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

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 (αß) pump that usually exports 3Na+ in exchange for 2 K+ per hydrolyzed ATP. Artemia express several NKA catalytic α-subunit subtypes. High-salinity adaptation increases abundance of α2KK, an isoform that contains two lysines (Lys308 and Lys758 in transmembrane segments TM4 and TM5, respectively) at positions where canonical NKAs have asparagines (Xenopus α1's Asn333 and Asn785). Using de novo transcriptome assembly and qPCR, we found that Artemia express two salinity-independent canonical α subunits (α1NN and α3NN), as well as two ß variants, in addition to the salinity-controlled α2KK. These ß subunits permitted heterologous expression of the α2KK pump 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 α2KK pumps and compared it to that of Xenopus α1 (and its α2KK-mimicking single- and double-lysine substitutions). The double substitution N333K/N785K confers α2KK-like characteristics to Xenopus α1, and mutant cycle analysis reveals energetic coupling between these two residues, illustrating how α2KK'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+-congener 86Rb+, 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 allowing Artemia to maintain steeper Na+ gradients in hypersaline environments.

The Na+/K+ pump dominates control of glycolysis in hippocampal dentate granule cells.

Cellular ATP that is consumed to perform energetically expensive tasks must be replenished by new ATP through the activation of metabolism. Neuronal stimulation, an energetically demanding process, transiently activates aerobic glycolysis, but the precise mechanism underlying this glycolysis activation has not been determined. We previously showed that neuronal glycolysis is correlated with Ca2+ influx, but is not activated by feedforward Ca2+ signaling (Díaz-García et al., 2021a). Since ATP-powered Na+ and Ca2+ pumping activities are increased following stimulation to restore ion gradients and are estimated to consume most neuronal ATP, we aimed to determine if they are coupled to neuronal glycolysis activation. By using two-photon imaging of fluorescent biosensors and dyes in dentate granule cell somas of acute mouse hippocampal slices, we observed that production of cytoplasmic NADH, a byproduct of glycolysis, is strongly coupled to changes in intracellular Na+, while intracellular Ca2+ could only increase NADH production if both forward Na+/Ca2+ exchange and Na+/K+ pump activity were intact. Additionally, antidromic stimulation-induced intracellular [Na+] increases were reduced >50% by blocking Ca2+ entry. These results indicate that neuronal glycolysis activation is predominantly a response to an increase in activity of the Na+/K+ pump, which is strongly potentiated by Na+ influx through the Na+/Ca2+ exchanger during extrusion of Ca2+ following stimulation.

Structure and function of H+/K+ pump mutants reveal Na+/K+ pump mechanisms.

Ion-transport mechanisms evolve by changing ion-selectivity, such as switching from Na+ to H+ selectivity in secondary-active transporters or P-type-ATPases. Here we study primary-active transport via P-type ATPases using functional and structural analyses to demonstrate that four simultaneous residue substitutions transform the non-gastric H+/K+ pump, a strict H+-dependent electroneutral P-type ATPase, into a bona fide Na+-dependent electrogenic Na+/K+ pump. Conversion of a H+-dependent primary-active transporter into a Na+-dependent one provides a prototype for similar studies of ion-transport proteins. Moreover, we solve the structures of the wild-type non-gastric H+/K+ pump, a suitable drug target to treat cystic fibrosis, and of its Na+/K+ pump-mimicking mutant in two major conformations, providing insight on how Na+ binding drives a concerted mechanism leading to Na+/K+ pump phosphorylation.

A high-throughput multiparameter screen for accelerated development and optimization of soluble genetically encoded fluorescent biosensors.

