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\"Mark and Steph have a relatively happy family with their young daughter in sunny Cape Town until one day when armed men in balaclavas break in to their home. Left traumatized but physically unharmed, Mark and Steph are unable to return to normal and live in constant fear. When a friend suggests a restorative vacation abroad via a popular house swapping website, it sounds like the perfect plan. They find a genial, artistic couple with a charming apartment in Paris who would love to come to Cape Town. Mark and Steph can't resist the idyllic, light-strewn pictures, and the promise of a romantic getaway. But once they arrive in Paris, they quickly realize that nothing is as advertised\"--Amazon.com.

Light-driven proton-pumping rhodopsins are widely distributed in many microorganisms. They convert sunlight energy into proton gradients that serve as energy source of the cell. Here we report a new functional class of a microbial rhodopsin, a light-driven sodium ion pump. We discover that the marine flavobacterium Krokinobacter eikastus possesses two rhodopsins, the first, KR1, being a prototypical proton pump, while the second, KR2, pumps sodium ions outward. Rhodopsin KR2 can also pump lithium ions, but converts to a proton pump when presented with potassium chloride or salts of larger cations. These data indicate that KR2 is a compatible sodium ion–proton pump, and spectroscopic analysis showed it binds sodium ions in its extracellular domain. These findings suggest that light-driven sodium pumps may be as important in situ as their proton-pumping counterparts. Light-driven proton-pumping rhodopsins are widely distributed in microorganisms and convert sunlight energy into proton gradients. Inoue et al . report the discovery of a light-driven sodium ion pump from marine bacteria.

In muscle, digoxin inhibits Na+,K+‐ATPase (NKA) whereas acute exercise can increase NKA gene expression, consistent with training‐induced increased NKA content. We investigated whether oral digoxin increased NKA isoform mRNA expression (qPCR) in muscle at rest, during and post‐exercise in 10 healthy adults, who received digoxin (DIG, 0.25 mg per day) or placebo (CON) for 14 days, in a randomised, double‐blind and cross‐over design. Muscle was biopsied at rest, after cycling 20 min (10 min each at 33%, then 67% V̇O2peak ${{\\dot{V}}_{{{\\mathrm{O}}}_2}{\\mathrm{peak}}}$ ), then to fatigue at 90% V̇O2peak ${{\\dot{V}}_{{{\\mathrm{O}}}_2}{\\mathrm{peak}}}$and 3 h post‐exercise. No differences were found between DIG and CON for NKA α1–3 or β1–3 isoform mRNA. Both α1 (354%, P = 0.001) and β3 mRNA (P = 0.008) were increased 3 h post‐exercise, with α2 and β1–2 mRNA unchanged, whilst α3 mRNA declined at fatigue (−43%, P = 0.045). In resting muscle, total β mRNA (∑(β1+β2+β3)) increased in DIG (60%, P = 0.025) and also when transcripts for each isoform were normalised to CON then either summed (P = 0.030) or pooled (n = 30, P = 0.034). In contrast, total α mRNA (∑(α1+α2+α3), P = 0.348), normalised then summed (P = 0.332), or pooled transcripts (n = 30, P = 0.717) did not differ with DIG. At rest, NKA α1–2 and β1–2 protein abundances were unchanged by DIG. Post‐exercise, α1 and β1–2 proteins were unchanged, but α2 declined at 3 h (19%, P = 0.020). In conclusion, digoxin did not modify gene expression of individual NKA isoforms at rest or with exercise, indicating NKA gene expression was maintained consistent with protein abundances. However, elevated resting muscle total β mRNA with digoxin suggests a possible underlying β gene‐stimulatory effect. Highlights What is the central question of this study? Na+,K+‐ATPase (NKA) in muscle is important for Na+/K+ homeostasis. We investigated whether the NKA‐inhibitor digoxin stimulates increased NKA gene expression in muscle and exacerbates NKA gene responses to exercise in healthy adults. What is the main finding and its importance? Digoxin did not modify exercise effects on muscle NKA α1–3 and β1–3 gene transcripts, which comprised increased post‐exercise α1 and β3 mRNA and reduced α3 mRNA during exercise. However, in resting muscle, digoxin increased NKA total β isoform mRNA expression. Despite inhibitory‐digoxin or acute exercise stressors, NKA gene regulation in muscle is consistent with the maintenance of NKA protein contents.

