ZnT1 induces a crosstalk between T-type and L-type calcium channels through interactions with Raf-1 kinase and the calcium channel ß2 subunit.

ZnT1 is a major zinc transporter that regulates cellular zinc homeostasis. We have previously shown that ZnT1 has additional functions that are independent of its activity as a Zn2+ extruder. These include inhibition of the L-type calcium channel (LTCC) through interaction with the auxiliary ß-subunit of the LTCC and activation of the Raf-ERK signaling leading to augmented activity of the T-type calcium channel (TTCC). Our findings indicate that ZnT1 increases TTCC activity by enhancing the trafficking of the channel to the plasma membrane. LTCC and TTCC are co-expressed in many tissues and have different functions in a variety of tissues. In the current work, we investigated the effect of the voltage-gated calcium channel (VGCC) ß-subunit and ZnT1 on the crosstalk between LTCC and TTCC and their functions. Our results indicate that the ß-subunit inhibits the ZnT1-induced augmentation of TTCC function. This inhibition correlates with the VGCC ß-subunit-dependent reduction in ZnT1-induced activation of Ras-ERK signaling. The effect of ZnT1 is specific, as the presence of the ß-subunit did not change the effect of endothelin-1 (ET-1) on TTCC surface expression. These findings document a novel regulatory function of ZnT1 serving as a mediator in the crosstalk between TTCC and LTCC. Overall, we demonstrate that ZnT1 binds and regulates the activity of the ß-subunit of VGCC and Raf-1 kinase and modulates surface expression of the LTCC and TTCC catalytic subunits, consequently modulating the activity of these channels.

Student responses to creative coding in biomedical science education.

Biomedical science students need to learn to code. Graduates face a future where they will be better prepared for research higher degrees and the workforce if they can code. Embedding coding in a biomedical curriculum comes with challenges. First, biomedical science students often experience anxiety learning quantitative and computational thinking skills and second biomedical faculty often lack expertise required to teach coding. In this study, we describe a creative coding approach to building coding skills in students using the packages of Processing and Arduino. Biomedical science students were taught by an interdisciplinary faculty team from Medicine and Health, Science and Architecture, Design and Planning. We describe quantitative and qualitative responses of students to this approach. Cluster analysis revealed a diversity of student responses, with a large majority of students who supported creative coding in the curriculum, a smaller but vocal cluster, who did not support creative coding because either the exercises were not sufficiently challenging or were too challenging and believed coding should not be in a Biomedical Science curriculum. We describe how two creative coding platforms, Processing and Arduino, embedded and used to visualize human physiological data, and provide responses to students, including those minority of students, who are opposed to coding in the curriculum This study found a variety of students responses in a final year capstone course of an undergraduate Biomedical Science degree where future pathways for students are either in research higher degrees or to the workforce with a future which will be increasingly data driven.

P2Y6 receptors are involved in mediating the effect of inactivated avian influenza virus H5N1 on IL-6 & CXCL8 mRNA expression in respiratory epithelium.

One of the key pathophysiologies of H5N1 infection is excessive proinflammatory cytokine response (cytokine storm) characterized by increases in IFN-ß, TNF-α, IL-6, CXCL10, CCL4, CCL2 and CCL5 in the respiratory tract. H5N1-induced cytokine release can occur via an infection-independent mechanism, however, detail of the cellular signaling involved is poorly understood. To elucidate this mechanism, the effect of inactivated (ß-propiolactone-treated) H5N1 on the cytokine and chemokine mRNA expression in 16HBE14o- human respiratory epithelial cells was investigated. We found that the inactivated-H5N1 increased mRNA for IL-6 and CXCL8 but not TNF-α, CCL5 or CXCL10. This effect of the inactivated-H5N1 was inhibited by sialic acid receptor inhibitor (α-2,3 sialidase), adenosine diphosphatase (apyrase), P2Y receptor (P2YR) inhibitor (suramin), P2Y6R antagonist (MRS2578), phospholipase C inhibitor (U73122), protein kinase C inhibitors (BIM and Gö6976) and cell-permeant Ca2+ chelator (BAPTA-AM). Inhibitors of MAPK signaling, including of ERK1/2 (PD98059), p38 MAPK (SB203580) and JNK (SP600125) significantly suppressed the inactivated-H5N1-induced mRNA expression of CXCL8. On the other hand, the inactivated-H5N1-induced mRNA expression of IL-6 was inhibited by SB203580, but not PD98059 or SP600125, whereas SN-50, an inhibitor of NF-κB, inhibited the effect of virus on mRNA expression of both of IL-6 and CXCL8. Taken together, our data suggest that, without infection, inactivated-H5N1 induces mRNA expression of IL-6 and CXCL8 by a mechanism, or mechanisms, requiring interaction between viral hemagglutinin and α-2,3 sialic acid receptors at the cell membrane of host cells, and involves activation of P2Y6 purinergic receptors.