Genetically encoded fluorescent biosensors are powerful tools used to track chemical processes in intact biological systems. However, the development and optimization of biosensors remains a challenging and labor-intensive process, primarily due to technical limitations of methods for screening candidate biosensors. Here we describe a screening modality that combines droplet microfluidics and automated fluorescence imaging to provide an order of magnitude increase in screening throughput. Moreover, unlike current techniques that are limited to screening for a single biosensor feature at a time (e.g. brightness), our method enables evaluation of multiple features (e.g. contrast, affinity, specificity) in parallel. Because biosensor features can covary, this capability is essential for rapid optimization. We use this system to generate a high-performance biosensor for lactate that can be used to quantify intracellular lactate concentrations. This biosensor, named LiLac, constitutes a significant advance in metabolite sensing and demonstrates the power of our screening approach.

The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle.

When neurons engage in intense periods of activity, the consequent increase in energy demand can be met by the coordinated activation of glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation. However, the trigger for glycolytic activation is unknown and the role for Ca2+ in the mitochondrial responses has been debated. Using genetically encoded fluorescent biosensors and NAD(P)H autofluorescence imaging in acute hippocampal slices, here we find that Ca2+ uptake into the mitochondria is responsible for the buildup of mitochondrial NADH, probably through Ca2+ activation of dehydrogenases in the TCA cycle. In the cytosol, we do not observe a role for the Ca2+/calmodulin signaling pathway, or AMPK, in mediating the rise in glycolytic NADH in response to acute stimulation. Aerobic glycolysis in neurons is triggered mainly by the energy demand resulting from either Na+ or Ca2+ extrusion, and in mouse dentate granule cells, Ca2+ creates the majority of this demand.

FXYD protein isoforms differentially modulate human Na/K pump function.

Tight regulation of the Na/K pump is essential for cellular function because this heteromeric protein builds and maintains the electrochemical gradients for Na+ and K+ that energize electrical signaling and secondary active transport. We studied the regulation of the ubiquitous human α1ß1 pump isoform by five human FXYD proteins normally located in muscle, kidney, and neurons. The function of Na/K pump α1ß1 expressed in Xenopus oocytes with or without FXYD isoforms was evaluated using two-electrode voltage clamp and patch clamp. Through evaluation of the partial reactions in the absence of K+ but presence of Na+ in the external milieu, we demonstrate that each FXYD subunit alters the equilibrium between E1P(3Na) and E2P, the phosphorylated conformations with Na+ occluded and free from Na+, respectively, thereby altering the apparent affinity for Na+. This modification of Na+ interaction shapes the small effects of FXYD proteins on the apparent affinity for external K+ at physiological Na+. FXYD6 distinctively accelerated both the Na+-deocclusion and the pump-turnover rates. All FXYD isoforms altered the apparent affinity for intracellular Na+ in patches, an effect that was observed only in the presence of intracellular K+. Therefore, FXYD proteins alter the selectivity of the pump for intracellular ions, an effect that could be due to the altered equilibrium between E1 and E2, the two major pump conformations, and/or to small changes in ion affinities that are exacerbated when both ions are present. Lastly, we observed a drastic reduction of Na/K pump surface expression when it was coexpressed with FXYD1 or FXYD6, with the former being relieved by injection of PKA's catalytic subunit into the oocyte. Our results indicate that a prominent effect of FXYD1 and FXYD6, and plausibly other FXYDs, is the regulation of Na/K pump trafficking.

Na/K Pump Mutations Associated with Primary Hyperaldosteronism Cause Loss of Function.

