Orai1α, but not Orai1β, co-localizes with TRPC1 and is required for its plasma membrane location and activation in HeLa cells

The identification of two variants of the canonical pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel Orai1, Orai1α and Orai1β, in mammalian cells arises the question whether they exhibit different functional characteristics. Orai1α and Orai1β differ in the N-terminal 63 amino acids, exclusive of Orai1α, and show different sensitivities to Ca2+-dependent inactivation, as well as distinct ability to form arachidonate-regulated channels. We have evaluated the role of both Orai1 variants in the activation of TRPC1 in HeLa cells. We found that Orai1α and Orai1β are required for the maintenance of regenerative Ca2+ oscillations, while TRPC1 plays a role in agonist-induced Ca2+ influx but is not essential for Ca2+ oscillations. Using APEX2 proximity labeling, co-immunoprecipitation and the fluorescence of G-GECO1.2 fused to Orai1α our results indicate that agonist stimulation and Ca2+ store depletion enhance Orai1α–TRPC1 interaction. Orai1α is essential for TRPC1 plasma membrane location and activation. Thus, TRPC1 function in HeLa cells depends on Ca2+ influx through Orai1α exclusively.

Role of blue light in bactericidal effect against meat-borne pathogens and freshness maintaining of beef

Beef is rich in various nutrients while easily spoils due to contamination by pathogens, thus it is of great significance to develop a bactericidal method to inactivate meat-borne pathogens and meanwhile maintain the freshness of beef. For the first time, the present study investigated the bactericidal effect of blue light (BL) at 415 nm against four meat-borne pathogens (methicillin-resistant Staphylococcus aureus , Escherichia coli , Salmonella Typhimurium and Listeria monocytogenes ) in vitro and inoculated on the surface of fresh beef, respectively. When the non-illuminated beef was used as control, the population of the four pathogens did not change significantly ( P > 0.05), while BL-illuminated beef showed dose-dependent inactivation effect in both in vitro and in vivo studies. The experiments on beef cuts showed that 109.44 J/cm 2 of BL inactivated 90% of inoculated cells for the tested strains ( P < 0.05), and the impact of BL inactivation could be sustained in 7 days of cold storage. Notably, changes of lipid oxidation rate, water holding capacity and cooking loss value between the control and beef illuminated by 109.44 J/cm 2 at the same time were scarcely detected during the storage. BL had a minor but insignificant influence on surface color and free amino acid content. Moreover, the pH of illuminated beef increased slower ( P < 0.05) than that of non-illuminated beef. The present work demonstrated that BL could be a novel bactericidal and freshness-maintaining method for fresh beef.

Structural Basis for Rapid Voltage Dependent Inactivation of HERG Potassium Channels

The exquisite fine tuning of biological electrical signalling is mediated by variations in the rates of opening and closing of different ion channels(1). In addition to open and closed conformations, ion channels can exist in an inactivated state, which prevents conduction in the presence of a prolonged activating stimulus(2). Human ether-a-go-go related gene (HERG) K+ channels undergo uniquely rapid and voltage dependent inactivation(3-5), which confers upon them a critical role in protecting against cardiac arrhythmias and sudden death(6). Previous structural studies have captured only the open state of the HERG channel(7,8). Here, we have exploited the K+ sensitivity of HERG inactivation to determine structures of both the conductive state and the elusive inactivated state of HERG. We show that hERG inactivation is facilitated by two competing networks of hydrogen bonds behind the selectivity filter that enable rapid and voltage dependent flipping of the valine carbonyls in the centre of the selectivity filter. Our data also explains how changes in extracellular K+ affects the distribution between conductive and inactivated states(9,10) and thereby explains why hypokalaemia reduces HERG channel activity thereby increasing the risk of cardiac arrhythmias(11).

