Molecular determinants for cold adaptation in an Antarctic Na+/K+-ATPase.

Enzymes from ectotherms living in chronically cold environments have evolved structural innovations to overcome the effects of temperature on catalysis. Cold adaptation of soluble enzymes is driven by changes within their primary structure or the aqueous milieu. For membrane-embedded enzymes, like the Na+/K+-ATPase, the situation is different because changes to the lipid bilayer in which they operate may also be relevant. Although much attention has been focused on thermal adaptation within lipid bilayers, relatively little is known about the contribution of structural changes within membrane-bound enzymes themselves. The identification of specific mutations that confer temperature compensation is complicated by the presence of neutral mutations, which can be more numerous. In the present study, we identified specific amino acids in a Na+/K+-ATPase from an Antarctic octopus that underlie cold resistance. Our approach was to generate chimeras between an Antarctic clone and a temperate ortholog and then study their temperature sensitivities in Xenopus oocytes using an electrophysiological approach. We identified 12 positions in the Antarctic Na+/K+-ATPase that, when transferred to the temperate ortholog, were sufficient to confer cold tolerance. Furthermore, although all 12 Antarctic mutations were required for the full phenotype, a single leucine in the third transmembrane segment (M3) imparted most of it. Mutations that confer cold resistance are mostly in transmembrane segments, at positions that face the lipid bilayer. We propose that the interface between a transmembrane enzyme and the lipid bilayer is a critical determinant of temperature sensitivity and, accordingly, has been a prime evolutionary target for thermal adaptation.

Cation leak through the ATP1A3 pump causes spasticity and intellectual disability.

ATP1A3 encodes the α3 subunit of the sodium-potassium ATPase, one of two isoforms responsible for powering electrochemical gradients in neurons. Heterozygous pathogenic ATP1A3 variants produce several distinct neurological syndromes, yet the molecular basis for phenotypic variability is unclear. We report a novel recurrent variant, ATP1A3(NM_152296.5):c.2324C>T; p.(Pro775Leu), in nine individuals associated with the primary clinical features of progressive or non-progressive spasticity and developmental delay/intellectual disability. No patients fulfil diagnostic criteria for ATP1A3-associated syndromes, including alternating hemiplegia of childhood, rapid-onset dystonia-parkinsonism or cerebellar ataxia-areflexia-pes cavus-optic atrophy-sensorineural hearing loss (CAPOS), and none were suspected of having an ATP1A3-related disorder. Uniquely among known ATP1A3 variants, P775L causes leakage of sodium ions and protons into the cell, associated with impaired sodium binding/occlusion kinetics favouring states with fewer bound ions. These phenotypic and electrophysiologic studies demonstrate that ATP1A3:c.2324C>T; p.(Pro775Leu) results in mild ATP1A3-related phenotypes resembling complex hereditary spastic paraplegia or idiopathic spastic cerebral palsy. Cation leak provides a molecular explanation for this genotype-phenotype correlation, adding another mechanism to further explain phenotypic variability and highlighting the importance of biophysical properties beyond ion transport rate in ion transport diseases.

Transient Electrical Currents Mediated by the Na+/K+-ATPase: A Tour from Basic Biophysics to Human Diseases.

The Na+/K+-ATPase is a chemical molecular machine responsible for the movement of Na+ and K+ ions across the cell membrane. These ions are moved against their electrochemical gradients, so the protein uses the free energy of ATP hydrolysis to transport them. In fact, the Na+/K+-ATPase is the single largest consumer of energy in most cells. In each pump cycle, the protein sequentially exports 3Na+ out of the cell, then imports 2K+ into the cell at an approximate rate of 200 cycles/s. In each half cycle of the transport process, there is a state in which ions are stably trapped within the permeation pathway of the protein by internal and external gates in their closed states. These gates are required to open alternately; otherwise, passive ion diffusion would be a wasteful end of the cell's energy. Once one of these gates open, ions diffuse from their binding sites to the accessible milieu, which involves moving through part of the electrical field across the membrane. Consequently, ions generate transient electrical currents first discovered more than 30 years ago. They have been studied in a variety of preparations, including native and heterologous expression systems. Here, we review three decades' worth of work using these transient electrical signals to understand the kinetic transitions of the movement of Na+ and K+ ions through the Na+/K+-ATPase and propose the significance that this work might have to the understanding of the dysfunction of human pump orthologs responsible for some newly discovered neurological pathologies.

