Our research aimed to elucidate the mechanism by which aurintricarboxylic acid (ATA) inhibits plasma membrane Ca2+-ATPase (PMCA), a crucial enzyme responsible for calcium transport. Given the pivotal role of PMCA in cellular calcium homeostasis, understanding how it is inhibited by ATA holds significant implications for potentially regulating physiopathological cellular processes in which this pump is involved. Our experimental findings revealed that ATA employs multiple modes of action to inhibit PMCA activity, which are influenced by ATP but also by the presence of calcium and magnesium ions. Specifically, magnesium appears to enhance this inhibitory effect. Our experimental and in-silico results suggest that, unlike those reported in other proteins, ATA complexed with magnesium (ATA·Mg) is the molecule that inhibits PMCA. In summary, our study presents a novel perspective and establishes a solid foundation for future research efforts aimed at the development of new pharmacological molecules both for PMCA and other proteins.

High serum estrogen concentrations are associated with asthma development and severity, suggesting a link between estradiol and airway hyperresponsiveness (AHR). 17β-estradiol (E2) has non-genomic effects via Ca2+ regulatory mechanisms; however, its effect on the plasma membrane Ca2+-ATPases (PMCA1 and 4) and sarcoplasmic reticulum Ca2+-ATPase (SERCA) is unknown. Hence, in the present study, we aim to demonstrate if E2 favors AHR by increasing intracellular Ca2+ concentrations in guinea pig airway smooth muscle (ASM) through a mechanism involving Ca2+-ATPases. In guinea pig ASM, Ca2+ microfluorometry, muscle contraction, and Western blot were evaluated. Then, we performed molecular docking analysis between the estrogens and Ca2+ ATPases. In tracheal rings, E2 produced AHR to carbachol. In guinea pig myocytes, acute exposure to physiological levels of E2 modified the transient Ca2+ peak induced by caffeine to a Ca2+ plateau. The incubation with PMCA inhibitors (lanthanum and carboxyeosin, CE) partially reversed the E2-induced sustained plateau in the caffeine response. In contrast, cyclopiazonic acid (SERCA inhibitor), U-0126 (an inhibitor of ERK 1/2), and choline chloride did not modify the Ca2+ plateau produced by E2. The mitochondrial uniporter activity and the capacitative Ca2+ entry were unaffected by E2. In guinea pig ASM, Western blot analysis demonstrated PMCA1 and PMCA4 expression. The results from the docking modeling demonstrate that E2 binds to both plasma membrane ATPases. In guinea pig tracheal smooth muscle, inhibiting the PMCA with CE, induced hyperresponsiveness to carbachol. 17β-estradiol produces hyperresponsiveness by inhibiting the PMCA in the ASM and could be one of the mechanisms responsible for the increase in asthmatic crisis in women.

In electrochemotherapy, permeabilization of the cell membrane by electric pulses increases the anti-tumour effect of chemotherapeutics. In calcium electroporation, chemotherapy is replaced by calcium chloride with obvious benefits. This study explores the effect and underlying mechanisms of calcium electroporation on basal cell carcinomas using either high- or low-frequency electroporation. Low-risk primary basal cell carcinomas were treated in local anaesthesia with intratumoral calcium chloride followed by electroporation with high (167 kHz) or low (5 kHz) frequencies. Non-complete responders were retreated after 3 months. The primary endpoint was tumour response 3 months after last calcium electroporation. Plasma membrane calcium ATPase was examined in various cell lines as plasma membrane calcium ATPase levels have been associated with calcium electroporation efficacy. Twenty-two out of 25 included patients complete the study and 7 of these (32%) achieved complete response at 3 months with no difference in efficacy between high- and low-frequency pulses. High-frequency calcium electroporation was significantly less painful (p=0.03). Plasma membrane calcium ATPase was increased 16-32-fold in basal cell carcinoma cell lines compared with 4 other cancer cell lines. Calcium electroporation for low-risk basal cell carcinomas does not fulfil the requirements of a new dermatological basal cell carcinoma treatment but may be useful as adjuvant treatment to surgery in more advanced basal cell carcinomas. The elevated PMCA levels in basal cell carcinomas may contribute to low efficacy.

