CNNM proteins selectively bind to the TRPM7 channel to stimulate divalent cation entry into cells

Magnesium is essential for cellular life, but how it is homeostatically controlled still remains poorly understood. Here, we report that members of CNNM family, which have been controversially implicated in both cellular Mg2+ influx and efflux, selectively bind to the TRPM7 channel to stimulate divalent cation entry into cells. Coexpression of CNNMs with the channel markedly increased uptake of divalent cations, which is prevented by an inactivating mutation to the channel’s pore. Knockout (KO) of Trpm7 in cells or application of the TRPM7 channel inhibitor NS8593 also interfered with CNNM-stimulated divalent cation uptake. Conversely, KO of CNNM3 and CNNM4 in HEK-293 cells significantly reduced TRPM7-mediated divalent cation entry, without affecting TRPM7 protein expression or its cell surface levels. Furthermore, we found that cellular overexpression of phosphatases of regenerating liver (PRLs), known CNNMs binding partners, stimulated TRPM7-dependent divalent cation entry and that CNNMs were required for this activity. Whole-cell electrophysiological recordings demonstrated that deletion of CNNM3 and CNNM4 from HEK-293 cells interfered with heterologously expressed and native TRPM7 channel function. We conclude that CNNMs employ the TRPM7 channel to mediate divalent cation influx and that CNNMs also possess separate TRPM7-independent Mg2+ efflux activities that contribute to CNNMs’ control of cellular Mg2+ homeostasis.

Scale Mitigation for Field Implementation of Alkaline-Surfactant-Polymer ASP Flooding in a Heterogeneous High Temperature Carbonate Reservoir with High Divalent Cation Concentration in Formation Water

An alkaline-surfactant-polymer (ASP) pilot in a regular five spot well pattern is underway in the Sabriyah Mauddud (SAMA) reservoir in Kuwait. High divalent cation concentrations in formation water and high carbonate concentration of the ASP formulation makes the formation of calcite scale a concern. The main objective of this study is to investigate the severity of the calcium carbonate (CaCO3) scaling issues in the central producer in pursuit of a risk mitigation strategy to treat the potential scale deposition and reduce the flow assurance challenges. Calcite scaling risk in terms of Saturation Ratio (SR) and scale mass (in mg/L of produced water) in the pilot producer is potentially very severe and the probability of forming calcium carbonate scale at the production well is high. Produced Ca2+ concentration is high (> 800 mg/l), which makes the equilibrated calcite SR severe (> 500) and results in significant amount of scale mass precipitation. Different flooding strategies were modelled to evaluate a variety of flood design options to mitigate scale risks (varying slug size, Na2CO3 concentration, and volume of softened pre-flush brine), with marginal impact on scale formation. When the high permeability contrast of the different layers is reduced (to mimic gel injection), calcite SR and precipitated scale mass is significantly reduced to manageable levels. The option of injecting a weak acid in the production well downhole can suppress most of the expected calcite scale through reduction of the brine pH in the produced fluid stream for the ASP flood. Weak acid concentrations in the range of 4,000 to 5,000 mg/l are forecast to mitigate scale formation.

Crystal structures and X-ray powder diffraction data for Cs2NiSi5O12, RbGaSi2O6, and CsGaSi2O6 synthetic leucite analogues

Leucites are tetrahedrally coordinated silicate framework structures with some of the silicon framework cations partially replaced by divalent or trivalent cations. These structures have general formulae A2BSi5O12 and ACSi2O6; where A is a monovalent alkali metal cation, B is a divalent cation, and C is a trivalent cation. In this paper, we report the Rietveld refinements of three more synthetic leucite analogues with stoichiometries of Cs2NiSi5O12, RbGaSi2O6, and CsGaSi2O6. Cs2NiSi5O12 is Ia $\bar{3}$ d cubic and is isostructural with Cs2CuSi5O12. RbGaSi2O6 is I41/a tetragonal and is isostructural with KGaSi2O6. CsGaSi2O6 is $I\bar{4}3d$ cubic and is isostructural with RbBSi2O6.

