Understanding the mechanisms of sodium (Na⁺) influx, effective compartmentalization, and efflux in higher plants is crucial to manipulate Na⁺ accumulation and assure the maintenance of low Na⁺ ...concentration in the cytosol and, hence, plant tolerance to salt stress. Na⁺ influx across the plasma membrane in the roots occur mainly via nonselective cation channels (NSCCs). Na⁺ is compartmentalized into vacuoles by Na⁺/H⁺ exchangers (NHXs). Na⁺ efflux from the plant roots is mediated by the activity of Na⁺/H⁺ antiporters catalyzed by the salt overly sensitive 1 (SOS1) protein. In animals, ouabain (OU)-sensitive Na⁺, K⁺-ATPase (a P-type ATPase) mediates sodium efflux. The evolution of P-type ATPases in higher plants does not exclude the possibility of sodium efflux mechanisms similar to the Na⁺, K⁺-ATPase-dependent mechanisms characteristic of animal cells. Using novel fluorescence imaging and spectrofluorometric methodologies, an OU-sensitive sodium efflux system has recently been reported to be physiologically active in roots. This review summarizes and analyzes the current knowledge on Na⁺ influx, compartmentalization, and efflux in higher plants in response to salt stress.
Phosphorylation is a widely used, reversible means of regulating enzymatic activity. Among the important phosphorylation targets are the Na
+,K
+- and H
+,K
+-ATPases that pump ions against their ...chemical gradients to uphold ionic concentration differences over the plasma membrane. The two pumps are very homologous, and at least one of the phosphorylation sites is conserved, namely a cAMP activated protein kinase (PKA) site, which is important for regulating pumping activity, either by changing the cellular distribution of the ATPases or by directly altering the kinetic properties as supported by electrophysiological results presented here. We further review the other proposed pump phosphorylations.
The molecular activity of Na,K-ATPase and other P2 ATPases like Ca2+-ATPase is influenced by the lipid environment via both general (physical) and specific (chemical) interactions. Whereas the ...general effects of bilayer structure on membrane protein function are fairly well described and understood, the importance of the specific interactions has only been realized within the last decade due particularly to the growing field of membrane protein crystallization, which has shed new light on the molecular details of specific lipid–protein interactions. It is a remarkable observation that specific lipid–protein interactions seem to be evolutionarily conserved, and conformations of specifically bound lipids at the lipid–protein surface within the membrane are similar in crystal structures determined with different techniques and sources of the protein, despite the rather weak lipid–protein interaction energy. Studies of purified detergent-soluble recombinant αβ or αβFXYD Na,K-ATPase complexes reveal three separate functional effects of phospholipids and cholesterol with characteristic structural selectivity. The observations suggest that these three effects are exerted at separate binding sites for phophatidylserine/cholesterol (stabilizing), polyunsaturated phosphatidylethanolamine (stimulatory), and saturated PC or sphingomyelin/cholesterol (inhibitory), which may be located within three lipid-binding pockets identified in recent crystal structures of Na,K-ATPase. The findings point to a central role of direct and specific interactions of different phospholipids and cholesterol in determining both stability and molecular activity of Na,K-ATPase and possible implications for physiological regulation by membrane lipid composition. This article is part of a special issue titled “Lipid–Protein Interactions.”
•General and specific interactions of lipids and Na,K-ATPase.•Specific lipid-binding sites in Na,K-ATPase E1 and E2 crystal structures.•Functional effects of specifically bound phospholipids and cholesterol on Na,K-ATPase activity.•Stimulatory, inhibitory and stabilizing effects of specifically bound lipids on Na,K-ATPase.•Bulk, annular and specifically bound lipids in Na,K-ATPase.
Cadmium (Cd) is an extremely toxic metal, capable of severely damaging several organs, including the brain. Studies have shown that Cd disrupts intracellular free calcium (Ca(2+)i) homeostasis, ...leading to apoptosis in a variety of cells including primary murine neurons. Calcium is a ubiquitous intracellular ion which acts as a signaling mediator in numerous cellular processes including cell proliferation, differentiation, and survival/death. However, little is known about the role of calcium signaling in Cd-induced apoptosis in neuronal cells. Thus we investigated the role of calcium signaling in Cd-induced apoptosis in primary rat cerebral cortical neurons. Consistent with known toxic properties of Cd, exposure of cerebral cortical neurons to Cd caused morphological changes indicative of apoptosis and cell death. It also induced elevation of Ca(2+)i and inhibition of Na(+)/K(+)-ATPase and Ca(2+)/Mg(2+)-ATPase activities. This Cd-induced elevation of Ca(2+)i was suppressed by an IP3R inhibitor, 2-APB, suggesting that ER-regulated Ca(2+) is involved. In addition, we observed elevation of reactive oxygen species (ROS) levels, dysfunction of cytochrome oxidase subunits (COX-I/II/III), depletion of mitochondrial membrane potential (ΔΨm), and cleavage of caspase-9, caspase-3 and poly (ADP-ribose) polymerase (PARP) during Cd exposure. Z-VAD-fmk, a pan caspase inhibitor, partially prevented Cd-induced apoptosis and cell death. Interestingly, apoptosis, cell death and these cellular events induced by Cd were blocked by BAPTA-AM, a specific intracellular Ca(2+) chelator. Furthermore, western blot analysis revealed an up-regulated expression of Bcl-2 and down-regulated expression of Bax. However, these were not blocked by BAPTA-AM. Thus Cd toxicity is in part due to its disruption of intracellular Ca(2+) homeostasis, by compromising ATPases activities and ER-regulated Ca(2+), and this elevation in Ca(2+) triggers the activation of the Ca(2+)-mitochondria apoptotic signaling pathway. This study clarifies the signaling events underlying Cd neurotoxicity, and suggests that regulation of Cd-disrupted Ca(2+)i homeostasis may be a new strategy for prevention of Cd-induced neurodegenerative diseases.