Macropinocytosis (MPC) is a large-scale endocytosis pathway that involves actin-dependent membrane ruffle formation and subsequent ruffle closure to generate macropinosomes for the uptake of fluid-phase cargos. MPC is categorized into two types: constitutive and stimuli-induced. Constitutive MPC in macrophages relies on extracellular Ca2+ sensing by a calcium-sensing receptor. However, the link between stimuli-induced MPC and Ca2+ remains unclear. Here, we find that both intracellular and extracellular Ca2+ are required for epidermal growth factor (EGF)-induced MPC in A431 human epidermoid carcinoma cells. Through investigation of mammalian homologs of coelomocyte uptake defective (CUP) genes, we identify ATP2B4, encoding for a Ca2+ pump called the plasma membrane calcium ATPase 4 (PMCA4), as a Ca2+-related regulator of EGF-induced MPC. Knockout (KO) of ATP2B4, as well as depletion of extracellular/intracellular Ca2+, inhibited ruffle closure and macropinosome formation, without affecting ruffle formation. We demonstrate the importance of PMCA4 activity itself, independent of interactions with other proteins via its C-terminus known as a PDZ domain-binding motif. Additionally, we show that ATP2B4-KO reduces EGF-stimulated Ca2+ oscillation during MPC. Our findings suggest that EGF-induced MPC requires ATP2B4-dependent Ca2+ dynamics.

OBJECTIVE: This study sought to elucidate the primary ATP-dependent mechanisms involved in clearing cytosolic Ca2+ in neurons and determine the predominant ATP-generating pathway-glycolysis or tricarboxylic acid cycle/oxidative phosphorylation (TCA/OxPhos)-associated with these mechanisms in hippocampal pyramidal neurons.
METHODS: Our investigation involved evaluating basal Ca2+ levels and analyzing the kinetic characteristics of evoked neuronal Ca2+ transients after selectively combined the inhibition/blockade of key ATP-dependent mechanisms with the suppression of either TCA/OxPhos or glycolytic ATP sources.
RESULTS: Our findings unveiled that the plasma membrane Ca2+ ATPase (PMCA) serves as the principal ATP-dependent mechanism for clearance cytosolic Ca2+ in hippocampal pyramidal neurons, both during rest and neuronal activity. Remarkably, during cellular activity, PMCA relies on ATP derived from glycolysis, challenging the traditional notion of neuronal reliance on TCA/OxPhos for ATP. Other mechanisms for Ca2+ clearance in pyramidal neurons, such as SERCA and NCX, appear to be dependent on TCA/OxPhos. Interestingly, at rest, the ATP required to fuel PMCA and SERCA, the two main mechanisms to keep resting Ca2+, seems to originate from a source other than glycolysis or the TCA/OxPhos.
CONCLUSIONS: These findings underscore the vital role of glycolysis in bolstering PMCA neuronal function to uphold Ca2+ homeostasis. Moreover, they elucidate the varying dependencies of cytoplasmic Ca2+ clearance mechanisms on distinct energy sources for their operation.