The sodium pump (Na⁺, K⁺-ATPase, NKA) is vital for animal cells, as it actively maintains Na⁺ and K⁺ electrochemical gradients across the cell membrane. It is a target of cardiotonic steroids (CTSs) such as ouabain and digoxin. As CTSs are almost unique strong inhibitors specific to NKA, a wide range of derivatives has been developed for potential therapeutic use. Several crystal structures have been published for NKA-CTS complexes, but they fail to explain the largely different inhibitory properties of the various CTSs. For instance, although CTSs are thought to inhibit ATPase activity by binding to NKA in the E2P state, we do not know if large conformational changes accompany binding, as no crystal structure is available for the E2P state free of CTS. Here, we describe crystal structures of the BeF₃⁻ complex of NKA representing the E2P ground state and then eight crystal structures of seven CTSs, including rostafuroxin and istaroxime, two new members under clinical trials, in complex with NKA in the E2P state. The conformations of NKA are virtually identical in all complexes with and without CTSs, showing that CTSs bind to a preformed cavity in NKA. By comparing the inhibitory potency of the CTSs measured under four different conditions, we elucidate how different structural features of the CTSs result in different inhibitory properties. The crystal structures also explain K⁺-antagonism and suggest a route to isoform specific CTSs.

Background Salinization is a primary abiotic stress constraining global plant growth and production. Weedy rice, though highly homologous to cultivated rice, is more salt tolerant during seed germination and seedling growth; we hypothesize that this is owing to ionic homeostasis and changes in the expression of genes encoding ion transport regulators. Results The four different genotypes of weedy ( JYGY-1 and JYFN-4 ) and cultivated ( Nipponbare and 9311 ) rice have different salt-tolerance during seed germination and seedling vegetative growth under salt stress. In this study, Na + and Ca 2+ content increased in weedy and cultivated rice genotypes under salt stress while K + and Mg 2+ decreased; however, JYGY-1 had the lowest Na + /K + ratio of assessed genotypes. Genes in the high-affinity K + transporter ( HKT ) and tonoplast sodium-hydrogen exchanger ( NHX ) families, and salt overly sensitive 1 ( OsSOS1 ) have more than 98% homology in amino acid sequences between weedy and cultivated rice genotypes. Under salt stress, the HKT family members were differentially expressed in the roots and shoots of four different genotypes. However, the NHX family transcripts were markedly up-regulated in all genotypes, but there are significant differences between different genotypes. OsSOS1 was significantly up-regulated in roots, especially in JYGY-1 genotype. Conclusions The results showed that different genotypes had different germination and nutrient survival under salt stress, which was related to the difference of ion content and the difference of a series of ion transport gene expression. At the same time this study will provide new insight into the similarities and differences in ion homeostasis and gene regulatory mechanisms between weedy and cultivated rice under salt stress, which can aid in novel rice breeding and growth strategies.

The catalytic α-subunits of both the Na + ,K + -ATPase and the gastric H + ,K + -ATPase possess lysine-rich N-termini which project into the cytoplasm. Due to conflicting experimental results, it is currently unclear whether the N-termini play a role in ion pump function or regulation, and, if they do, by what mechanism. Comparison of the lysine frequencies of the N-termini of both proteins with those of all of their extramembrane domains showed that the N-terminal lysine frequencies are far higher than one would expect simply from exposure to the aqueous solvent. The lysine frequency was found to vary significantly between different vertebrate classes, but this is due predominantly to a change in N-terminal length. As evidenced by a comparison between fish and mammals, an evolutionary trend towards an increase of the length of the N-terminus of the H + ,K + -ATPase on going from an ancestral fish to mammals could be identified. This evolutionary trend supports the hypothesis that the N-terminus is important in ion pump function or regulation. In placental mammals, one of the lysines is replaced by serine (Ser-27), which is a target for protein kinase C. In most other animal species, a lysine occupies this position and hence no protein kinase C target is present. Interaction with protein kinase C is thus not the primary role of the lysine-rich N-terminus. The disordered structure of the N-terminus may, via increased flexibility, facilitate interaction with another binding partner, e.g. the surrounding membrane, or help to stabilise particular enzyme conformations via the increased entropy it produces. Graphical Abstract