The plasma membrane H+‐ATPase of fungi and plants is a single polypeptide of fewer than 1,000 residues that extrudes protons from the cell against a large electric and concentration gradient. The ...minimalist structure of this nanomachine is in stark contrast to that of the large multi‐subunit FOF1 ATPase of mitochondria, which is also a proton pump, but under physiological conditions runs in the reverse direction to act as an ATP synthase. The plasma membrane H+‐ATPase is a P‐type ATPase, defined by having an obligatory phosphorylated reaction cycle intermediate, like cation pumps of animal membranes, and thus, this pump has a completely different mechanism to that of FOF1 ATPases, which operates by rotary catalysis. The work that led to these insights in plasma membrane H+‐ATPases of fungi and plants has a long history, which is briefly summarized in this review.
The effect of boron (B) deficiency on mediating the contribution of H+-ATPase in the uptake and assimilation of exogenous cyanide (CN−) is investigated. Under CN− treatments, rice seedlings with ...B-deficient (-B) conditions exhibited significantly higher CN− uptake and assimilation rates than B-supplemented (+B) seedlings, whereas NH4+ uptake and assimilation rates were slightly higher in -B rice seedlings than in +B. In this connection, the expression pattern of genes encoding β-CAS, ST, and H+-ATPase was assessed to unravel their role in the current scenario. The abundances of three β-CAS isogenes (OsCYS-D1, OsCYS-D2, and OsCYS–C1) in rice tissues are upregulated from both “CN−-B” and “CN−+B” treatments, however, only OsCYS–C1 in roots from the “CN−-B” treatments was significantly upregulated than “CN−+B” treatments. Expression patterns of ST-related genes (OsStr9, OsStr22, and OsStr23) are tissue specific, in which significantly higher upregulation of ST-related genes was observed in shoots from “CN−-B” treatments than “CN−+B” treatments. Expression pattern of 7 selected H+-ATPase isogenes, OsA1, OSA2, OsA3, OsA4, OsA7, OsA8, and OsA9 are quite tissue specific between “CN−+B” and “CN−-B” treatments. Among these, OsA4 and OsA7 genes were highly activated in the uptake and assimilation of exogenous CN− in -B nutrient solution. These results indicated that B deficiency disturbs the pattern of N cycles in CN−-treated rice seedlings, where activation of ST during CN− assimilation decreases the flux of the innate pool of NH4+ produced from CN− assimilation by the β-CAS pathway in plants. Collectively, the B deficiency increased the uptake and assimilation of exogenous CN− through activating H+-ATPase.
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•B deficiency increased the uptake and assimilation of CN− by rice seedlings.•β-CAS and ST pathways are highly activated in –B rice seedlings than in +B rice seedlings.•Higher activation of ST in –B rice seedlings decreases the innate flux of NH4+ produced from the β-CAS pathway.•B deficiency disturbs the pattern of N nutritional cycles in CN−-treated rice seedlings.
P-type ATPases are found in all kingdoms of life and constitute a wide range of cation transporters, primarily for H
+
, Na
+
, K
+
, Ca
2+
, and transition metal ions such as Cu(I), Zn(II), and ...Cd(II). They have been studied through a wide range of techniques, and research has gained very significant insight on their transport mechanism and regulation. Here, we review the structure, function, and dynamics of P2-ATPases including Ca
2+
-ATPases and Na,K-ATPase. We highlight mechanisms of functional transitions that are associated with ion exchange on either side of the membrane and how the functional cycle is regulated by interaction partners, autoregulatory domains, and off-cycle states. Finally, we discuss future perspectives based on emerging techniques and insights.