A Structural Model of the Inactivation Gate of Voltage-Activated Potassium Channels.

After opening, the Shaker voltage-gated potassium (KV) channel rapidly inactivates when one of its four N-termini enters and occludes the channel pore. Although it is known that the tip of the N-terminus reaches deep into the central cavity, the conformation adopted by this domain during inactivation and the nature of its interactions with the rest of the channel remain unclear. Here, we use molecular dynamics simulations coupled with electrophysiology experiments to reveal the atomic-scale mechanisms of inactivation. We find that the first six amino acids of the N-terminus spontaneously enter the central cavity in an extended conformation, establishing hydrophobic contacts with residues lining the pore. A second portion of the N-terminus, consisting of a long 24 amino acid α-helix, forms numerous polar contacts with residues in the intracellular entryway of the T1 domain. Double mutant cycle analysis revealed a strong relationship between predicted interatomic distances and empirically observed thermodynamic coupling, establishing a plausible model of the transition of KV channels to the inactivated state.

Interactions of divalent cations with calcium binding sites of BK channels reveal independent motions within the gating ring.

Large-conductance voltage- and calcium-activated K+ (BK) channels are key physiological players in muscle, nerve, and endocrine function by integrating intracellular Ca2+ and membrane voltage signals. The open probability of BK channels is regulated by the intracellular concentration of divalent cations sensed by a large structure in the BK channel called the "gating ring," which is formed by four tandems of regulator of conductance for K+ (RCK1 and RCK2) domains. In contrast to Ca2+ that binds to both RCK domains, Mg2+, Cd2+, or Ba2+ interact preferentially with either one or the other. Interaction of cations with their binding sites causes molecular rearrangements of the gating ring, but how these motions occur remains elusive. We have assessed the separate contributions of each RCK domain to the cation-induced gating-ring structural rearrangements, using patch-clamp fluorometry. Here we show that Mg2+ and Ba2+ selectively induce structural movement of the RCK2 domain, whereas Cd2+ causes motions of RCK1, in all cases substantially smaller than those elicited by Ca2+ By combining divalent species interacting with unique sites, we demonstrate that RCK1 and RCK2 domains move independently when their specific binding sites are occupied. Moreover, binding of chemically distinct cations to both RCK domains is additive, emulating the effect of fully occupied Ca2+ binding sites.

State-dependent FRET reports calcium- and voltage-dependent gating-ring motions in BK channels.

Large-conductance voltage- and calcium-dependent potassium channels (BK, "Big K+") are important controllers of cell excitability. In the BK channel, a large C-terminal intracellular region containing a "gating-ring" structure has been proposed to transduce Ca(2+) binding into channel opening. Using patch-clamp fluorometry, we have investigated the calcium and voltage dependence of conformational changes of the gating-ring region of BK channels, while simultaneously monitoring channel conductance. Fluorescence resonance energy transfer (FRET) between fluorescent protein inserts indicates that Ca(2+) binding produces structural changes of the gating ring that are much larger than those predicted by current X-ray crystal structures of isolated gating rings.

Regulation of Ion Channel and Transporter Function Through RNA Editing.

A large proportion of the recoding events mediated by RNA editing are in mRNAs that encode ion channels and transporters. The effects of these events on protein function have been characterized in only a few cases. In even fewer instances are the mechanistic underpinnings of these effects understood. This review focuses on how RNA editing affects protein function and higher order physiology. In mammals, particular attention is given to the GluA2, an ionotropic glutamate receptor subunit, and K(v) 1.1, a voltage-dependent K+ channel, because they are particularly well understood. In K(v) addition, work on cephalopod K+ channels and Na+/K+-ATPases has also provided important clues on the rules used by RNA editing to regulate excitability. Finally, we discuss some of the emerging targets for editing and how this process may be used to regulate nervous function in response to a variable environment.

Energy landscape of the reactions governing the Na+ deeply occluded state of the Na+/K+-ATPase in the giant axon of the Humboldt squid.

