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1. Phosphatidylinositol 3, 5‐bisphosphate regulates Ca ²⁺ transport during yeast vacuolar fusion through the Ca ²⁺ ATPase Pmc1

   https://doi.org/10.1111/tra.12736  

   Miner, Gregory E.; Sullivan, Katherine D.; Zhang, Chi; Rivera‐Kohr, David; Guo, Annie; Hurst, Logan R.; Ellis, Ez C.; Starr, Matthew L.; Jones, Brandon C.; Fratti, Rutilio A. (June 2020, Traffic)

   Abstract The transport of Ca²⁺across membranes precedes the fusion and fission of various lipid bilayers. Yeast vacuoles under hyperosmotic stress become fragmented through fission events that requires the release of Ca²⁺stores through the TRP channel Yvc1. This requires the phosphorylation of phosphatidylinositol‐3‐phosphate (PI3P) by the PI3P‐5‐kinase Fab1 to produce transient PI(3,5)P₂pools. Ca²⁺is also released during vacuole fusion upontrans‐SNARE complex assembly, however, its role remains unclear. The effect of PI(3,5)P₂on Ca²⁺flux during fusion was independent of Yvc1. Here, we show that while low levels of PI(3,5)P₂were required for Ca²⁺uptake into the vacuole, increased concentrations abolished Ca²⁺efflux. This was as shown by the addition of exogenous dioctanoyl PI(3,5)P₂or increased endogenous production of by the hyperactivefab1^T2250Amutant. In contrast, the lack of PI(3,5)P₂on vacuoles from the kinase deadfab1^EEEmutant showed delayed and decreased Ca²⁺uptake. The effects of PI(3,5)P₂were linked to the Ca²⁺pump Pmc1, as its deletion rendered vacuoles resistant to the effects of excess PI(3,5)P₂. Experiments with Verapamil inhibited Ca²⁺uptake when added at the start of the assay, while adding it after Ca²⁺had been taken up resulted in the rapid expulsion of Ca²⁺. Vacuoles lacking both Pmc1 and the H⁺/Ca²⁺exchanger Vcx1 lacked the ability to take up Ca²⁺and instead expelled it upon the addition of ATP. Together these data suggest that a balance of efflux and uptake compete during the fusion pathway and that the levels of PI(3,5)P₂can modulate which path predominates. 

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2. The vacuolar Ca ²⁺ transporter CATION EXCHANGER 2 regulates cytosolic calcium homeostasis, hypoxic signaling, and response to flooding in Arabidopsis thaliana

   https://doi.org/10.1111/nph.19274  

   Bakshi, Arkadipta; Choi, Won‐Gyu; Kim, Su‐Hwa; Gilroy, Simon (September 2023, New Phytologist)

   Summary Flooding represents a major threat to global agricultural productivity and food security, but plants are capable of deploying a suite of adaptive responses that can lead to short‐ or longer‐term survival to this stress. One cellular pathway thought to help coordinate these responses is via flooding‐triggered Ca²⁺signaling.We have mined publicly available transcriptomic data from Arabidopsis subjected to flooding or low oxygen stress to identify rapidly upregulated, Ca²⁺‐related transcripts. We then focused on transporters likely to modulate Ca²⁺signals. Candidates emerging from this analysis includedAUTOINHIBITED Ca²⁺ATPASE 1andCATION EXCHANGER 2. We therefore assayed mutants in these genes for flooding sensitivity at levels from growth to patterns of gene expression and the kinetics of flooding‐related Ca²⁺changes.Knockout mutants inCAX2especially showed enhanced survival to soil waterlogging coupled with suppressed induction of many marker genes for hypoxic response and constitutive activation of others.CAX2mutants also generated larger and more sustained Ca²⁺signals in response to both flooding and hypoxic challenges.CAX2 is a Ca²⁺transporter located on the tonoplast, and so these results are consistent with an important role for vacuolar Ca²⁺transport in the signaling systems that trigger flooding response. 

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3. Design of a light and Ca ²⁺ switchable organic–peptide hybrid

   https://doi.org/10.1073/pnas.2411316122  

   Khaleel, Zinah_Hilal; No, Young_Hyun; Kim, Nam_Hyeong; Bae, Do_Hyun; Wu, Yibing; Kim, Suhyeon; Choi, Hojae; Lee, Da_Eun; Jeong, Se_Yun; Ko, Yoon-Joo; et al (January 2025, Proceedings of the National Academy of Sciences)

