Regulation of Epidermal Growth Factor Receptor Signaling by Endocytosis and Intracellular Trafficking

Ligand activation of the epidermal growth factor receptor (EGFR) leads to its rapid internalization and eventual delivery to lysosomes. This process is thought to be a mechanism to attenuate signaling, but signals could potentially be generated after endocytosis. To directly evaluate EGFR signaling during receptor trafficking, we developed a technique to rapidly and selectively isolate internalized EGFR and associated molecules with the use of reversibly biotinylated anti-EGFR antibodies. In addition, we developed antibodies specific to tyrosine-phosphorylated EGFR. With the use of a combination of fluorescence imaging and affinity precipitation approaches, we evaluated the state of EGFR activation and substrate association during trafficking in epithelial cells. We found that after internalization, EGFR remained active in the early endosomes. However, receptors were inactivated before degradation, apparently due to ligand removal from endosomes. Adapter molecules, such as Shc, were associated with EGFR both at the cell surface and within endosomes. Some molecules, such as Grb2, were primarily found associated with surface EGFR, whereas others, such as Eps8, were found only with intracellular receptors. During the inactivation phase, c-Cbl became EGFR associated, consistent with its postulated role in receptor attenuation. We conclude that the association of the EGFR with different proteins is compartment specific. In addition, ligand loss is the proximal cause of EGFR inactivation. Thus, regulated trafficking could potentially influence the pattern as well as the duration of signal transduction.     

细胞内胆固醇水平调节研究进展

细胞内胆固醇水平动态平衡是细胞发挥生理功能的重要保障.破坏细胞内胆固醇水平动态平衡不仅增加心血管系统疾病患病风险,而且与许多代谢性疾病相关.细胞内胆固醇水平主要受胆固醇生物合成、摄取、流出和酯化的调节.3-羟基-3-甲基-戊二酰基辅酶A还原酶、角鲨烯单加氧酶和固醇调节元件结合蛋白2是胆固醇合成关键因子.尼曼-匹克C1型...     

Regulation of alpha1 Na/K-ATPase expression by cholesterol

We have reported that α1 Na/K-ATPase regulates the trafficking of caveolin-1 and consequently alters cholesterol distribution in the plasma membrane. Here, we report the reciprocal regulation of α1 Na/K-ATPase by cholesterol. Acute exposure of LLC-PK1 cells to methyl β-cyclodextrin led to parallel decreases in cellular cholesterol and the expression of α1 Na/K-ATPase. Cholesterol repletion fully reversed the effect of methyl β-cyclodextrin. Moreover, inhibition of intracellular cholesterol trafficking to the plasma membrane by compound U18666A had the same effect on α1 Na/K-ATPase. Similarly, the expression of α1, but not α2 and α3, Na/K-ATPase was significantly reduced in the target organs of Niemann-Pick type C mice where the intracellular cholesterol trafficking is blocked. Mechanistically, decreases in the plasma membrane cholesterol activated Src kinase and stimulated the endocytosis and degradation of α1 Na/K-ATPase through Src- and ubiquitination-dependent pathways. Thus, the new findings, taken together with what we have already reported, revealed a previously unrecognized feed-forward mechanism by which cells can utilize the Src-dependent interplay among Na/K-ATPase, caveolin-1, and cholesterol to effectively alter the structure and function of the plasma membrane.     

Regulation of caveolin-1 membrane trafficking by the Na/K-ATPase

Here, we show that the Na/K-ATPase interacts with caveolin-1 (Cav1) and regulates Cav1 trafficking. Graded knockdown of Na/K-ATPase decreases the plasma membrane pool of Cav1, which results in a significant reduction in the number of caveolae on the cell surface. These effects are independent of the pumping function of Na/K-ATPase, and instead depend on interaction between Na/K-ATPase and Cav1 mediated by an N-terminal caveolin-binding motif within the ATPase α1 subunit. Moreover, knockdown of the Na/K-ATPase increases basal levels of active Src and stimulates endocytosis of Cav1 from the plasma membrane. Microtubule-dependent long-range directional trafficking in Na/K-ATPase–depleted cells results in perinuclear accumulation of Cav1-positive vesicles. Finally, Na/K-ATPase knockdown has no effect on processing or exit of Cav1 from the Golgi. Thus, the Na/K-ATPase regulates Cav1 endocytic trafficking and stabilizes the Cav1 plasma membrane pool.     

