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1. Non‐selective cation channel activity of aquaporin AtPIP2;1 regulated by Ca 2+ and pH

   https://doi.org/10.1111/pce.12832  

   Byrt, Caitlin S. ; Zhao, Manchun ; Kourghi, Mohamad ; Bose, Jayakumar ; Henderson, Sam W. ; Qiu, Jiaen ; Gilliham, Matthew ; Schultz, Carolyn ; Schwarz, Manuel ; Ramesh, Sunita A. ; et al ( October 2016 , Plant, Cell & Environment)

   The aquaporin AtPIP2;1 is an abundant plasma membrane intrinsic protein inArabidopsis thalianathat is implicated in stomatal closure, and is highly expressed in plasma membranes of root epidermal cells. When expressed inXenopus laevisoocytes, AtPIP2;1 increased water permeability and induced a non‐selective cation conductance mainly associated with Na⁺. A mutation in the water pore, G103W, prevented both the ionic conductance and water permeability of PIP2;1. Co‐expression of AtPIP2;1 with AtPIP1;2 increased water permeability but abolished the ionic conductance. AtPIP2;2 (93% identical to AtPIP2;1) similarly increased water permeability but not ionic conductance. The ionic conductance was inhibited by the application of extracellular Ca²⁺and Cd²⁺, with Ca²⁺giving a biphasic dose–response with a prominent IC₅₀of 0.32 mм comparable with a previous report of Ca²⁺sensitivity of a non‐selective cation channel (NSCC) in Arabidopsis root protoplasts. Low external pH also inhibited ionic conductance (IC₅₀pH 6.8).Xenopusoocytes andSaccharomyces cerevisiaeexpressing AtPIP2;1 accumulated more Na⁺than controls. Establishing whether AtPIP2;1 has dual ion and water permeabilityin plantawill be important in understanding the roles of this aquaporin and if AtPIP2;1 is a candidate for a previously reported NSCC responsible for Ca²⁺and pH sensitive Na⁺entry into roots.

    

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2. A Na pump with reduced stoichiometry is up-regulated by brine shrimp in extreme salinities

   https://doi.org/10.1073/pnas.2313999120  

   Artigas, Pablo ; Meyer, Dylan J. ; Young, Victoria C. ; Spontarelli, Kerri ; Eastman, Jessica ; Strandquist, Evan ; Rui, Huan ; Roux, Benoît ; Birk, Matthew A. ; Nakanishi, Hanayo ; et al ( December 2023 , Proceedings of the National Academy of Sciences)

   Brine shrimp (Artemia) are the only animals to thrive at sodium concentrations above 4 M. Salt excretion is powered by the Na⁺,K⁺-ATPase (NKA), a heterodimeric (αβ) pump that usually exports 3Na⁺in exchange for 2 K⁺per hydrolyzed ATP.Artemiaexpress several NKA catalytic α-subunit subtypes. High-salinity adaptation increases abundance of α2_KK, an isoform that contains two lysines (Lys308 and Lys758 in transmembrane segments TM4 and TM5, respectively) at positions where canonical NKAs have asparagines (Xenopusα1’s Asn333 and Asn785). Using de novo transcriptome assembly and qPCR, we found thatArtemiaexpress two salinity-independent canonical α subunits (α1_NNand α3_NN), as well as two β variants, in addition to the salinity-controlled α2_KK. These β subunits permitted heterologous expression of the α2_KKpump and determination of its CryoEM structure in a closed, ion-free conformation, showing Lys758 residing within the ion-binding cavity. We used electrophysiology to characterize the function of α2_KKpumps and compared it to that ofXenopusα1 (and its α2_KK-mimicking single- and double-lysine substitutions). The double substitution N333K/N785K confers α2_KK-like characteristics toXenopusα1, and mutant cycle analysis reveals energetic coupling between these two residues, illustrating how α2_KK’s Lys308 helps to maintain high affinity for external K⁺when Lys758 occupies an ion-binding site. By measuring uptake under voltage clamp of the K⁺-congener⁸⁶Rb⁺, we prove that double-lysine-substituted pumps transport 2Na⁺and 1 K⁺per catalytic cycle. Our results show how the two lysines contribute to generate a pump with reduced stoichiometry allowingArtemiato maintain steeper Na⁺gradients in hypersaline environments.

    

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3. Role of a conserved ion-binding site tyrosine in ion selectivity of the Na+/K+ pump

   https://doi.org/10.1085/jgp.202113039  

   Spontarelli, Kerri ; Infield, Daniel T. ; Nielsen, Hang N. ; Holm, Rikke ; Young, Victoria C. ; Galpin, Jason D. ; Ahern, Christopher A. ; Vilsen, Bente ; Artigas, Pablo ( June 2022 , Journal of General Physiology)