Light-driven sodium pumps actively transport small cations across cellular membranes 1 . These pumps are used by microorganisms to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved 2 , 3 , it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient. Here, using the Swiss X-ray Free Electron Laser 4 , we have collected serial crystallographic data at ten pump–probe delays from femtoseconds to milliseconds. High-resolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data, in combination with quantum chemical calculations, indicate that a sodium ion binds transiently close to the retinal within one millisecond. In the last structural intermediate, at 20 milliseconds after activation, we identified a potential second sodium-binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes. Crystallographic ‘snapshots’ taken at intervals of femtoseconds to milliseconds after activation show how a light-activated sodium pump carries sodium ions across the cell membrane.

Morris Brown and colleagues identify somatic mutations in ATP1A1 and CACNA1D in aldosterone-producing adenomas with features resembling zonaglomerulosa cells. They further show that the ATP1A1 mutations cause inward leak currents under physiological conditions, whereas the CACNA1D mutations induce a shift of voltage-dependent gating to more negative potentials and suppress channel inactivation. At least 5% of individuals with hypertension have adrenal aldosterone-producing adenomas (APAs). Gain-of-function mutations in KCNJ5 and apparent loss-of-function mutations in ATP1A1 and ATP2A3 were reported to occur in APAs 1 , 2 . We find that KCNJ5 mutations are common in APAs resembling cortisol-secreting cells of the adrenal zona fasciculata but are absent in a subset of APAs resembling the aldosterone-secreting cells of the adrenal zona glomerulosa 3 . We performed exome sequencing of ten zona glomerulosa–like APAs and identified nine with somatic mutations in either ATP1A1 , encoding the Na + /K + ATPase α1 subunit, or CACNA1D , encoding Ca v 1.3. The ATP1A1 mutations all caused inward leak currents under physiological conditions, and the CACNA1D mutations induced a shift of voltage-dependent gating to more negative voltages, suppressed inactivation or increased currents. Many APAs with these mutations were <1 cm in diameter and had been overlooked on conventional adrenal imaging. Recognition of the distinct genotype and phenotype for this subset of APAs could facilitate diagnosis.

The light-driven sodium-pumping rhodopsin KR2 from Krokinobacter eikastus is the only non-proton cation active transporter with demonstrated potential for optogenetics. However, the existing structural data on KR2 correspond exclusively to its ground state, and show no sodium inside the protein, which hampers the understanding of sodium-pumping mechanism. Here we present crystal structure of the O-intermediate of the physiologically relevant pentameric form of KR2 at the resolution of 2.1 Å, revealing a sodium ion near the retinal Schiff base, coordinated by N112 and D116 of the characteristic NDQ triad. We also obtained crystal structures of D116N and H30A variants, conducted metadynamics simulations and measured pumping activities of putative pathway mutants to demonstrate that sodium release likely proceeds alongside Q78 towards the structural sodium ion bound between KR2 protomers. Our findings highlight the importance of pentameric assembly for sodium pump function, and may be used for rational engineering of enhanced optogenetic tools. The Na + -pumping KR2 rhodopsin from Krokinobacter eikastus is a light-driven non-proton cation pump whose mechanism of pumping remains to be understood. Here authors solved crystal structures of the O-intermediate state of the pentameric form of KR2 and its D116N and H30A mutants, which sheds light on the mechanism of non-proton cation light-driven pumping.