Isoform Specificity of the Na/K-ATPase Association and Regulation by Phospholemman

Phospholemman (PLM) phosphorylation mediates enhanced Na/K-ATPase (NKA) function during adrenergic stimulation of the heart. Multiple NKA isoforms exist, and their function/regulation may differ. We combined fluorescence resonance energy transfer (FRET) and functional measurements to investigate isoform specificity of the NKA-PLM interaction. FRET was measured as the increase in the donor fluorescence (CFP-NKA-α1 or CFP-NKA-α2) during progressive acceptor (PLM-YFP) photobleach in HEK-293 cells. Both pairs exhibited robust FRET (maximum of 23.6 ± 3.4% for NKA-α1 and 27.5 ± 2.5% for NKA-α2). Donor fluorescence depended linearly on acceptor fluorescence, indicating a 1:1 PLM:NKA stoichiometry for both isoforms. PLM phosphorylation induced by cAMP-dependent protein kinase and protein kinase C activation drastically reduced the FRET with both NKA isoforms. However, submaximal cAMP-dependent protein kinase activation had less effect on PLM-NKA-α2 versus PLM-NKA-α1. Surprisingly, ouabain virtually abolished NKA-PLM FRET but only partially reduced co-immunoprecipitation. PLM-CFP also showed FRET to PLM-YFP, but the relationship during progressive photobleach was highly nonlinear, indicating oligomers involving ≥3 monomers. Using cardiac myocytes from wild-type mice and mice where NKA-α1 is ouabain-sensitive and NKA-α2 is ouabain-resistant, we assessed the effects of PLM phosphorylation on NKA-α1 and NKA-α2 function. Isoproterenol enhanced internal Na⁺ affinity of both isoforms (K_½ decreased from 18.1 ± 2.0 to 11.5 ± 1.9 mm for NKA-α1 and from 16.4 ± 2.5 to 10.4 ± 1.5 mm for NKA-α2) without altering maximum transport rate (V_max). Protein kinase C activation also decreased K_½ for both NKA-α1 and NKA-α2 (to 9.4 ± 1.0 and 9.1 ± 1.1 mm, respectively) but increased V_max only for NKA-α2 (1.9 ± 0.4 versus 1.2 ± 0.5 mm/min). In conclusion, PLM associates with and modulates both NKA-α1 and NKA-α2 in a comparable but not identical manner.Cardiac Na/K-ATPase (NKA)³ regulates intracellular Na⁺, which in turn affects intracellular Ca²⁺ and contractility via Na⁺/Ca²⁺ exchange. Members of the FXYD family of small, single membrane-spanning proteins, including phospholemman (PLM) and the NKA γ-subunit (1), have emerged recently as tissue-specific regulators of NKA. PLM is the only FXYD protein known to be highly expressed in cardiac myocytes and is also unique within the family in that it is phosphorylated at two or more sites by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) (2, 3). In the heart, PLM is a major phosphorylation target for both PKA and PKC.Co-immunoprecipitation experiments have demonstrated that PLM is physically associated with NKA (4–8), and this is not affected by PLM phosphorylation (6, 7). We have shown recently (9) that PLM and NKA are in very close proximity, such that fluorescence resonance energy transfer (FRET) occurs. PLM phosphorylation by either PKA or PKC reduces the FRET significantly, suggesting that although PLM and NKA are not physically dissociated upon phosphorylation, their interaction is altered. PLM inhibits NKA (4, 8, 10, 11), mostly by reducing the affinity of the pump for internal Na⁺. PLM phosphorylation relieves this inhibition and thus mediates the enhancement of NKA function by α- and β-adrenergic stimulation in mouse ventricular myocytes (10, 11).There are multiple NKA isoforms in cardiac myocytes. NKA-α1 is the dominant, ubiquitous isoform, whereas NKA-α2 and NKA-α3 are present in relatively small amounts and in a species-dependent manner (12). For instance, the adult rodent heart expresses NKA-α1 and NKA-α2, although dogs and monkeys do not have the NKA-α2 subunit (13). In humans all three NKA-α isoforms can be detected (14). It has been suggested that NKA-α2 and NKA-α3 are located mainly in the T-tubules, at the junctions with the sarcoplasmic reticulum, where they could regulate local Na⁺/Ca²⁺ exchange and thus cardiac myocyte Ca²⁺. There is rather convincing evidence supporting such a model in the smooth muscle (15). However, things are less clear in the heart. The functional density of NKA-α2 is significantly higher in the T-tubules (versus external sarcolemma) in cardiac myocytes from both rats (16, 17) and mice (18), but their precise localization with respect to the junctions with the sarcoplasmic reticulum is not known. Based on Ca²⁺ transients from heterozygous NKA-α1^+/− and NKA-α2^+/− mice, James et al. (19) concluded that NKA-α2 is involved in cardiac myocyte Ca²⁺ regulation, whereas NKA-α1 is not. Further support for this idea came from the observation that replacing mouse NKA-α2 with a low affinity mutant leads to a loss of glycoside inotropy (20), and increased expression of NKA-α2 decreased the Na⁺/Ca²⁺ exchange current and Ca²⁺ transients (21). However, other findings challenge the preferential role of NKA-α2 in regulating intracellular Ca²⁺ and contractility. Moseley et al. (22) showed that NKA-α1^+/− mice were severely compromised, and Dostanic et al. (23) showed that NKA-α1 is also physically and functionally associated with the Na⁺/Ca²⁺ exchanger.In this context, it is important to determine whether NKA-α1 and NKA-α2 interact differently with PLM. The data available so far on this are contradictory. We have found (7) that NKA-α1, NKA-α2, and NKA-α3 isoforms co-immunoprecipitate PLM, both unphosphorylated and phosphorylated, in rabbit heart. In contrast, Silverman et al. (8) reported that NKA-α1 but not NKA-α2 co-immunoprecipitate with PLM in ventricular myocytes from guinea pig. The functional data are also contradictory. PLM was found to reduce the affinity for Na⁺ of both NKA-α1 and NKA-α2 isoforms in a heterologous expression system (4), whereas Silverman et al. (8) reported that forskolin-induced PLM phosphorylation results in a higher NKA-α1-mediated current and no change in the current generated by NKA-α2.Here we used two methods to investigate whether the interaction and functional effects of PLM on NKA are NKA-α isoform-specific. First, we used FRET to assess the interaction between PLM-YFP and CFP-NKA-α1/CFP-NKA-α2 transfected in HEK-293 cells and how PLM phosphorylation by PKA and PKC affects this interaction. Second, we measured NKA function in myocytes isolated from wild-type (WT) mice and mice where NKA isoforms have swapped ouabain affinities (SWAP; NKA-α1 is ouabain-sensitive, whereas NKA-α2 is ouabain-resistant) (23). In this way we could test the effect of β-adrenergic stimulation separately on NKA-α1 and NKA-α2 isoforms in the native myocyte environment, as an indicator of the functional interaction with PLM. Our results indicate that NKA-α1 and NKA-α2 interact similarly with PLM, and this interaction is equally affected by PLM phosphorylation.     