Zinc transport and the inhibition of the L-type calcium channel are two separable functions of ZnT-1.

Traditionally, proteins are considered to perform a single role, be it as an enzyme, a channel, a transporter or as a structural scaffold. However, recent studies have described moonlighting proteins that perform distinct and independent functions; for example, TRPM7 is both an ion channel and a kinase. ZnT-1 is a member of the Carrier Diffusion Facilitator family that is expressed throughout the phylogenetic tree from bacteria to humans. Since its cloning in 1995, ZnT-1 is considered a major extruder of Zn2+ based on its capability to protect cells against zinc toxicity. Recently, we reported that ZnT-1 inhibits the L-type calcium channel (LTCC), a major Zn2+ and Ca2+ entry pathway. Here we show that ZnT-1 is a dual-function protein by demonstrating that its abilities to exchange Zn2+/H+ and to inhibit the LTCC are independent of each other and are mediated by different parts of the protein. Specifically, mutations in the membrane-spanning helices that render ZnT-1 unable to transport zinc do not prevent it from inhibiting the LTCC. Moreover, a fragment consisting of the intracellular ZnT-1 C-terminal, which lacks all ion-transfer segments, inhibits the LTCC as efficiently as wild-type ZnT-1. Our data therefore indicates that ZnT-1 performs two structurally independent functions related to zinc homeostasis.

ZnT-1 extrudes zinc from mammalian cells functioning as a Zn(2+)/H(+) exchanger.

ZnT-1 is a Cation Diffusion Facilitator (CDF) family protein, and is present throughout the phylogenetic tree from bacteria to humans. Since its original cloning in 1995, ZnT-1 has been considered to be the major Zn(2+) extruding transporter, based on its ability to protect cells against zinc toxicity. However, experimental evidence for ZnT-1 induced Zn(2+) extrusion was not convincing. In the present study, based on the 3D crystal structure of the ZnT-1 homologue, YiiP, that predicts a homodimer that utilizes the H(+) electrochemical gradient to facilitate Zn(2+) efflux, we demonstrate ZnT-1 dependent Zn(2+) efflux from HEK 293T cells using FluoZin-3 and Fura 2 by single cell microscope based fluorescent imaging. ZnT-1 facilitates zinc efflux in a sodium-independent, pH-driven and calcium-sensitive manner. Moreover, substitution of two amino acids in the putative zinc binding domain of ZnT-1 led to nullification of Zn(2+) efflux and rendered the mutated protein incapable of protecting cells against Zn(2+) toxicity. Our results demonstrate that ZnT-1 extrudes zinc from mammalian cells by functioning as a Zn(2+)/H(+) exchanger.

Increased gut permeability and microbiota change associate with mesenteric fat inflammation and metabolic dysfunction in diet-induced obese mice.

We investigated the relationship between gut health, visceral fat dysfunction and metabolic disorders in diet-induced obesity. C57BL/6J mice were fed control or high saturated fat diet (HFD). Circulating glucose, insulin and inflammatory markers were measured. Proximal colon barrier function was assessed by measuring transepithelial resistance and mRNA expression of tight-junction proteins. Gut microbiota profile was determined by 16S rDNA pyrosequencing. Tumor necrosis factor (TNF)-α and interleukin (IL)-6 mRNA levels were measured in proximal colon, adipose tissue and liver using RT-qPCR. Adipose macrophage infiltration (F4/80⁺) was assessed using immunohistochemical staining. HFD mice had a higher insulin/glucose ratio (P = 0.020) and serum levels of serum amyloid A3 (131%; P = 0.008) but reduced circulating adiponectin (64%; P = 0.011). In proximal colon of HFD mice compared to mice fed the control diet, transepithelial resistance and mRNA expression of zona occludens 1 were reduced by 38% (P<0.001) and 40% (P = 0.025) respectively and TNF-α mRNA level was 6.6-fold higher (P = 0.037). HFD reduced Lactobacillus (75%; P<0.001) but increased Oscillibacter (279%; P = 0.004) in fecal microbiota. Correlations were found between abundances of Lactobacillus (r = 0.52; P = 0.013) and Oscillibacter (r = -0.55; P = 0.007) with transepithelial resistance of the proximal colon. HFD increased macrophage infiltration (58%; P = 0.020), TNF-α (2.5-fold, P<0.001) and IL-6 mRNA levels (2.5-fold; P = 0.008) in mesenteric fat. Increased macrophage infiltration in epididymal fat was also observed with HFD feeding (71%; P = 0.006) but neither TNF-α nor IL-6 was altered. Perirenal and subcutaneous adipose tissue showed no signs of inflammation in HFD mice. The current results implicate gut dysfunction, and attendant inflammation of contiguous adipose, as salient features of the metabolic dysregulation of diet-induced obesity.