   The design of organic–peptide hybrids has the potential to combine our vast knowledge of protein design with small molecule engineering to create hybrid structures with complex functions. Here, we describe the computational design of a photoswitchable Ca²⁺-binding organic–peptide hybrid. The designed molecule, designated Ca²⁺-binding switch (CaBS), combines an EF-hand motif from classical Ca²⁺-binding proteins such as calmodulin with a photoswitchable group that can be reversibly isomerized between a spiropyran (SP) and merocyanine (MC) state in response to different wavelengths of light. The MC/SP group acts both as a photoswitch as well as an optical sensor of Ca²⁺binding. Photoconversion of the SP to the corresponding MC unmasks an acidic phenol, which CaBS uses as an integral part of both its Ca²⁺-binding site as well as its tertiary and quaternary structure. By design, the SP state of CaBS is monomeric, while the Ca²⁺-bound form of the MC state is an obligate dimer, with two Ca²⁺-binding sites formed at the interface of a domain-swapped dimer. Thus, light and Ca²⁺were expected to serve as an “AND gate” that powers a change in backbone structure/dynamics, oligomerization state, and fluorescence properties of the designed molecule. CaBS was designed using Rosetta and molecular dynamics simulations, and experimentally characterized by nuclear magnetic resonance, isothermal titration calorimetry, and optical titrations. These data illustrate the potential of combining small molecule engineering with de novo protein design to develop sensors whose conformation, association state, and optical properties respond to multiple environmental cues. 

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4. Plant “helper” immune receptors are Ca ²⁺ -permeable nonselective cation channels

   https://doi.org/10.1126/science.abg7917  

   Jacob, Pierre; Kim, Nak Hyun; Wu, Feihua; El-Kasmi, Farid; Chi, Yuan; Walton, William G.; Furzer, Oliver J.; Lietzan, Adam D.; Sunil, Sruthi; Kempthorn, Korina; et al (June 2021, Science)

   Plant nucleotide-binding leucine-rich repeat receptors (NLRs) regulate immunity and cell death. InArabidopsis, a subfamily of “helper” NLRs is required by many “sensor” NLRs. Active NRG1.1 oligomerized, was enriched in plasma membrane puncta, and conferred cytoplasmic calcium ion (Ca²⁺) influx in plant and human cells. NRG1.1-dependent Ca²⁺influx and cell death were sensitive to Ca²⁺channel blockers and were suppressed by mutations affecting oligomerization or plasma membrane enrichment. Ca²⁺influx and cell death mediated by NRG1.1 and ACTIVATED DISEASE RESISTANCE 1 (ADR1), another helper NLR, required conserved negatively charged N-terminal residues. Whole-cell voltage-clamp recordings demonstrated thatArabidopsishelper NLRs form Ca²⁺-permeable cation channels to directly regulate cytoplasmic Ca²⁺levels and consequent cell death. Thus, helper NLRs transduce cell death signals directly. 

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5. Effects of mavacamten on Ca ²⁺ sensitivity of contraction as sarcomere length varied in human myocardium

   https://doi.org/10.1111/bph.15271  

   Awinda, Peter O.; Bishaw, Yemeserach; Watanabe, Marissa; Guglin, Maya A.; Campbell, Kenneth S.; Tanner, Bertrand C. W. (October 2020, British Journal of Pharmacology)

   Background and PurposeHeart failure can reflect impaired contractile function at the myofilament level. In healthy hearts, myofilaments become more sensitive to Ca²⁺as cells are stretched. This represents a fundamental property of the myocardium that contributes to the Frank–Starling response, although the molecular mechanisms underlying the effect remain unclear. Mavacamten, which binds to myosin, is under investigation as a potential therapy for heart disease. We investigated how mavacamten affects the sarcomere‐length dependence of Ca²⁺‐sensitive isometric contraction to determine how mavacamten might modulate the Frank–Starling mechanism. Experimental ApproachMulticellular preparations from the left ventricular‐free wall of hearts from organ donors were chemically permeabilized and Ca²⁺activated in the presence or absence of 0.5‐μM mavacamten at 1.9 or 2.3‐μm sarcomere length (37°C). Isometric force and frequency‐dependent viscoelastic myocardial stiffness measurements were made. Key ResultsAt both sarcomere lengths, mavacamten reduced maximal force and Ca²⁺sensitivity of contraction. In the presence and absence of mavacamten, Ca²⁺sensitivity of force increased as sarcomere length increased. This suggests that the length‐dependent activation response was maintained in human myocardium, even though mavacamten reduced Ca²⁺sensitivity. There were subtle effects of mavacamten reducing force values under relaxed conditions (pCa 8.0), as well as slowing myosin cross‐bridge recruitment and speeding cross‐bridge detachment under maximally activated conditions (pCa 4.5). Conclusion and ImplicationsMavacamten did not eliminate sarcomere length‐dependent increases in the Ca²⁺sensitivity of contraction in myocardial strips from organ donors at physiological temperature. Drugs that modulate myofilament function may be useful therapies for cardiomyopathies. 

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