junction protein ZO-1 in MDCK (Madin Darby Canine Kidney) epithelial cells and in neonatal rat lens. The NHS-1A isoform however is a cytoplasmic protein. Both Nhs isoforms are expressed during mouse development. Immunolabelling of developing mouse with the anti-NHS antibody that detects both isoforms revealed the protein in the developing head including the eye and brain. It was primarily expressed in epithelium including neural epithelium and certain vascular endothelium but only weakly expressed in mesenchymal cells. In the epithelium and vascular endothelium the protein associated with the cell membrane and co-localized with ZO-1, which indirectly indicates expression of the Nhs-A isoform in these structures. Membrane localization of the protein in the lens vesicle similarly supports Nhs-A expression. In conclusion, the NHS-A isoform of NHS is a novel interactor of ZO-1 and may have a role at tight junctions. This isoform is important in mammalian development especially of the organs in the head.     

Genomic analysis of SARS-CoV-2 reveals local viral evolution in Ghana

The confirmed case fatality rate for the coronavirus disease 2019 (COVID-19) in Ghana has dropped from a peak of 2% in March to be consistently below 1% since May 2020. Globally, case fatality rates have been linked to the strains/clades of circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within a specific country. Here we present 46 whole genomes of SARS-CoV-2 circulating in Ghana, from two separate sequencing batches: 15 isolates from the early epidemic (March 12–April 1 2020) and 31 from later time-points ( 25–27 May 2020). Sequencing was carried out on an Illumina MiSeq system following an amplicon-based enrichment for SARS-CoV-2 cDNA. After genome assembly and quality control processes, phylogenetic analysis showed that the first batch of 15 genomes clustered into five clades: 19A, 19B, 20A, 20B, and 20C, whereas the second batch of 31 genomes clustered to only three clades 19B, 20A, and 20B. The imported cases (6/46) mapped to circulating viruses in their countries of origin, namely, India, Hungary, Norway, the United Kingdom, and the United States of America. All genomes mapped to the original Wuhan strain with high similarity (99.5–99.8%). All imported strains mapped to the European superclade A, whereas 5/9 locally infected individuals harbored the B4 clade, from the East Asian superclade B. Ghana appears to have 19B and 20B as the two largest circulating clades based on our sequence analyses. In line with global reports, the D614G linked viruses seem to be predominating. Comparison of Ghanaian SARS-CoV-2 genomes with global genomes indicates that Ghanaian strains have not diverged significantly from circulating strains commonly imported into Africa. The low level of diversity in our genomes may indicate lower levels of transmission, even for D614G viruses, which is consistent with the relatively low levels of infection reported in Ghana.     

Caveolin-3 Overexpression Attenuates Cardiac Hypertrophy via Inhibition of T-type Ca2+ Current Modulated by Protein Kinase Cα in Cardiomyocytes

Pathological cardiac hypertrophy is characterized by subcellular remodeling of the ventricular myocyte with a reduction in the scaffolding protein caveolin-3 (Cav-3), altered Ca²⁺ cycling, increased protein kinase C expression, and hyperactivation of calcineurin/nuclear factor of activated T cell (NFAT) signaling. However, the precise role of Cav-3 in the regulation of local Ca²⁺ signaling in pathological cardiac hypertrophy is unclear. We used cardiac-specific Cav-3-overexpressing mice and in vivo and in vitro cardiac hypertrophy models to determine the essential requirement for Cav-3 expression in protection against pharmacologically and pressure overload-induced cardiac hypertrophy. Transverse aortic constriction and angiotensin-II (Ang-II) infusion in wild type (WT) mice resulted in cardiac hypertrophy characterized by significant reduction in fractional shortening, ejection fraction, and a reduced expression of Cav-3. In addition, association of PKCα and angiotensin-II receptor, type 1, with Cav-3 was disrupted in the hypertrophic ventricular myocytes. Whole cell patch clamp analysis demonstrated increased expression of T-type Ca²⁺ current (I_{Ca, T}) in hypertrophic ventricular myocytes. In contrast, the Cav-3-overexpressing mice demonstrated protection from transverse aortic constriction or Ang-II-induced pathological hypertrophy with inhibition of I_{Ca, T} and intact Cav-3-associated macromolecular signaling complexes. siRNA-mediated knockdown of Cav-3 in the neonatal cardiomyocytes resulted in enhanced Ang-II stimulation of I_{Ca, T} mediated by PKCα, which caused nuclear translocation of NFAT. Overexpression of Cav-3 in neonatal myocytes prevented a PKCα-mediated increase in I_{Ca, T} and nuclear translocation of NFAT. In conclusion, we show that stable Cav-3 expression is essential for protecting the signaling mechanisms in pharmacologically and pressure overload-induced cardiac hypertrophy.     

Unique modulation of L-type Ca2+ channels by short auxiliary beta1d subunit present in cardiac muscle

Recent studies have identified a growing diversity of splice variants of auxiliary Ca2+ channel Ca(v)beta subunits. The Ca(v)beta(1d) isoform encodes a putative protein composed of the amino-terminal half of the full-length Ca(v)beta(1) isoform and thus lacks the known high-affinity binding site that recognizes the Ca2+ channel alpha1-subunit, the alpha-binding pocket. The present study investigated whether the Ca(v)beta(1d) subunit is expressed at the protein level in heart, and whether it exhibits any of the functional properties typical of full-length Ca(v)beta subunits. On Western blots, an antibody directed against the unique carboxyl terminus of Ca(v)beta(1d) identified a protein of the predicted molecular mass of 23 kDa from canine and human hearts. Immunocytochemistry and surface-membrane biotinylation experiments in transfected HEK-293 cells revealed that the full-length Ca(v)beta(1b) subunit promoted membrane trafficking of the pore-forming alpha1C (Ca(v)1.2)-subunit to the surface membrane, whereas the Ca(v)beta(1d) subunit did not. Whole cell patch-clamp analysis of transfected HEK-293 cells demonstrated no effect of coexpression of the Ca(v)beta(1d) with the alpha1C-subunit compared with the 15-fold larger currents and leftward shift in voltage-dependent activation induced by full-length Ca(v)beta(1b) coexpression. In contrast, cell-attached patch single-channel studies demonstrated that coexpression of either Ca(v)beta(1b) or Ca(v)beta(1d) significantly increased mean open probability four- to fivefold relative to the alpha1C-channels alone, but only Ca(v)beta(1b) coexpression increased the number of channels observed per patch. In conclusion, the Ca(v)beta(1d) isoform is expressed in heart and can modulate the gating of L-type Ca2+ channels, but it does not promote membrane trafficking of the channel complex.