Frog skin epithelium: electrolyte transport and chytridiomycosis.

One unique physiological characteristic of frogs is that their main route for intake of water is across the skin. In these animals, the skin acts in concert with the kidney and urinary bladder to maintain electrolyte homeostasis. Water absorption across the skin is driven by the osmotic gradient that develops as a consequence of solute transport. Our recent study demonstrated that chytridiomycosis, an infection of amphibian skin by the fungal pathogen, Batrachochytrium dendrobatidis, inhibits epithelial Na(+) channels, attenuating Na(+) absorption through the skin. In frogs that become severely affected by this fungus, systemic depletion of Na(+), K(+) and Cl(-) is thought to cause deterioration of cardiac electrical function, leading to cardiac arrest. Here we review the ion transport mechanisms of frog skin, and discuss the effect of chytridiomycosis on these mechanisms.

Regulation of the epithelial Na+ channel by the RH domain of G protein-coupled receptor kinase, GRK2, and Galphaq/11.

The G protein-coupled receptor kinase (GRK2) belongs to a family of protein kinases that phosphorylates agonist-activated G protein-coupled receptors, leading to G protein-receptor uncoupling and termination of G protein signaling. GRK2 also contains a regulator of G protein signaling homology (RH) domain, which selectively interacts with α-subunits of the Gq/11 family that are released during G protein-coupled receptor activation. We have previously reported that kinase activity of GRK2 up-regulates activity of the epithelial sodium channel (ENaC) in a Na(+) absorptive epithelium by blocking Nedd4-2-dependent inhibition of ENaC. In the present study, we report that GRK2 also regulates ENaC by a mechanism that does not depend on its kinase activity. We show that a wild-type GRK2 (wtGRK2) and a kinase-dead GRK2 mutant ((K220R)GRK2), but not a GRK2 mutant that lacks the C-terminal RH domain (ΔRH-GRK2) or a GRK2 mutant that cannot interact with Gαq/11/14 ((D110A)GRK2), increase activity of ENaC. GRK2 up-regulates the basal activity of the channel as a consequence of its RH domain binding the α-subunits of Gq/11. We further found that expression of constitutively active Gαq/11 mutants significantly inhibits activity of ENaC. Conversely, co-expression of siRNA against Gαq/11 increases ENaC activity. The effect of Gαq on ENaC activity is not due to change in ENaC membrane expression and is independent of Nedd4-2. These findings reveal a novel mechanism by which GRK2 and Gq/11 α-subunits regulate the activity ENaC.

Negative regulation of Ca(2+) influx during P2Y(2) purinergic receptor activation is mediated by Gbetagamma-subunits.

We have previously reported that P2Y(2) purinoceptors and muscarinic M(3) receptors trigger Ca(2+) responses in HT-29 cells that differ in their timecourse, the Ca(2+) response to P2Y(2) receptor activation being marked by a more rapid decline of intracellular Ca(2+) concentration ([Ca(2+)](i)) after the peak response and that this rapid decline of [Ca(2+)](i) was slowed in cells expressing heterologous beta-adrenergic receptor kinase (betaARK). In the present study, we demonstrate that, during P2Y(2) receptor activation, betaARK expression increases the rate of Gd(3+)-sensitive Mn(2+) influx, a measure of the rate of store-operated Ca(2+) entry from the extracellular space, during P2Y(2) activation and that this effect of betaARK is mimicked by exogenous alpha-subunits of G(q), G(11) and G(i2). The effect of betaARK on the rate of Mn(2+) influx is thus attributable to its ability to scavenge G protein betagamma-subunits released during activation of P2Y(2) receptor. We further find that the effect of betaARK on the rate of Mn(2+) influx during P2Y(2) receptor activation can be overcome by arachidonic acid. In addition, the UTP-induced Mn(2+) influx rate was significantly increased by inhibitors of phospholipase A(2) (PLA(2)) and an siRNA directed against PLA(2)beta, but not by an siRNA directed against PLA(2)alpha or by inhibitors of arachidonic acid metabolism. These findings provide evidence for the existence of a P2Y(2) receptor-activated signalling system that acts in parallel with depletion of intracellular Ca(2+) stores to inhibit Ca(2+) influx across the cell membrane. This signalling process is mediated via Gbetagamma and involves PLA(2)beta and arachidonic acid.