Formation of Two Intramolecular Disulfide Bonds Is Necessary for ApoA-I-dependent Cholesterol Efflux Mediated by ABCA1

ABCA1 plays a major role in cholesterol homeostasis and high density lipoprotein (HDL) metabolism. ABCA1 contains disulfide bond(s) between its N- and C-terminal halves, but it remains unclear whether disulfide bond formation is important for the functions of ABCA1 and which cysteines are involved in disulfide bond formation. To answer these questions, we constructed >30 ABCA1 mutants in which 16 extracellular domain (ECD) cysteines were replaced with serines and examined disulfide bond formation, apoA-I binding, and HDL formation in these mutants. From the single cysteine replacements, two cysteines (Cys⁷⁵ and Cys³⁰⁹) in ECD1 were found to be essential for apoA-I binding. In contrast, in ECD2, only Cys¹⁴⁷⁷ was found to be essential for HDL formation, and no single cysteine replacement impaired apoA-I binding. The concurrent replacement of two cysteines, Cys¹⁴⁶³ and Cys¹⁴⁶⁵, impaired apoA-I binding and HDL formation, suggesting that four of five extracellular cysteines (Cys⁷⁵, Cys³⁰⁹, Cys¹⁴⁶³, Cys¹⁴⁶⁵, and Cys¹⁴⁷⁷) are involved in these functions of ABCA1. Trypsin digestion experiments suggested that one disulfide bond is not sufficient and that two intramolecular disulfide bonds (between Cys⁷⁵ and Cys³⁰⁹ in ECD1 and either Cys¹⁴⁶³ or Cys¹⁴⁶⁵ and Cys¹⁴⁷⁷ in ECD2) are required for ABCA1 to be fully functional.Maintenance of cellular cholesterol homeostasis is important for normal human physiology; its disruption can lead to a variety of pathological conditions, including cardiovascular disease (1). ABCA1 (ATP-binding cassette protein A1), a key factor in cholesterol homeostasis, mediates the secretion of cellular free cholesterol and phospholipids to an extracellular acceptor, apoA-I, to form high density lipoprotein (HDL)² (2, 3). HDL formation is the only known pathway for the elimination of excess cholesterol from peripheral cells. Defects in ABCA1 cause Tangier disease (4–6), in which patients have a near absence of circulating HDL, prominent cholesterol ester accumulation in tissue macrophages, and premature atherosclerotic vascular disease (1, 7).ABCA1 is a member of the ABCA subclass of ABC transporters, which contain the basic architecture of the “full-length” ABC transporters organized into two tandemly arranged halves. Each half contains several transmembrane α-helices (TMs), which provide a translocation pathway, followed by a cytoplasmic nucleotide-binding domain, which hydrolyze ATP. In the case of “half-size” ABC transporters, such as ABCG1, ABCD1, TAP1/TAP2 (transporter associated with antigen processing), and the bacterial homolog Sav1866, they dimerize to form the full transporter. Crystallographic analysis of the bacterial homolog Sav1866 revealed that the TMs of one subunit are closely related to the TMs of the other subunit, forming two “wings” in an outward-facing conformation (8).When ABCA1 is partially digested by trypsin, ABCA1 is cleaved at site A, just C-terminal to TM6, and at site B, just N-terminal to TM7, to produce four fragments of 170 and 150 kDa and subsequently of 125 and 110 kDa (Fig. 1A) (9). When these fragments are analyzed by SDS-PAGE under nonreducing conditions, they co-migrate with undigested ABCA1. These results suggest that the N- and C-terminal halves of ABCA1 are connected by disulfide bond(s), as reported for ABCA4 (ABCR) (10). The ABCA subclass is distinguished from other ABC transporter subclasses by the presence of large extracellular domains (ECDs) (Fig. 1A) (11, 12). ECD1 and ECD2 of ABCA1 contain nine and five cysteine residues, respectively, and each connecting loop between TM5 and TM6 and between TM11 and TM12 contains a cysteine residue. These cysteine residues were assigned numbers, C1 to C16, based on their distance from the N terminus (Fig. 1A). These cysteine residues are well conserved among ABCA1, ABCA4, and ABCA7 (Fig. 1B). All of the cysteine residues in ECD1 are conserved between ABCA1 and ABCA4, and seven cysteine residues (except C4 and C5) are conserved in ABCA7. All five of the cysteine residues in ECD2 are conserved between ABCA1 and ABCA4, and three cysteine residues (except C11 and C12) are conserved in ABCA7. Because ABCA7, like ABCA1, mediates apoA-I-dependent lipid efflux (13, 14), conserved cysteine residues might be important for its function. Indeed, the Tangier disease mutation C1477R has been reported to abolish apoA-I binding and HDL formation (15–17), and several missense mutations in cysteine residues within ECD1 (C54Y, C75G) and ECD2 (C1488R, C1490Y) of ABCA4 have been linked to Stargardt disease (18–21). It remains unclear, however, whether disulfide bond formation is important for the proper folding and/or the functions of ABCA subclass proteins.Open in a separate windowFIGURE 1.Structural features of ABCA1. A, topological model for human ABCA1. ABCA1 consists of 12 transmembrane α-helices (TM1–TM12) and two large ECDs. ECD1 and ECD2 contain nine and five cysteine residues, respectively, and each connecting loop between TM5 and TM6 and between TM11 and TM12 contains a cysteine residue. These cysteine residues were assigned numbers, from C1 to C16, based on their distance from the N terminus. ABCA1 is cleaved at sites A and B by limited trypsin digestion. B, amino acid sequence alignment of ECDs of human ABCA1, ABCA4, and ABCA7. Conserved cysteine residues are indicated in black boxes.In this study, we analyzed which cysteine residues are involved in disulfide bond formation and examined whether disulfide bond formation is necessary for the functions of ABCA1. Cysteine substitution experiments suggested that two disulfide bonds are formed between C2 and C6 in ECD1 and between either C13 or C14 and C15 in ECD2 and that this two-disulfide bond formation is necessary for apoA-I-dependent cholesterol efflux by ABCA1.     