The plasma membrane Ca2+-ATPase (PMCA) is crucial for the fine tuning of intracellular calcium levels in eukaryotic cells. In this study, we show the presence of CARC sequences in all human and rat PMCA isoforms and we performed further analysis by molecular dynamics simulations. This analysis focuses on PMCA1, containing three CARC motifs, and PMCA4, with four CARC domains. In PMCA1, two CARC motifs reside within transmembrane domains, while the third is situated at the intracellular interface. The simulations depict more stable RMSD values and lower RMSF fluctuations in the presence of cholesterol, emphasizing its potential stabilizing effect. In PMCA4, a distinct dynamic was found. Notably, the total energy differences between simulations with cholesterol and phospholipids are pronounced in PMCA4 compared to PMCA1. RMSD values for PMCA4 indicate a more energetically favorable conformation in the presence of cholesterol, suggesting a robust interaction between CARCs and this lipid in the membranes. Furthermore, RMSF analysis for CARCs in both PMCA isoforms exhibit lower values in the presence of cholesterol compared to POPC alone. The analysis of H-bond occupancy and total energy values strongly suggests the potential interaction of CARCs with cholesterol. Given the crucial role of PMCAs in physiological calcium regulation and their involvement in diverse pathological processes, this study underscores the significance of CARC motifs and their interaction with cholesterol in elucidating PMCA function. These insights into the energetic preferences associated with CARC-cholesterol interactions offer valuable implications for understanding PMCA function in maintaining calcium homeostasis and addressing potential associated pathologies.

Epileptic seizures are seen as a result of changing excitability balance depending on the deterioration in synaptic plasticity in the brain. Neuroplastin, and its related molecules which are known to play a role in synaptic plasticity, neurotransmitter activities that provide balance of excitability and, different neurological diseases, have not been studied before in epilepsy. In this study, a total of 34 Sprague-Dawley male and female rats, 2 months old, weighing 250-300 g were used. The epilepsy model in rats was made via pentylenetetrazole (PTZ). After the completion of the experimental procedure, the brain tissue of the rats were taken and the histopathological changes in the hippocampus and cortex parts and the brain stem were investigated, as well as the immunoreactivity of the proteins related to the immunohistochemical methods. As a result of the histopathological evaluation, it was determined that neuron degeneration and the number of dilated blood vessels in the hippocampus, frontal cortex, and brain stem were higher in the PTZ status epilepticus (SE) groups than in the control groups. It was observed that neuroplastin and related proteins TNF receptor-associated factor 6 (TRAF6), Gamma amino butyric acid type A receptors [(GABA(A)], and plasma membrane Ca2+ ATPase (PMCA) protein immunoreactivity levels increased especially in the male hippocampus, and only AMPA receptor subunit type 1 (GluA1) immunoreactivity decreased, unlike other proteins. We believe this may be caused by a problem in the mechanisms regulating the interaction of neuroplastin and GluA1 and may cause problems in synaptic plasticity in the experimental epilepsy model. It may be useful to elucidate this mechanism and target GluA1 when determining treatment strategies.

Single-nucleotide polymorphisms (SNP) of ATP2B1 gene are associated with essential hypertension but their association with resistant hypertension (RHT) remains unexplored. The authors examined the relationship between ATP2B1 SNPs and RHT by genotyping 12 SNPs in ATP2B1 gene of 1124 Japanese individuals with lifestyle-related diseases. Patients with RHT had inadequate blood pressure (BP) control using three antihypertensive drugs or used ≥4 antihypertensive drugs. Patients with controlled hypertension had BP controlled using ≤3 antihypertensive drugs. The association between each SNP and RHT was analyzed by logistic regression. The final cohort had 888 (79.0%) and 43 (3.8%) patients with controlled hypertension and RHT, respectively. Compared with patients homozygous for the minor allele of each SNP in ATP2B1, a significantly higher number of patients carrying the major allele at 10 SNPs exhibited RHT (most significant at rs1401982: 5.8% vs. 0.8%, p = .014; least significant at rs11105378: 5.7% vs. 0.9%, p = .035; most nonsignificant at rs12817819: 5.1% vs. 10%, p = .413). After multivariate adjustment for age, sex, systolic BP, and other confounders, the association remained significant for rs2681472 and rs1401982 (OR: 7.60, p < .05 and OR: 7.62, p = .049, respectively). Additionally, rs2681472 and rs1401982 were in linkage disequilibrium with rs11105378. This study identified two ATP2B1 SNPs associated with RHT in the Japanese population. rs1401982 was most closely associated with RHT, and major allele carriers of rs1401982 required significantly more antihypertensive medications. Analysis of ATP2B1 SNPs in patients with hypertension can help in early prediction of RHT and identification of high-risk patients who are more likely to require more antihypertensive medications.