The activity of the epithelial sodium channels is regulated by caveolin-1 via a Nedd4-2-dependent mechanism.

It has recently been shown that the epithelial Na(+) channel (ENaC) is compartmentalized in caveolin-rich lipid rafts and that pharmacological depletion of membrane cholesterol, which disrupts lipid raft formation, decreases the activity of ENaC. Here we show, for the first time, that a signature protein of caveolae, caveolin-1 (Cav-1), down-regulates the activity and membrane surface expression of ENaC. Physical interaction between ENaC and Cav-1 was also confirmed in a coimmunoprecipitation assay. We found that the effect of Cav-1 on ENaC requires the activity of Nedd4-2, a ubiquitin protein ligase of the Nedd4 family, which is known to induce ubiquitination and internalization of ENaC. The effect of Cav-1 on ENaC requires the proline-rich motifs at the C termini of the beta- and gamma-subunits of ENaC, the binding motifs that mediate interaction with Nedd4-2. Taken together, our data suggest that Cav-1 inhibits the activity of ENaC by decreasing expression of ENaC at the cell membrane via a mechanism that involves the promotion of Nedd4-2-dependent internalization of the channel.

Regulation of epithelial Na+ channels by aldosterone: role of Sgk1.

1. The epithelial sodium channel (ENaC) is tightly regulated by hormonal and humoral factors, including cytosolic ion concentration and glucocorticoid and mineralocorticoid hormones. Many of these regulators of ENaC control its activity by regulating its surface expression via neural precursor cell-expressed developmentally downregulated (gene 4) protein (Nedd4-2). 2. During the early phase of aldosterone action, Nedd4-2-dependent downregulation of ENaC is inhibited by the serum- and glucocorticoid-induced kinase 1 (Sgk1). 3. Sgk1 phosphorylates Nedd4-2. Subsequently, phosphorylated Nedd4-2 binds to the 14-3-3 protein and, hence, reduces binding of Nedd4-2 to ENaC. 4. Nedd4-2 is also phosphorylated by protein kinase B (Akt1). Both Sgk1 and Akt1 are part of the insulin signalling pathway that increases transepithelial Na(+) absorption by inhibiting Nedd4-2 and activating ENaC.

Value of noncontact mapping for identifying left ventricular scar in an ovine model.

BACKGROUND: We assessed the hypothesis that "virtual electrograms" from a noncontact mapping system (EnSite 3000) could be used to localize myocardial scar. METHODS AND RESULTS: Myocardial infarctions were induced in sheep by inflating an angioplasty balloon in the left anterior descending coronary artery for 3 hours. Scar mapping was performed on 8 sheep without inducible ventricular tachycardia by use of the noncontact mapping system and a 256-channel contact mapping system. Transmural mapping needles were inserted into myocardial regions that were (1) scarred, (2) peripheral to the scar, and (3) distant from the scar. Unipolar electrograms were exported from both systems and analyzed on a personal computer workstation. The percentage of myocardial scarring at each needle site was assessed histologically. Pearson's correlation was used to assess the degree of association between various electrogram characteristics and the presence of myocardial scarring. The only noncontact electrogram characteristic that showed any association with the presence of myocardial scarring was the negative slope duration (contact, r=0.62, P<0.001; noncontact, r=0.23, P=0.004). The other electrogram characteristics studied were electrogram maximal deflection (contact, r=0.38, P<0.001; noncontact, r=0.03, P=0.75) and minimal slope (contact, r=0.42, P<0.001; noncontact, r=0.05, P=0.54). CONCLUSIONS: Noncontact electrograms do not reliably identify ventricular scar. Alternative strategies such as importing computed tomography images into the geometry should be used when scar localization is important.

Cooled intramural needle catheter ablation creates deeper lesions than irrigated tip catheter ablation.