Regulation of Na,K-ATPase function in the lens

The two cell types in the lens, epithelium and fiber, have a very different specific activity of Na,K-ATPase; activity is much higher in the epithelium. However, judged by Western blot, fibers and epithelium express a similar amount of both Na,K-ATPase alpha and beta subunit proteins. Na,K-ATPase protein abundance does not tally with Na,K-ATPase activity. Studies were conducted to examine whether protein synthesis plays a role in maintenance of the high Na,K-ATPase activity in lens epithelium. An increase of cytoplasmic sodium was found to increase Na,K-ATPase protein expression in the epithelium, but not in the fibers. The findings illustrate the ability of lens epithelium to synthesize new Na,K-ATPase protein as a way to boost Na,K-ATPase in response to cell damage or pathological events. Methionine incorporation studies suggested Na,K-ATPase synthesis may also play a role in day to day preservation of high Na,K-ATPase activity. Na,K-ATPase protein in lens epithelial cells appeared to be continually synthesized and degraded. Experiments with cycloheximide suggest that specific activity of Na,K-ATPase in the lens epithelium may depend on the ability of the cells to continuously synthesize fresh Na,K-ATPase proteins. However, other factors such as phosphorylation of Na,K-ATPase alpha subunit may also influence Na,K-ATPase activity. When intact lenses were exposed to the agonist thrombin, Na,K-ATPase activity was diminished, but the response was suppressed by inhibitors of the Src family of non-receptor tyrosine kinases. Thrombin elicited tyrosine phosphorylation of lens epithelium membrane proteins, including a 100 kDa protein band thought to be the Na,K-ATPase alpha 1 subunit. It remains to be determined whether a tyrosine phosphorylation mechanism contributes to the low activity of Na,K-ATPase in lens fibers.     