The vacuolar (H+)-ATPases (V-ATPases) are ATP-dependent proton pumps that acidify intracellular compartments and are also present at the plasma membrane. They function in such processes as membrane ...traffic, protein degradation, virus and toxin entry, bone resorption, pH homeostasis, and tumor cell invasion. V-ATPases are large multisubunit complexes, composed of an ATP-hydrolytic domain (V1) and a proton translocation domain (V0), and operate by a rotary mechanism. This review focuses on recent insights into their structure and mechanism, the mechanisms that regulate V-ATPase activity (particularly regulated assembly and trafficking), and the role of V-ATPases in processes such as cell signaling and cancer. These developments have highlighted the potential of V-ATPases as a therapeutic target in a variety of human diseases.
Recent structural studies of the V-ATPase have identified several novel features, including the presence of highly tilted α-helices in subunit a of the integral V0 domain that participate in proton translocation across the membrane.
Regulated assembly of the V-ATPase from its peripheral V1 and integral V0 domains represents an important mechanism of controlling its activity in vivo and has been shown to be under control of various signaling pathways, including protein kinase A (PKA) and PI-3 kinase.
The V-ATPase has been found to function in a variety of cellular signaling pathways, including the mechanistic target of rapamycin complex 1 (mTORC1), AMP-activated protein kinase (AMPK), Wnt, and Notch.
The V-ATPase is emerging as important for cancer cell survival and invasion, making it a potential target in the development of anticancer drugs.
Nanocarriers with positive surface charges are known for their toxicity which has limited their clinical appli- cations. The mechanism underlying their toxicity, such as the induction of inflammatory ...response, remains largely unknown. In the present study we found that injection of cationic nanocarriers, including cationic liposomes, PEI, and chitosan, led to the rapid appearance of necrotic cells. Cell necrosis induced by cationic nanocarriers is dependent on their positive surface charges, but does not require RIP1 and Mlkl. Instead, intracellular Na^＋ overload was found to accompany the cell death. Depletion of Na^＋ in culture medium or pretreatment of cells with the Na^＋/K^＋- ATPase cation-binding site inhibitor ouabain, protected cells from cell necrosis. Moreover, treatment with cationic nanocarriers inhibited Na^＋/K^＋-ATPase activity both in vitro and in vivo. The computational simulation showed that cationic carriers could interact with cation-binding site of Na^＋/K^＋-ATPase. Mice pretreated with a small dose of ouabain showed improved survival after injection of a lethal dose of cationic nanocarriers. Further analyses suggest that cell necrosis induced by cationic nanocarriers and the resulting leakage of mitochondrial DNA could trigger severe inflammation in vivo, which is mediated by a pathway involving TLR9 and MyD88 signaling. Taken together, our results reveal a novel mechanism whereby cationic nanocarriers induce acute cell necrosis through the interaction with Na^＋/K^＋-ATPase, with the subsequent exposure of mitochondrial damage-associated molecular patterns as a key event that mediates the inflammatory responses. Our study has important implications for evaluating the biocompatibility of nanocarriers and designing better and safer ones for drug delivery.
Na,K‐ATPase is a ubiquitous molecule contributing to the asymmetrical distribution of Na+ and K+ ions across the plasma membrane and maintenance of the membrane potential, a prerequisite of neuronal ...activity. Na,K‐ATPase comprises three subunits (α, β, and FXYD). The α subunit has four isoforms in mice, with three of them (α1, α2, and α3) expressed in the brain. However, the functional and biological significances of the different brain isoforms remain to be fully elucidated. Recent studies have revealed the association of Atp1a3, a gene encoding α3 subunit, with neurological disorders. To map the cellular distributions of the α subunit isoforms and their coexpression patterns, we evaluated the mRNA expression of Atp1a1, Atp1a2, and Atp1a3 by in situ hybridization in the mouse brain. Atp1a1 and Atp1a3 were expressed in neurons, whereas Atp1a2 was almost exclusively expressed in glial cells. Most neurons coexpressed Atp1a1 and Atp1a3, with highly heterogeneous expression levels across the brain regions and neuronal subtypes. We identified parvalbumin (PV)‐expressing GABAergic neurons in the hippocampus, somatosensory cortex, and retrosplenial cortex as an example of a neuronal subtype expressing low Atp1a1 and high Atp1a3. The expression of Atp1b isoforms was also heterogeneous across brain regions and cellular subtypes. The PV‐expressing neurons expressed a high level of Atp1b1 and a low level of Atp1b2 and Atp1b3. These findings provide basic information on the region‐ and neuronal‐subtype‐dependent expression of Na,K‐ATPase α and β subunit isoforms, as well as a rationale for the selective involvement of neurons expressing high levels of Atp1a3 in neurological disorders.
Na,K‐ATPase comprises α (Atp1a), β (Atp1b), and FXYD subunits, and each subunit has isoforms. In situ hybridization for α and β subunit isoforms revealed regional‐ and neuronal‐subtype‐specific expression of the isoforms in the mouse brain. The photograph shows Atp1a1 (red) and Atp1a3 (green) expression in the hippocampal dentate gyrus (stained with DAPI, blue).
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