Endocardial radiofrequency ablation of the left ventricle does not create transmural lesions reliably even with active electrode cooling. The authors developed a prototype catheter with an internally cooled needle electrode that could be advanced an adjustable distance into the myocardium. Freshly excised hearts from eight male sheep were perfused and superfused using oxygenated ovine blood. Ablations were performed for 2 minutes using the prototype catheter and a conventional endocardial 5-mm irrigated tip ablation catheter at target temperatures of 80 degrees C and 50 degrees C, respectively. The prototype catheter needle was inserted 12 mm deep for all ablations. The maximal power and irrigation rate was 50 W, 20 mL/min for the irrigated tip catheter and 20 W, 10 mL/min for the intramural needle catheter. Intramural needle lesions were significantly deeper (13.5 +/- 2.3 vs 9.1 +/- 1.3 mm, P < 0.01) but less wide (8.7 +/- 1.5 vs 12.7 +/- 1.9 mm, P < 0.01) than irrigated tip lesions. Popping occurred during 12 (37%) of the 32 irrigated tip ablations. Popping did not occur during intramural needle ablation. The cooled intramural needle ablation catheter creates lesions that are significantly deeper than irrigated tip catheters with less tissue boiling. In contrast to irrigated tip ablation, electrode temperature monitoring can be used to determine if a lesion has been created during intramural needle ablation. The cooled intramural needle ablation lesions were of a clinically useful width, addressing one of the main recognized deficiencies of intramural needle ablation.

Noncontact mapping of the left ventricle: insights from validation with transmural contact mapping.

It is not clear whether the noncontact electrograms obtained using the EnSite system in the left ventricle resemble most closely endocardial, intramural, or epicardial contact electrograms or a summation of transmural electrograms. This study compared unipolar virtual electrograms from the EnSite system with unipolar contact electrograms from transmural plunge needle electrodes using a 256-channel mapping system. The study also evaluated the effects of differing activation sites (endocardial, intramural, or epicardial). A grid of 50-60 plunge needles was positioned in the left ventricles of eight male sheep. Each needle had four electrodes to record from the endocardium, two intramural sites, and the epicardium. Correlations between contact and noncontact electrograms were calculated on 32,242 electrograms. Noncontact electrograms correlated equally well in morphology and accuracy of timing with endocardial (0.88 +/- 0.15), intramural (0.87 +/- 0.15), epicardial (0.88 +/- 0.15), and transmural summation contact electrograms (0.89 +/- 0.14) during sinus rhythm, endocardial pacing, and epicardial pacing. There was a nonlinear relationship between noncontact electrogram accuracy as measured by correlation with the contact electrogram and distance from the multielectrode array (MEA): beyond 40 mm accuracy decreased rapidly. The accuracy of noncontact electrograms also decreased with increasing distance from the equator of the MEA. Virtual electrograms from noncontact mapping of normal left ventricles probably represent a summation of transmural activation. Noncontact mapping has similar accuracy with either endocardial or epicardial sites of origin of electrical activity provided the MEA is within 40 mm of the recording site.

Catheter intramural needle radiofrequency ablation creates deeper lesions than irrigated tip catheter ablation.

Radiofrequency ablation of the left ventricle using an endocardially placed electrode is unable to reliably create transmural lesions even with active electrode cooling. To produce deeper radiofrequency lesions, the authors developed and tested a prototype intramural needle ablation catheter that had a distal 1.1-mm diameter straight needle that could be advanced 12 mm into the myocardium. Freshly excised hearts from eight male sheep were perfused and superfused with oxygenated ovine blood. Ablations were performed for 60 seconds with the prototype catheter and a conventional 5-mm irrigated tip ablation catheter at target temperatures of 90 degrees C and 50 degrees C, respectively. The ablation lesions were bisected and stained with blue tetrazolium to assess lesion geometry. The irrigated tip ablation catheter required significantly more power than the intramural needle ablation catheter (37.7 +/- 7.3 vs 6.4 +/- 2.1 W, P < 0.01). Intramural needle lesions were significantly deeper (12.5 +/- 3.0 mm vs 8.3 +/- 2.1 mm, P < 0.01) but less wide (3.9 +/- 1.1 mm vs 11.5 +/- 2.0 mm, P < 0.01) than irrigated tip lesions. There was a high incidence of crater formation (74%), popping (45%), and myocardial charring (29%) during irrigated tip ablation; these phenomena were not observed during intramural needle ablation. The intramural needle ablation catheter creates significantly deeper but narrower lesions without evidence of tissue boiling. This technology may be particularly useful for ablation of ventricular tachycardia originating from regions where tissue depth is increased, like the ventricular septum.