Pro219 is an electrostatic color determinant in the light-driven sodium pump KR2

Color tuning in animal and microbial rhodopsins has attracted the interest of many researchers, as the color of their common retinal chromophores is modulated by the amino acid residues forming the chromophore cavity. Critical cavity amino acid residues are often called “color switches”, as the rhodopsin color is effectively tuned through their substitution. Well-known color switches are the L/Q and A/TS switches located in the C and G helices of the microbial rhodopsin structure respectively. Recently, we reported on a third G/P switch located in the F helix of the light-driven sodium pumps of KR2 and JsNaR causing substantial spectral red-shifts in the latter with respect to the former. In order to investigate the molecular-level mechanism driving such switching function, here we present an exhaustive mutation, spectroscopic and computational investigation of the P219X mutant set of KR2. To do so, we study the changes in the absorption band of the 19 possible mutants and construct, semi-automatically, the corresponding hybrid quantum mechanics/molecular mechanics models. We found that the P219X feature a red-shifted light absorption with the only exception of P219R. The analysis of the corresponding models indicate that the G/P switch induces red-shifting variations via electrostatic interactions, while replacement-induced chromophore geometrical (steric) distortions play a minor role. However, the same analysis indicates that the P219R blue-shifted variant has a more complex origin involving both electrostatic and steric changes accompanied by protonation state and hydrogen bond networks modifications. These results make it difficult to extract simple rules or formulate theories for predicting how a switch operates without considering the atomistic details and environmental consequences of the side chain replacement.

Emergency Use of Targeted Osmotic Lysis for the Treatment of a Patient with Aggressive Late-Stage Squamous Cell Carcinoma of the Cervix

Upregulation of voltage-gated sodium channels (VGSCs) and Na+/K+-ATPase (sodium pumps) is common across most malignant carcinomas. Targeted osmotic lysis (TOL) is a developing technology in which the concomitant stimulation of VGSCs and pharmacological blockade of sodium pumps causes rapid selective osmotic lysis of carcinoma cells. This treatment of cervical carcinoma is evidence that TOL is a safe, well-tolerated and effective treatment for aggressive advanced carcinomas that has the potential to extend life without compromising its quality. TOL is likely to have broad application for the treatment of advanced-stage carcinomas.

Molecular mechanism of light-driven sodium pumping

ABSTRACTMicrobial rhodopsins appeared to be the most abundant light-harvesting proteins on the Earth and are the major contributes to the solar energy captured in the sea. They possess highly diverse biological functions. Explosion of research on microbial rhodopsins led to breakthroughs in their applications, in particular, in neuroscience.An unexpected new discovery was a Na+-pumping KR2 rhodopsin from Krokinobacter eikastus, the first light-driven non-proton cation pump. A fundamental difference between proton and other cation pumps is that non-proton pumps cannot use tunneling or Grotthuss mechanism for the ion translocation and, therefore, Na+ pumping cannot be understood in the framework of classical proton pump, like bacteriorhodopsin. Extensive studies on the molecular mechanism of KR2 failed to reveal mechanism of pumping. The existing high-resolution structures relate only to the ground state of the protein and revealed no Na+ inside the protein, which is unusual for active ion transporters.KR2 is only known non proton cation transporter with demonstrated remarkable potential for optogenetic applications and, therefore, elucidation of the mechanism of cation transport is important. To understand conception of cation pumping we solved crystal structures of the functionally key O-intermediate state of physiologically relevant pentameric form of KR2 and its D116N and H30A key mutants at high resolution and performed additional functional studies.The structure of the O-state reveals a sodium ion near the retinal Schiff base coordinated by N112 and D116 residues of the characteristic (for the whole family) NDQ triad. The structural and functional data show that cation uptake and release are driven by a switching mechanism. Surprisely, Na+ pathway in KR2 is lined with the chain of polar pores/cavities, similarly to the channelrhodopsin-2. Using Parinello fast molecular dynamics approach we obtained a molecular movie of a probable ion release.Our data provides insight into the mechanism of a non-proton cation light-driven pumping, strongly suggest close relation of sodium pumps to channel rhodopsins and, we believe, expand the present knowledge of rhodopsin world. Certainly they might be used for engineering of cation pumps and ion channels for optogenetics.