The Na(+)/K(+) pump is a nearly ubiquitous membrane protein in animal cells that uses the free energy of ATP hydrolysis to alternatively export 3Na(+) from the cell and import 2K(+) per cycle. This exchange of ions produces a steady-state outwardly directed current, which is proportional in magnitude to the turnover rate. Under certain ionic conditions, a sudden voltage jump generates temporally distinct transient currents mediated by the Na(+)/K(+) pump that represent the kinetics of extracellular Na(+) binding/release and Na(+) occlusion/deocclusion transitions. For many years, these events have escaped a proper thermodynamic treatment due to the relatively small electrical signal. Here, taking the advantages offered by the large diameter of the axons from the squid Dosidicus gigas, we have been able to separate the kinetic components of the transient currents in an extended temperature range and thus characterize the energetic landscape of the pump cycle and those transitions associated with the extracellular release of the first Na(+) from the deeply occluded state. Occlusion/deocclusion transition involves large changes in enthalpy and entropy as the ion is exposed to the external milieu for release. Binding/unbinding is substantially less costly, yet larger than predicted for the energetic cost of an ion diffusing through a permeation pathway, which suggests that ion binding/unbinding must involve amino acid side-chain rearrangements at the site.

ATP1A3 Disease Spectrum Includes Paroxysmal Weakness and Encephalopathy Not Triggered by Fever.

Background and Objectives: Heterozygous pathogenic variants in ATP1A3, which encodes the catalytic alpha subunit of neuronal Na+/K+-ATPase, cause primarily neurologic disorders with widely variable features that can include episodic movement deficits. One distinctive presentation of ATP1A3-related disease is recurrent fever-triggered encephalopathy. This can occur with generalized weakness and/or ataxia and is described in the literature as relapsing encephalopathy with cerebellar ataxia. This syndrome displays genotype-phenotype correlation with variants at p.R756 causing temperature sensitivity of ATP1A3. We report clinical and in vitro functional evidence for a similar phenotype not triggered by fever but associated with protein loss-of-function. Methods: We describe the phenotype of an individual with de novo occurrence of a novel heterozygous ATP1A3 variant, NM_152296.5:c.388_390delGTG; p.(V130del). We confirmed the pathogenicity of p.V130del by cell survival complementation assay in HEK293 cells and then characterized its functional impact on enzymatic ion transport and extracellular sodium binding by two-electrode voltage clamp electrophysiology in Xenopus oocytes. To determine whether variant enzymes reach the cell surface, we surface-biotinylated oocytes expressing N-tagged ATP1A3. Results: The proband is a 7-year-old boy who has had 2 lifetime episodes of paroxysmal weakness, encephalopathy, and ataxia not triggered by fever. He had speech regression and intermittent hand tremors after the second episode but otherwise spontaneously recovered after episodes and is at present developmentally appropriate. The p.V130del variant was identified on clinical trio exome sequencing, which did not reveal any other variants possibly associated with the phenotype. p.V130del eliminated ATP1A3 function in cell survival complementation assay. In Xenopus oocytes, p.V130del variant Na+/K+-ATPases showed complete loss of ion transport activity and marked abnormalities of extracellular Na+ binding at room temperature. Despite this clear loss-of-function effect, surface biotinylation under the same conditions revealed that p.V130del variant enzymes were still present at the oocyte's cell membrane. Discussion: This individual's phenotype expands the clinical spectrum of ATP1A3-related recurrent encephalopathy to include presentations without fever-triggered events. The total loss of ion transport function with p.V130del, despite enzyme presence at the cell membrane, indicates that haploinsufficiency can cause relatively mild phenotypes in ATP1A3-related disease.

ATP1A1-linked diseases require a malfunctioning protein product from one allele.