BACKGROUND: LncRNA ATP2B1-AS1 (ATP2B1-AS1) is involved in the occurrence and development of various diseases, while the relationship between lung adenocarcinoma (LUAD) and ATP2B1-AS1 is unclear. This study was to investigate the expression of ATP2B1-AS1 in LUAD and its influence on survival and prognosis of patients.
METHODS: LUAD tissue samples from patients participating in this study were collected, and the expression levels of ATP2B1-AS1 and miR-141-3p in LUAD sampleswere detected by real-time quantitative polymerase chain reaction (RT-qPCR). The effect of ATP2B1-AS1 on the growth of A549 cells was investigated through cell counting kit-8 (CCK-8) and transwell experiments. Besides, the prognostic value of ATP2B1-AS1 in LUAD was assessed via Kaplan-Meier curve and multivariate Cox regression.
RESULTS: ATP2B1-AS1 was downregulated in LUAD tissues and cells, whereas miR-141-3p was upregulated. After pcDNA3.1-ATP2B1-AS1 was transfected into A549 cells, the proliferation ability of A549 cells was decreased, and the migration level and invasion of A549 cells were also inhibited. High expression of ATP2B1-AS1 sponge miR-141-3p exerted prognostic value.
CONCLUSIONS: ATP2B1-AS1 sponge miR-141-3p alleviated the progression of LUAD, and ATP2B1-AS1 may be deemed as a prognostic marker for LUAD.

BACKGROUND: It is valuable to analyze the genome-wide association studies (GWAS) data for a complex disease phenotype in the context of the protein-protein interaction (PPI) network, as the related pathophysiology results from the function of interacting polyprotein pathways. The analysis may include the design and curation of a phenotype-specific GWAS meta-database incorporating genotypic and eQTL data linking to PPI and other biological datasets, and the development of systematic workflows for PPI network-based data integration toward protein and pathway prioritization. Here, we pursued this analysis for blood pressure (BP) regulation.
METHODS: The relational scheme of the implemented in Microsoft SQL Server BP-GWAS meta-database enabled the combined storage of: GWAS data and attributes mined from GWAS Catalog and the literature, Ensembl-defined SNP-transcript associations, and GTEx eQTL data. The BP-protein interactome was reconstructed from the PICKLE PPI meta-database, extending the GWAS-deduced network with the shortest paths connecting all GWAS-proteins into one component. The shortest-path intermediates were considered as BP-related. For protein prioritization, we combined a new integrated GWAS-based scoring scheme with two network-based criteria: one considering the protein role in the reconstructed by shortest-path (RbSP) interactome and one novel promoting the common neighbors of GWAS-prioritized proteins. Prioritized proteins were ranked by the number of satisfied criteria.
RESULTS: The meta-database includes 6687 variants linked with 1167 BP-associated protein-coding genes. The GWAS-deduced PPI network includes 1065 proteins, with 672 forming a connected component. The RbSP interactome contains 1443 additional, network-deduced proteins and indicated that essentially all BP-GWAS proteins are at most second neighbors. The prioritized BP-protein set was derived from the union of the most BP-significant by any of the GWAS-based or the network-based criteria. It included 335 proteins, with ~ 2/3 deduced from the BP PPI network extension and 126 prioritized by at least two criteria. ESR1 was the only protein satisfying all three criteria, followed in the top-10 by INSR, PTN11, CDK6, CSK, NOS3, SH2B3, ATP2B1, FES and FINC, satisfying two. Pathway analysis of the RbSP interactome revealed numerous bioprocesses, which are indeed functionally supported as BP-associated, extending our understanding about BP regulation.
CONCLUSIONS: The implemented workflow could be used for other multifactorial diseases.