   The essential transmembrane Na+ and K+ gradients in animal cells are established by the Na+/K+ pump, a P-type ATPase that exports three Na+ and imports two K+ per ATP hydrolyzed. The mechanism by which the Na+/K+ pump distinguishes between Na+ and K+ at the two membrane sides is poorly understood. Crystal structures identify two sites (sites I and II) that bind Na+ or K+ and a third (site III) specific for Na+. The side chain of a conserved tyrosine at site III of the catalytic α-subunit (Xenopus-α1 Y780) has been proposed to contribute to Na+ binding by cation–π interaction. We substituted Y780 with natural and unnatural amino acids, expressed the mutants in Xenopus oocytes and COS-1 cells, and used electrophysiology and biochemistry to evaluate their function. Substitutions disrupting H-bonds impaired Na+ interaction, while Y780Q strengthened it, likely by H-bond formation. Utilizing the non-sense suppression method previously used to incorporate unnatural derivatives in ion channels, we were able to analyze Na+/K+ pumps with fluorinated tyrosine or phenylalanine derivatives inserted at position 780 to diminish cation–π interaction strength. In line with the results of the analysis of mutants with natural amino acid substitutions, the results with the fluorinated derivatives indicate that Na+–π interaction with the phenol ring at position 780 contributes minimally, if at all, to the binding of Na+. All Y780 substitutions decreased K+ apparent affinity, highlighting that a state-dependent H-bond network is essential for the selectivity switch at sites I and II when the pump changes conformational state.

    

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4. Oncogenic KRAS alters splicing factor phosphorylation and alternative splicing in lung cancer

   https://doi.org/10.1186/s12885-022-10311-1  

   Lo, April ; McSharry, Maria ; Berger, Alice H. ( December 2022 , BMC Cancer)

   Alternative RNA splicing is widely dysregulated in cancers including lung adenocarcinoma, where aberrant splicing events are frequently caused by somatic splice site mutations or somatic mutations of splicing factor genes. However, the majority of mis-splicing in cancers is unexplained by these known mechanisms. We hypothesize that the aberrant Ras signaling characteristic of lung cancers plays a role in promoting the alternative splicing observed in tumors.

   We recently performed transcriptome and proteome profiling of human lung epithelial cells ectopically expressing oncogenic KRAS and another cancer-associated Ras GTPase, RIT1. Unbiased analysis of phosphoproteome data identified altered splicing factor phosphorylation in KRAS-mutant cells, so we performed differential alternative splicing analysis using rMATS to identify significantly altered isoforms in lung epithelial cells. To determine whether these isoforms were uniquely regulated by KRAS, we performed a large-scale splicing screen in which we generated over 300 unique RNA sequencing profiles of isogenic A549 lung adenocarcinoma cells ectopically expressing 75 different wild-type or variant alleles across 28 genes implicated in lung cancer.

   Mass spectrometry data showed widespread downregulation of splicing factor phosphorylation in lung epithelial cells expressing mutant KRAS compared to cells expressing wild-type KRAS. We observed alternative splicing in the same cells, with 2196 and 2416 skipped exon events in KRAS^G12Vand KRAS^Q61Hcells, respectively, 997 of which were shared (p < 0.001 by hypergeometric test). In the high-throughput splicing screen, mutant KRAS induced the greatest number of differential alternative splicing events, second only to the RNA binding protein RBM45 and its variant RBM45^M126I. We identified ten high confidence cassette exon events across multiple KRAS variants and cell lines. These included differential splicing of the Myc Associated Zinc Finger (MAZ). As MAZ regulates expression of KRAS, this splice variant may be a mechanism for the cell to modulate wild-type KRAS levels in the presence of oncogenic KRAS.

   Proteomic and transcriptomic profiling of lung epithelial cells uncovered splicing factor phosphorylation and mRNA splicing events regulated by oncogenic KRAS. These data suggest that in addition to widespread transcriptional changes, the Ras signaling pathway can promote post-transcriptional splicing changes that may contribute to oncogenic processes.

    

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5. Testing the selective sequestration hypothesis: Monarch butterflies preferentially sequester plant defences that are less toxic to themselves while maintaining potency to others

   https://doi.org/10.1111/ele.14340  

   Agrawal, Anurag A. ; Hastings, Amy P. ; Duplais, Christophe ( November 2023 , Ecology Letters)

   Herbivores that sequester toxins are thought to have cracked the code of plant defences. Nonetheless, coevolutionary theory predicts that plants should evolve toxic variants that also negatively impact specialists. We propose and test the selective sequestration hypothesis, that specialists preferentially sequester compounds that are less toxic to themselves while maintaining toxicity to enemies. Using chemically distinct plants, we show that monarch butterflies sequester only a subset of cardenolides from milkweed leaves that are less potent against their target enzyme (Na⁺/K⁺‐ATPase) compared to several dominant cardenolides from leaves. However, sequestered compounds remain highly potent against sensitive Na⁺/K⁺‐ATPases found in most predators. We confirmed this differential toxicity with mixtures of purified cardenolides from leaves and butterflies. The genetic basis of monarch adaptation to sequestered cardenolides was also confirmed with transgenicDrosophilathat were CRISPR‐edited with the monarch's Na⁺/K⁺‐ATPase. Thus, the monarch's selective sequestration appears to reduce self‐harm while maintaining protection from enemies.

    

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