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1. Transcriptional regulation of esophageal, intestinal, and branchial solute transporters by salinity, growth hormone, and cortisol in Atlantic salmon

   https://doi.org/10.1002/jez.2766  

   Breves, Jason P. ; Runiewicz, Ellie R. ; Richardson, Sierra G. ; Bradley, Serena E. ; Hall, Daniel J. ; McCormick, Stephen D. ( November 2023 , Journal of Experimental Zoology Part A: Ecological and Integrative Physiology)

   In marine habitats, Atlantic salmon (Salmo salar) imbibe seawater (SW) to replace body water that is passively lost to the ambient environment. By desalinating consumed SW, the esophagus enables solute‐linked water absorption across the intestinal epithelium. The processes underlying esophageal desalination in salmon and their hormonal regulation during smoltification and following SW exposure are unresolved. To address this, we considered whether two Na⁺/H⁺exchangers (Nhe2 and −3) expressed in the esophagus contribute to the uptake of Na⁺from lumenal SW. There were no seasonal changes in esophagealnhe2or−3expression during smoltification; however,nhe3increased following 48 h of SW exposure in May. Esophagealnhe2, −3, andgrowth hormone receptor b1were elevated in smolts acclimated to SW for 2.5 weeks. Treatment with cortisol stimulated branchial Na⁺/K⁺‐ATPase (Nka) activity, andNa⁺/K⁺/2Cl⁻cotransporter 1(nkcc1),cystic fibrosis transmembrane regulator 1 (cftr1), andnka‐α1bexpression. Esophagealnhe2, but notnhe3expression, was stimulated by cortisol. In anterior intestine, cortisol stimulatednkcc2, cftr2, andnka‐α1b. Our findings indicate that salinity stimulates esophagealnhe2and−3, and that cortisol coordinates the expression of esophageal, intestinal, and branchial solute transporters to support the SW adaptability of Atlantic salmon.

    

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2. Voltage-gated ion channels are expressed in the Malpighian tubules and anal papillae of the yellow fever mosquito ( Aedes aegypti ), and may regulate ion transport during salt and water imbalance

   https://doi.org/10.1242/jeb.246486  

   Farrell, Serena ; Dates, Jocelyne ; Ramirez, Nancy ; Hausknecht-Buss, Hannah ; Kolosov, Dennis ( February 2024 , Journal of Experimental Biology)

   Vectors of infectious disease include several species of Aedes mosquitoes. The life cycle of Aedes aegypti, the yellow fever mosquito, consists of a terrestrial adult and an aquatic larval life stage. Developing in coastal waters can expose larvae to fluctuating salinity, causing salt and water imbalance, which is addressed by two prime osmoregulatory organs – the Malpighian tubules (MTs) and anal papillae (AP). Voltage-gated ion channels (VGICs) have recently been implicated in the regulation of ion transport in the osmoregulatory epithelia of insects. In the current study, we: (i) generated MT transcriptomes of freshwater-acclimated and brackish water-exposed larvae of Ae. aegypti, (ii) detected expression of several voltage-gated Ca2+, K+, Na+ and non-ion-selective ion channels in the MTs and AP using transcriptomics, PCR and gel electrophoresis, (iii) demonstrated that mRNA abundance of many altered significantly following brackish water exposure, and (iv) immunolocalized CaV1, NALCN, TRP/Painless and KCNH8 in the MTs and AP of larvae using custom-made antibodies. We found CaV1 to be expressed in the apical membrane of MTs of both larvae and adults, and its inhibition to alter membrane potentials of this osmoregulatory epithelium. Our data demonstrate that multiple VGICs are expressed in osmoregulatory epithelia of Ae. aegypti and may play an important role in the autonomous regulation of ion transport.

    

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3. Cell death signaling in Anopheles gambiae initiated by Bacillus thuringiensis Cry4B toxin involves Na + /K + ATPase

   https://doi.org/10.1177/15353702231188072  

   Liu, Li ; Bulla, Jr, Lee A. ( August 2023 , Experimental Biology and Medicine)

   Identifying the mechanisms by which bacterial pathogens kill host cells is fundamental to understanding how to control and prevent human and animal disease. In the case of Bacillus thuringiensis (Bt), such knowledge is critical to using the bacterium to kill insect vectors that transmit human and animal disease. For the Cry4B toxin produced by Bt, its capacity to kill Anopheles gambiae, the primary mosquito vector of malaria, is the consequence of a variety of signaling activities. We show here that Cry4B, acting as first messenger, binds specifically to the bitopic cadherin BT-R₃G-protein-coupled receptor (GPCR) localized in the midgut of A. gambiae, activating the downstream second messenger cyclic adenosine monophosphate (cAMP). The direct result of the Cry4B–BT-R₃binding is the release of α_sfrom the heterotrimeric αβγ-G-protein complex and its activation of adenylyl cyclase (AC). The upshot is an increased level of cAMP, which activates protein kinase A (PKA). The functional impact of cAMP–PKA signaling is the stimulation of Na⁺/K⁺-ATPase (NKA) which serves as an Na⁺/K⁺pump to maintain proper gradients of extracellular Na⁺and intracellular K⁺. Increased level of cAMP amplifies NKA and upsets normal ion concentration gradients. NKA, as a scaffolding protein, accelerates the first messenger signal to the nucleus, generating additional BT-R₃molecules and promoting their exocytotic trafficking to the cell membrane. Accumulation of BT-R₃on the cell surface facilitates recruitment of additional toxin molecules which, in turn, amplify the original signal in a cascade-like manner. This report provides the first evidence of a bacterial toxin using NKA via AC/PKA signaling to execute cell death.

    

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4. 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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5. 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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