Characterization of divalent cation interactions with AASTY nanodiscs

Amphiphilic copolymers show great promise in extracting membrane proteins directly from lipid bilayers into 'native nanodiscs'. However, many such copolymers are polyanionic and sensitive to divalent cations, limiting their applicability towards Ca2+ or Mg2+ dependent proteins. Here, we characterize the Ca2+ and Mg2+ sensitivity of poly(acrylic acid-co-styrene) (AASTY) copolymers using analytical UV and fluorescent size exclusion chromatography, enabling us to separate signals from nanodiscs, copolymers, and soluble aggregates. Determination of free Ca2+ ion concentrations in the presence of copolymer shows that divalent cation tolerance is dependent on not only specific characteristics of a copolymer, but also on its concentration. We see that high ionic strength protects against aggregation facilitated by divalent cations, which is prominent in nanodiscs isolated from excess free copolymer through dialysis. Overall, we conclude that the behavior of amphiphilic copolymers in the presence of divalent cations is more complex than precipitation beyond a specific cation concentration.

Crystal structure of nafamostat dimesylate

Nafamostat dimesylate {systematic name: [amino({6-[(4-{[amino(iminiumyl)methyl]amino}phenyl)carbonyloxy]naphthalen-2-yl})methylidene]azanium bis(methanesulfonate)}, C19H19N5O22 +·2CH3O3S−, is a broad-spectrum serine protease inhibitor and has been applied clinically as an anticoagulant agent during hemodialysis and for treatment of severe acute pancreatitis (SAP). Since nafamostat contains flexible moieties, it is necessary to determine the conformation to understand the structure–activity relationships. The divalent cation has a screw-like motif. The guanidinium group is approximately perpendicular to the naphthyl ring system, subtending a dihedral angle of 84.30 (14)°. In the crystal, the nafamostat molecules form columnar structures surrounded by a hydrophilic region.

Modulation of the Cardiac Myocyte Action Potential by the Magnesium-Sensitive TRPM6 and TRPM7-like Current

The cardiac Mg2+-sensitive, TRPM6, and TRPM7-like channels remain undefined, especially with the uncertainty regarding TRPM6 expression in cardiomyocytes. Additionally, their contribution to the cardiac action potential (AP) profile is unclear. Immunofluorescence assays showed the expression of the TRPM6 and TRPM7 proteins in isolated pig atrial and ventricular cardiomyocytes, of which the expression was modulated by incubation in extracellular divalent cation-free conditions. In patch clamp studies of cells dialyzed with solutions containing zero intracellular Mg2+ concentration ([Mg2+]i) to activate the Mg2+-sensitive channels, raising extracellular [Mg2+] ([Mg2+]o) from the 0.9-mM baseline to 7.2 mM prolonged the AP duration (APD). In contrast, no such effect was observed in cells dialyzed with physiological [Mg2+]i. Under voltage clamp, in cells dialyzed with zero [Mg2+]i, depolarizing ramps induced an outward-rectifying current, which was suppressed by raising [Mg2+]o and was absent in cells dialyzed with physiological [Mg2+]i. In cells dialyzed with physiological [Mg2+]i, raising [Mg2+]o decreased the L-type Ca2+ current and the total delayed-rectifier current but had no effect on the APD. These results suggest a co-expression of the TRPM6 and TRPM7 proteins in cardiomyocytes, which are therefore the molecular candidates for the native cardiac Mg2+-sensitive channels, and also suggest that the cardiac Mg2+-sensitive current shortens the APD, with potential implications in arrhythmogenesis.

Ca2+-dependent liquid-liquid phase separation underlies intracellular Ca2+ stores.

Endoplasmic/sarcoplasmic reticulum Ca2+ stores are essential to myriad cellular processes, however, the structure of these stores is largely unknown and existing models neither explain observations made in vivo nor sufficiently account for physiological data. We investigate CASQ1 - the major Ca2+ binding protein of skeletal muscle - and discover Ca2+-dependent liquid-liquid phase separation activity. The intrinsic disorder of CASQ1 underlies this activity and is regulated via phosphorylation by the secretory pathway kinase FAM20C. This novel divalent cation driven condensation demonstrates liquid-liquid phase separation occurs within the endoplasmic/sarcoplasmic reticulum, mechanistically explains efficient Ca2+ buffering and storage, and represents a largely unexplored mechanism of divalent-cation driven protein association.