Heterozygous germline variants in ATP1A1, the gene encoding the α1 subunit of the Na+/K+-ATPase (NKA), have been linked to diseases including primary hyperaldosteronism and the peripheral neuropathy Charcot-Marie-Tooth disease (CMT). ATP1A1 variants that cause CMT induce loss-of-function of NKA. This heterodimeric (αß) enzyme hydrolyzes ATP to establish transmembrane electrochemical gradients of Na+ and K+ that are essential for electrical signaling and cell survival. Of the 4 catalytic subunit isoforms, α1 is ubiquitously expressed and is the predominant paralog in peripheral axons. Human population sequencing datasets indicate strong negative selection against both missense and protein-null ATP1A1 variants. To test whether haploinsufficiency generated by heterozygous protein-null alleles are sufficient to cause disease, we tested the neuromuscular characteristics of heterozygous Atp1a1+/- knockout mice and their wildtype littermates, while also evaluating if exercise increased CMT penetrance. We found that Atp1a1+/- mice were phenotypically normal up to 18 months of age. Consistent with the observations in mice, we report clinical phenotyping of a healthy adult human who lacks any clinical features of known ATP1A1-related diseases despite carrying a plasma-membrane protein-null early truncation variant, p.Y148*. Taken together, these results suggest that a malfunctioning gene product is required for disease induction by ATP1A1 variants and that if any pathology is associated with protein-null variants, they may display low penetrance or high age of onset.

Regulation of Na+/K+ ATPase transport velocity by RNA editing.

Because firing properties and metabolic rates vary widely, neurons require different transport rates from their Na(+)/K(+) pumps in order to maintain ion homeostasis. In this study we show that Na(+)/K(+) pump activity is tightly regulated by a novel process, RNA editing. Three codons within the squid Na(+)/K(+) ATPase gene can be recoded at the RNA level, and the efficiency of conversion for each varies dramatically, and independently, between tissues. At one site, a highly conserved isoleucine in the seventh transmembrane span can be converted to a valine, a change that shifts the pump's intrinsic voltage dependence. Mechanistically, the removal of a single methyl group specifically targets the process of Na(+) release to the extracellular solution, causing a higher turnover rate at the resting membrane potential.

Some aspects of the life of SARS-CoV-2 ORF3a protein in mammalian cells.

The accessory protein ORF3a, from SARS-CoV-2, plays a critical role in viral infection and pathogenesis. Here, we characterized ORF3a assembly, ion channel activity, subcellular localization, and interactome. At the plasma membrane, ORF3a exists mostly as monomers and dimers, which do not alter the native cell membrane conductance, suggesting that ORF3a does not function as a viroporin at the cell surface. As a membrane protein, ORF3a is synthesized at the ER and sorted via a canonical route. ORF3a overexpression induced an approximately 25% increase in cell death. By developing an APEX2-based proximity labeling assay, we uncovered proteins proximal to ORF3a, suggesting that ORF3a recruits some host proteins to weaken the cell. In addition, it exposed a set of mitochondria related proteins that triggered mitochondrial fission. Overall, this work can be an important instrument in understanding the role of ORF3a in the virus pathogenicity and searching for potential therapeutic treatments for COVID-19.

ATP1A1 -linked diseases require a malfunctioning protein product from one allele.

Heterozygous germline variants in ATP1A1 , the gene encoding the α1 subunit of the Na + /K + -ATPase (NKA), have been linked to diseases including primary hyperaldosteronism and the peripheral neuropathy Charcot-Marie-Tooth disease (CMT). ATP1A1 variants that cause CMT induce loss-of-function of NKA. This heterodimeric (αß) enzyme hydrolyzes ATP to establish transmembrane electrochemical gradients of Na + and K + that are essential for electrical signaling and cell survival. Of the 4 catalytic subunit isoforms, α1 is ubiquitously expressed and is the predominant paralog in peripheral axons. Human population sequencing datasets indicate strong negative selection against both missense and protein-null ATP1A1 variants. To test whether haploinsufficiency generated by heterozygous protein-null alleles are sufficient to cause disease, we tested the neuromuscular characteristics of heterozygous Atp1a1 +/- knockout mice and their wildtype littermates, while also evaluating if exercise increased CMT penetrance. We found that Atp1a1 +/- mice were phenotypically normal up to 18 months of age. Consistent with the observations in mice, we report clinical phenotyping of a healthy adult human who lacks any clinical features of known ATP1A1 -related diseases despite carrying a protein-null early truncation variant, p.Y148*. Taken together, these results suggest that a malfunctioning gene product is required for disease induction by ATP1A1 variants and that if any pathology is associated with protein-null variants, they may display low penetrance or high age of onset.