Primary hyperaldosteronism (Conn's syndrome), a common cause of secondary hypertension, is frequently produced by unilateral aldosterone-producing adenomas that carry mutations in ion-transporting genes, including ATP1A1, encoding the Na/K pump's α1 subunit. Whether Na/K pump mutant-mediated inward currents are required to depolarize the cell and increase aldosterone production remains unclear, as such currents were observed in four out of five mutants described so far. Here, we use electrophysiology and uptake of the K+ congener 86Rb+, to characterize the effects of eight additional Na/K pump mutations in transmembrane segments TM1 (delM102-L103, delL103-L104, and delM102-I106), TM4 (delI322-I325 and I327S), and TM9 (delF956-E961, delF959-E961, and delE960-L964), expressed in Xenopus oocytes. All deletion mutants induced abnormal inward currents of different amplitudes at physiological voltages, while I327S lacked such currents. A detailed functional characterization revealed that I327S significantly reduces intracellular Na+ affinity without altering affinity for external K+. 86Rb+-uptake experiments show that I327S dramatically impairs function under physiological concentrations of Na+ and K+. Since Na/K pumps in the adrenal cortex may be formed by association of α1 with ß3 instead of ß1 subunits, we evaluated whether G99R (another mutant without inward currents when associated with ß1) would show inward currents when associated with ß3. We found that the kinetic characteristics of either mutant or wild-type α1ß3 pumps expressed in Xenopus oocytes to be indistinguishable from those of α1ß1 pumps. The observed functional consequences of each hyperaldosteronism mutant point to the loss of Na/K pump function as the common feature of all mutants, which is sufficient to induce hyperaldosteronism.

On the effect of hyperaldosteronism-inducing mutations in Na/K pumps.

Primary aldosteronism, a condition in which too much aldosterone is produced and that leads to hypertension, is often initiated by an aldosterone-producing adenoma within the zona glomerulosa of the adrenal cortex. Somatic mutations of ATP1A1, encoding the Na/K pump α1 subunit, have been found in these adenomas. It has been proposed that a passive inward current transported by several of these mutant pumps is a "gain-of-function" activity that produces membrane depolarization and concomitant increases in aldosterone production. Here, we investigate whether the inward current through mutant Na/K pumps is large enough to induce depolarization of the cells that harbor them. We first investigate inward currents induced by these mutations in Xenopus Na/K pumps expressed in Xenopus oocytes and find that these inward currents are similar in amplitude to wild-type outward Na/K pump currents. Subsequently, we perform a detailed functional evaluation of the human Na/K pump mutants L104R, delF100-L104, V332G, and EETA963S expressed in Xenopus oocytes. By combining two-electrode voltage clamp with [3H]ouabain binding, we measure the turnover rate of these inward currents and compare it to the turnover rate for outward current through wild-type pumps. We find that the turnover rate of the inward current through two of these mutants (EETA963S and L104R) is too small to induce significant cell depolarization. Electrophysiological characterization of another hyperaldosteronism-inducing mutation, G99R, reveals the absence of inward currents under many different conditions, including in the presence of the regulator FXYD1 as well as with mammalian ionic concentrations and body temperatures. Instead, we observe robust outward currents, but with significantly reduced affinities for intracellular Na+ and extracellular K+ Collectively, our results point to loss-of-function as the common mechanism for the hyperaldosteronism induced by these Na/K pump mutants.

An in silico analysis of primary and secondary structure specificity determinants for human peptidylarginine deiminase types 2 and 4.

Human peptidylarginine deiminases (hPADs) are a family of five calcium-dependent enzymes that facilitate citrullination, which is the post-translational modification of peptidyl arginine to peptidyl citrulline. The isozymes hPAD2 and hPAD4 have been implicated in the development and progression of several autoimmune diseases, including rheumatoid arthritis and multiple sclerosis. To better characterize the primary and secondary structure determinants of citrullination specificity, we mined the literature for protein sequences susceptible to citrullination by hPAD2 or hPAD4. First, protein secondary structure classification (α-helix, ß-sheet, or coil) was predicted using the PSIPRED software. Next, we used motif-x and pLogo to extract and visualize statistically significant motifs within each data set. Within the data sets of peptides predicted to lie in coil regions, both hPAD2 and hPAD4 appear to favor citrullination of glycine-containing motifs, while distinct hydrophobic motifs were identified for hPAD2 citrullination sites predicted to reside within α-helical and ß-sheet regions. Additionally, we identified potential substrate overlap between coil region citrullination and arginine methylation. Together, these results confirm the importance and offer some insight into the role of secondary structure elements for citrullination specificity, and provide biological context for the existing hPAD specificity and arginine post-translational modification literature.