Bidirectional regulation of calcium release–activated calcium (CRAC) channel by SARAF

Store-operated calcium entry (SOCE) through the Ca2+ release–activated Ca2+ (CRAC) channel is a central mechanism by which cells generate Ca2+ signals and mediate Ca2+-dependent gene expression. The molecular basis for CRAC channel regulation by the SOCE-associated regulatory factor (SARAF) remained insufficiently understood. Here we found that following ER Ca2+ depletion, SARAF facilitates a conformational change in the ER Ca2+ sensor STIM1 that relieves an activation constraint enforced by the STIM1 inactivation domain (ID; aa 475–483) and promotes initial activation of STIM1, its translocation to ER–plasma membrane junctions, and coupling to Orai1 channels. Following intracellular Ca2+ rise, cooperation between SARAF and the STIM1 ID controls CRAC channel slow Ca2+-dependent inactivation. We further show that in T lymphocytes, SARAF is required for proper T cell receptor evoked transcription. Taking all these data together, we uncover a dual regulatory role for SARAF during both activation and inactivation of CRAC channels and show that SARAF fine-tunes intracellular Ca2+ responses and downstream gene expression in cells.

Snapin Specifically Up-Regulates Cav1.3 Ca2+ Channel Variant with a Long Carboxyl Terminus

Ca2+ entry through Cav1.3 Ca2+ channels plays essential roles in diverse physiological events. We employed yeast-two-hybrid (Y2H) assays to mine novel proteins interacting with Cav1.3 and found Snapin2, a synaptic protein, as a partner interacting with the long carboxyl terminus (CTL) of rat Cav1.3L variant. Co-expression of Snapin with Cav1.3L/Cavβ3/α2δ2 subunits increased the peak current density or amplitude by about 2-fold in HEK-293 cells and Xenopus oocytes, without affecting voltage-dependent gating properties and calcium-dependent inactivation. However, the Snapin up-regulation effect was not found for rat Cav1.3S containing a short CT (CTS) in which a Snapin interaction site in the CTL was deficient. Luminometry and electrophysiology studies uncovered that Snapin co-expression did not alter the membrane expression of HA tagged Cav1.3L but increased the slope of tail current amplitudes plotted against ON-gating currents, indicating that Snapin increases the opening probability of Cav1.3L. Taken together, our results strongly suggest that Snapin directly interacts with the CTL of Cav1.3L, leading to up-regulation of Cav1.3L channel activity via facilitating channel opening probability.

Role of Ascorbic Acid in the Extraction and Quantification of Potato Polyphenol Oxidase Activity

The ability to accurately measure the activity of polyphenol oxidase (PPO) in complex matrices is essential. A problem encountered when using spectrophotometric methods is interference due to ascorbic acid (AA), often used as an enzyme “protecting agent” during PPO extraction. This study focuses on the nature of AA’s effect on spectrophotometric determinations of PPO activity as well as enzyme extraction. Potato extracts and semi-purified PPO were used as enzyme sources. The inactivation of PPO attributed to AA is substrate-mediated. The extent of AA-dependent inactivation of PPO in model systems varied between substrates. AA only slows mechanism-based inactivation of PPO induced by catechol, possibly owing to the prevention of quinone formation. AA minimally protects PPO activity during enzyme extraction. The problem associated with AA in PPO assay could be circumvented by using ascorbate oxidase to remove AA when catechol is the primary substrate or by using chlorogenic acid as the primary substrate.

Enzymatic Hydrogen Electrosynthesis at Enhanced Current Density Using a Redox Polymer

High-temperature tolerant enzymes offer multiple advantages over enzymes from mesophilic organisms for the industrial production of sustainable chemicals due to high specific activities and stabilities towards fluctuations in pH, heat, and organic solvents. The production of molecular hydrogen (H2) is of particular interest because of the multiple uses of hydrogen in energy and chemicals applications, and the ability of hydrogenase enzymes to reduce protons to H2 at a cathode. We examined the activity of Hydrogen-Dependent CO2 Reductase (HDCR) from the thermophilic bacterium Thermoanaerobacter kivui when immobilized in a redox polymer, cobaltocene-functionalized polyallylamine (Cc-PAA), on a cathode for enzyme-mediated H2 formation from electricity. The presence of Cc-PAA increased reductive current density 340-fold when used on an electrode with HDCR at 40 °C, reaching unprecedented current densities of up to 3 mA·cm−2 with minimal overpotential and high faradaic efficiency. In contrast to other hydrogenases, T. kivui HDCR showed substantial reversibility of CO-dependent inactivation, revealing an opportunity for usage in gas mixtures containing CO, such as syngas. This study highlights the important potential of combining redox polymers with novel enzymes from thermophiles for enhanced electrosynthesis.