Ouabain binding site in a functioning Na+/K+ ATPase.

The Na(+)/K(+) ATPase is an almost ubiquitous integral membrane protein within the animal kingdom. It is also the selective target for cardiotonic derivatives, widely prescribed inhibitors for patients with heart failure. Functional studies revealed that ouabain-sensitive residues distributed widely throughout the primary sequence of the protein. Recently, structural work has brought some consensus to the functional observations. Here, we use a spectroscopic approach to estimate distances between a fluorescent ouabain and a lanthanide binding tag (LBT), which was introduced at five different positions in the Na(+)/K(+) ATPase sequence. These five normally functional LBT-Na(+)/K(+) ATPase constructs were expressed in the cell membrane of Xenopus laevis oocytes, operating under physiological internal and external ion conditions. The spectroscopic data suggest two mutually exclusive distances between the LBT and the fluorescent ouabain. From the estimated distances and using homology models of the LBT-Na(+)/K(+) ATPase constructs, approximate ouabain positions could be determined. Our results suggest that ouabain binds at two sites along the ion permeation pathway of the Na(+)/K(+) ATPase. The external site (low apparent affinity) occupies the same region as previous structural findings. The high apparent affinity site is, however, slightly deeper toward the intracellular end of the protein. Interestingly, in both cases the lactone ring faces outward. We propose a sequential ouabain binding mechanism that is consistent with all functional and structural studies.

Structural basis of Na(+)/K(+)-ATPase adaptation to marine environments.

Throughout evolution, enzymes have adapted to perform in different environments. The Na(+)/K(+) pump, an enzyme crucial for maintaining ionic gradients across cell membranes, is strongly influenced by the ionic environment. In vertebrates, the pump sees much less external Na(+) (100-160 mM) than it does in osmoconformers such as squid (450 mM), which live in seawater. If the extracellular architecture of the squid pump were identical to that of vertebrates, then at the resting potential, the pump's function would be severely compromised because the negative voltage would drive Na(+) ions back to their binding sites, practically abolishing forward transport. Here we show that four amino acids that ring the external mouth of the ion translocation pathway are more positive in squid, thereby reducing the pump's sensitivity to external Na(+) and explaining how it can perform optimally in the marine environment.

Disease mutations of human α3 Na+/K+-ATPase define extracellular Na+ binding/occlusion kinetics at ion binding site III.

Na+/K+-ATPase, which creates transmembrane electrochemical gradients by exchanging 3 Na+ for 2 K+, is central to the pathogenesis of neurological diseases such as alternating hemiplegia of childhood. Although Na+/K+-ATPase has 3 distinct ion binding sites I-III, the difficulty of distinguishing ion binding events at each site from the others hinders kinetic study of these transitions. Here, we show that binding of Na+ at each site in the human α3 Na+/K+-ATPase can be resolved using extracellular Na+-mediated transient currents. When Na+/K+-ATPase is constrained to bind and release only Na+, three kinetic components: fast, medium, and slow, can be isolated, presumably corresponding to the protein dynamics associated with the binding (or release depending on the voltage step direction) and the occlusion (or deocclusion) of each of the 3 Na+. Patient-derived mutations of residues which coordinate Na+ at site III exclusively impact the slow component, demonstrating that site III is crucial for deocclusion and release of the first Na+ into the extracellular milieu. These results advance understanding of Na+/K+-ATPase mutation pathogenesis and provide a foundation for study of individual ions' binding kinetics.

Comparative description of the mRNA expression profile of Na+ /K+ -ATPase isoforms in adult mouse nervous system.