Intracellular Requirements for Passive Proton Transport through the Na+,K+-ATPase.

The Na+,K+-ATPase (NKA or Na/K pump) hydrolyzes one ATP to exchange three intracellular Na+ (Na+i) for two extracellular K+ (K+o) across the plasma membrane by cycling through a set of reversible transitions between phosphorylated and dephosphorylated conformations, alternately opening ion-binding sites externally (E2) or internally (E1). With subsaturating [Na+]o and [K+]o, the phosphorylated E2P conformation passively imports protons generating an inward current (IH), which may be exacerbated in NKA-subunit mutations associated with human disease. To elucidate the mechanisms of IH, we studied the effects of intracellular ligands (transported ions, nucleotides, and beryllium fluoride) on IH and, for comparison, on transient currents measured at normal Na+o (QNa). Utilizing inside-out patches from Xenopus oocytes heterologously expressing NKA, we observed that 1) in the presence of Na+i, IH and QNa were both activated by ATP, but not ADP; 2) the [Na+]i dependence of IH in saturating ATP showed K0.5,Na = 1.8 ± 0.2 mM and the [ATP] dependence at saturating [Na+]i yielded K0.5,ATP = 48 ± 11 µM (in comparison, Na+i-dependent QNa yields K0.5,Na = 0.8 ± 0.2 mM and K0.5,ATP = 0.43 ± 0.03 µM; 3) ATP activated IH in the presence of K+i (∼15% of the IH observed in Na+i) only when Mg2+i was also present; and 4) beryllium fluoride induced maximal IH even in the absence of nucleotide. These data indicate that IH occurs when NKA is in an externally open E2P state with nucleotide bound, a conformation that can be reached through forward Na/K pump phosphorylation of E1, with Na+i and ATP, or by backward binding of K+i to E1, which drives the pump to the occluded E2(2K), where free Pi (at the micromolar levels found in millimolar ATP solutions) promotes external release of occluded K+ by backdoor NKA phosphorylation. Maximal IH through beryllium-fluorinated NKA indicates that this complex mimics ATP-bound E2P states.

Importance of the Voltage Dependence of Cardiac Na/K ATPase Isozymes.

Cardiac cells express more than one isoform of the Na, K-ATPase (NKA), the heteromeric enzyme that creates the Na(+) and K(+) gradients across the plasmalemma. Cardiac isozymes contain one catalytic α-subunit isoform (α1, α2, or α3) associated with an auxiliary ß-subunit isoform (ß1 or ß2). Past studies using biochemical approaches have revealed minor kinetic differences between isozymes formed by different α-ß isoform combinations; these results make it difficult to understand the physiological requirement for multiple isoforms. In intact cells, however, NKA enzymes operate in a more complex environment, which includes a substantial transmembrane potential. We evaluated the voltage dependence of human cardiac NKA isozymes expressed in Xenopus oocytes, and of native NKA isozymes in rat ventricular myocytes, using normal mammalian physiological concentrations of Na(+)o and K(+)o. We demonstrate that although α1 and α3 pumps are functional at all physiologically relevant voltages, α2ß1 pumps and α2ß2 pumps are inhibited by ∼75% and ∼95%, respectively, at resting membrane potentials, and only activate appreciably upon depolarization. Furthermore, phospholemman (FXYD1) inhibits pump function without significantly altering the pump's voltage dependence. Our observations provide a simple explanation for the physiological relevance of the α2 subunit (∼20% of total α subunits in rat ventricle): they act as a reserve and are recruited into action for extra pumping during the long-lasting cardiac action potential, where most of the Na(+) entry occurs. This strong voltage dependence of α2 pumps also helps explain how cardiotonic steroids, which block NKA pumps, can be a beneficial treatment for heart failure: by only inhibiting the α2 pumps, they selectively reduce NKA activity during the cardiac action potential, leading to an increase in systolic Ca(2+), due to reduced extrusion through the Na/Ca exchanger, without affecting resting Na(+) and Ca(2+) concentrations.