Uncultured Microbial Phyla Suggest Mechanisms for Multi-Thousand-Year Subsistence in Baltic Sea Sediments

ABSTRACTEnergy-starved microbes in deep marine sediments subsist at near-zero growth for thousands of years, yet the mechanisms for their subsistence are unknown because no model strains have been cultivated from most of these groups. We investigated Baltic Sea sediments with single-cell genomics, metabolomics, metatranscriptomics, and enzyme assays to identify possible subsistence mechanisms employed by unculturedAtribacteria,Aminicenantes,Actinobacteriagroup OPB41,Aerophobetes,Chloroflexi,Deltaproteobacteria,Desulfatiglans,Bathyarchaeota, andEuryarchaeotamarine group II lineages. Some functions appeared to be shared by multiple lineages, such as trehalose production and NAD+-consuming deacetylation, both of which have been shown to increase cellular life spans in other organisms by stabilizing proteins and nucleic acids, respectively. Other possible subsistence mechanisms differed between lineages, possibly providing them different physiological niches. Enzyme assays and transcripts suggested thatAtribacteriaandActinobacteriagroup OPB41 catabolized sugars, whereasAminicenantesandAtribacteriacatabolized peptides. Metabolite and transcript data suggested thatAtribacteriautilized allantoin, possibly as an energetic substrate or chemical protectant, and also possessed energy-efficient sodium pumps.Atribacteriasingle-cell amplified genomes (SAGs) recruited transcripts for full pathways for the production of all 20 canonical amino acids, and the gene for amino acid exporter YddG was one of their most highly transcribed genes, suggesting that they may benefit from metabolic interdependence with other cells. Subsistence of uncultured phyla in deep subsurface sediments may occur through shared strategies of using chemical protectants for biomolecular stabilization, but also by differentiating into physiological niches and metabolic interdependencies.IMPORTANCEMuch of life on Earth exists in a very slow-growing state, with microbes from deeply buried marine sediments representing an extreme example. These environments are like natural laboratories that have run multi-thousand-year experiments that are impossible to perform in a laboratory. We borrowed some techniques that are commonly used in laboratory experiments and applied them to these natural samples to make hypotheses about how these microbes subsist for so long at low activity. We found that some methods for stabilizing proteins and nucleic acids might be used by many members of the community. We also found evidence for niche differentiation strategies, and possibly cross-feeding, suggesting that even though they are barely growing, complex ecological interactions continue to occur over ultralong timescales.

Two sodium pumps in the hepatopancreas of the intertidal euryhaline crab Neohelice granulata: biochemical characteristics and differential modulation after feeding

No study has been done on the existence, biochemical characteristics, and modulation of K+-independent ouabain-insensitive Na+ ATPase activity (the second sodium pump) in the digestive tract of intertidal euryhaline crabs and moreover on the coexistence and modulation under distinct physiological and (or) environmental conditions of different sodium pumps. We determined the occurrence, characteristics, and responses at different times (0, 1, 24, 48, and 120 h) after feeding upon distinct salinities of Na+ ATPase activity and Na+/K+ ATPase in the hepatopancreas of Neohelice granulata (Dana, 1851), which is a model species. The stimulation by Na+ under total inhibition of Na+/K+ ATPase activity revealed the occurrence of Na+ ATPase activity that was totally inhibited by 2 mmol·L–1 furosemide, exhibits Michaelis–Menten kinetics for ATP (apparent Km = 0.52 ± 0.16 mmol·L–1), and highest activity at around pH 7.4. In crabs acclimated to 35 psu (osmoconforming conditions), Na+ ATPase activity was highly increased (about 15-fold) (532 ± 58 nmol Pi·mg protein−1·min−1) in the hepatopancreas 48 h after feeding. In 10 psu (hyper-regulating conditions), Na+ ATPase activity decreased in the hepatopancreas 24 h after feeding (7 ± 9 nmol Pi·mg protein−1·min−1) and recovered initial values after 48 h (24 ± 35 nmol Pi·mg protein−1·min−1). Unlike Na+ ATPase, Na+/K+ ATPase activity did not change after feeding at any salinity, suggesting the specific modulation of the second sodium pump and its role in postprandial adjustments in the hepatopancreas.

Sodium pump regulation of locomotor control circuits

Sodium pumps are ubiquitously expressed membrane proteins that extrude three Na+ ions in exchange for two K+ ions, using ATP as an energy source. Recent studies have illuminated additional, dynamic roles for sodium pumps in regulating the excitability of neuronal networks in an activity-dependent fashion. We review their role in a novel form of short-term memory within rhythmic locomotor networks. The data we review derives mainly from recent studies on Xenopus tadpoles and neonatal mice. The role and underlying mechanisms of pump action broadly match previously published data from an invertebrate, the Drosophila larva. We therefore propose a highly conserved mechanism by which sodium pump activity increases following a bout of locomotion. This results in an ultraslow afterhyperpolarization (usAHP) of the membrane potential that lasts around 1 min, but which only occurs in around half the network neurons. This usAHP in turn alters network excitability so that network output is reduced in a locomotor interval-dependent manner. The pumps therefore confer on spinal locomotor networks a temporary memory trace of recent network performance.