Mutations in genes encoding Na+ /K+ -ATPase α1, α2, and α3 subunits cause a wide range of disabling neurological disorders, and dysfunction of Na+ /K+ -ATPase may contribute to neuronal injury in stroke and dementia. To better understand the pathogenesis of these diseases, it is important to determine the expression patterns of the different Na+ /K+ -ATPase subunits within the brain and among specific cell types. Using two available scRNA-Seq databases from the adult mouse nervous system, we examined the mRNA expression patterns of the different isoforms of the Na+ /K+ -ATPase α, ß and Fxyd subunits at the single-cell level among brain regions and various neuronal populations. We subsequently identified specific types of neurons enriched with transcripts for α1 and α3 isoforms and elaborated how α3-expressing neuronal populations govern cerebellar neuronal circuits. We further analyzed the co-expression network for α1 and α3 isoforms, highlighting the genes that positively correlated with α1 and α3 expression. The top 10 genes for α1 were Chn2, Hpcal1, Nrgn, Neurod1, Selm, Kcnc1, Snrk, Snap25, Ckb and Ccndbp1 and for α3 were Sorcs3, Eml5, Neurod2, Ckb, Tbc1d4, Ptprz1, Pvrl1, Kirrel3, Pvalb, and Asic2.

Physiological adaptation of an Antarctic Na+/K+-ATPase to the cold.

Because enzymatic activity is strongly suppressed by the cold, polar poikilotherms face significant adaptive challenges. For example, at 0°C the catalytic activity of a typical enzyme from a temperate organism is reduced by more than 90%. Enzymes embedded in the plasma membrane, such as the Na(+)/K(+)-ATPase, may be even more susceptible to the cold because of thermal effects on the lipid bilayer. Accordingly, adaptive changes in response to the cold may include adjustments to the enzyme or the surrounding lipid environment, or synergistic changes to both. To assess the contribution of the enzyme itself, we cloned orthologous Na(+)/K(+)-ATPase α-subunits from an Antarctic (Pareledone sp.; -1.8°C) and a temperate octopus (Octopus bimaculatus; ∼18°C), and compared their turnover rates and temperature sensitivities in a heterologous expression system. The primary sequences of the two pumps were found to be highly similar (97% identity), with most differences being conservative changes involving hydrophobic residues. The physiology of the pumps was studied using an electrophysiological approach in intact Xenopus oocytes. The voltage dependence of the pumps was equivalent. However, at room temperature the maximum turnover rate of the Antarctic pump was found to be 25% higher than that of the temperate pump. In addition, the Antarctic pump exhibited a lower temperature sensitivity, leading to significantly higher relative activity at lower temperatures. Orthologous Na(+)/K(+) pumps were then isolated from two tropical and two Arctic octopus. The temperature sensitivities of these pumps closely matched those of the temperate and Antarctic pumps, respectively. Thus, reduced thermal sensitivity appears to be a common mechanism driving cold adaptation in the Na(+)/K(+)-ATPase.

Gating at the selectivity filter in cyclic nucleotide-gated channels.

By opening and closing the permeation pathway (gating) in response to cGMP binding, cyclic nucleotide-gated (CNG) channels serve key roles in the transduction of visual and olfactory signals. Compiling evidence suggests that the activation gate in CNG channels is not located at the intracellular end of pore, as it has been established for voltage-activated potassium (K(V)) channels. Here, we show that ion permeation in CNG channels is tightly regulated at the selectivity filter. By scanning the entire selectivity filter using small cysteine reagents, like cadmium and silver, we observed a state-dependent accessibility pattern consistent with gated access at the middle of the selectivity filter, likely at the corresponding position known to regulate structural changes in KcsA channels in response to low concentrations of permeant ions.

Voltage profile along the permeation pathway of an open channel.

For ion channels, the transmembrane potential plays a critical role by acting as a driving force for permeant ions. At the microscopic level, the transmembrane potential is thought to decay nonlinearly across the ion permeation pathway because of the irregular three-dimensional shape of the channel's pore. By taking advantage of the current structural and functional understanding of cyclic nucleotide-gated channels, in this study we experimentally explore the transmembrane potential's distribution across the open pore. As a readout for the voltage drop, we engineered cysteine residues along the selectivity filter and scanned the sensitivity of their modification rates by Ag(+) to the transmembrane potential. The experimental data, which indicate that the majority of the electric field drops across the selectivity filter, are in good agreement with continuum electrostatic calculations using a homology model of an open CNG channel. By focusing the transmembrane potential across the selectivity filter, the electromotive driving force is coupled with the movement of permeant ions in the filter, maximizing the efficiency of this process.