Regulation of Na,K-ATPase during acute lung injury

A hallmark of acute lung injury is the accumulation of a protein rich edema which impairs gas exchange and leads to hypoxemia. The resolution of lung edema is effected by active sodium transport, mostly contributed by apical Na⁺ channels and the basolateral located Na,K-ATPase. It has been reported that the decrease of Na,K-ATPase function seen during lung injury is due to its endocytosis from the cell plasma membrane into intracellular pools. In alveolar epithelial cells exposed to severe hypoxia, we have reported that increased production of mitochondrial reactive oxygen species leads to Na,K-ATPase endocytosis and degradation. We found that this regulated process follows what is referred as the Phosphorylation–Ubiquitination–Recognition–Endocytosis–Degradation (PURED) pathway. Cells exposed to hypoxia generate reactive oxygen species which activate PKCζ which in turn phosphorylates the Na,K-ATPase at the Ser18 residue in the N-terminus of the α1-subunit leading the ubiquitination of any of the four lysines (K16, K17, K19, K20) adjacent to the Ser18 residue. This process promotes the α1-subunit recognition by the μ2 subunit of the adaptor protein-2 and its endocytosis trough a clathrin dependent mechanism. Finally, the ubiquitinated Na,K-ATPase undergoes degradation via a lysosome/proteasome dependent mechanism.     

Oxidative Resistance of Na/K-ATPase

1. Oxidative modification of Na/K-ATPase from brain and kidney has been studied. Brain enzyme has been found to be more sensitive than kidney enzyme to inhibition by both H₂O₂ and NaOCl.2. The inhibition of Na/K-ATPase correlates well with the decrease in a number of SH groups, suggesting that the latter belong mainly to ATPase protein and are essential for the enzyme activity. We suggest that the differences in the number, location, and accessibility of SH groups in Na/K-ATPase isozymes predict their oxidative stability.3. The hydrophilic natural antioxidant carnosine, the hydrophobic natural antioxidant -tocopherol, and the synthetic antioxidant ionol as well as the ferrous ion chelating agent deferoxamine were found to protect Na/K-ATPase from oxidation by different concentrations of H₂O₂. The data suggest that these antioxidants are effective due to their ability to neutralize or to prevent formation of hydroxyl radicals.     

Regulation of Tomato Fruit Polygalacturonase mRNA Accumulation by Ethylene: A Re-Examination

Polygalacturonase (PG) is the major enzyme responsible for pectin disassembly in ripening fruit. Despite extensive research on the factors regulating PG gene expression in fruit, there is conflicting evidence regarding the role of ethylene in mediating its expression. Transgenic tomato (Lycopersicon esculentum) fruits in which endogenous ethylene production was suppressed by the expression of an antisense 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene were used to re-examine the role of ethylene in regulating the accumulation of PG mRNA, enzyme activity, and protein during fruit ripening. Treatment of transgenic antisense ACC synthase mature green fruit with ethylene at concentrations as low as 0.1 to 1 μL/L for 24 h induced PG mRNA accumulation, and this accumulation was higher at concentrations of ethylene up to 100 μL/L. Neither PG enzyme activity nor PG protein accumulated during this 24-h period of ethylene treatment, indicating that translation lags at least 24 h behind the accumulation of PG mRNA, even at high ethylene concentrations. When examined at concentrations of 10 μL/L, PG mRNA accumulated within 6 h of ethylene treatment, indicating that the PG gene responds rapidly to ethylene. Treatment of transgenic tomato fruit with a low level of ethylene (0.1 μL/L) for up to 6 d induced levels of PG mRNA, enzyme activity, and protein after 6 d, which were comparable to levels observed in ripening wild-type fruit. A similar level of internal ethylene (0.15 μL/L) was measured in transgenic antisense ACC synthase fruit that were held for 28 d after harvest. In these fruit PG mRNA, enzyme activity, and protein were detected. Collectively, these results suggest that PG mRNA accumulation is ethylene regulated, and that the low threshold levels of ethylene required to promote PG mRNA accumulation may be exceeded, even in transgenic antisense ACC synthase tomato fruit.     

Regulation of Na,K-ATPase by synaptosomal factors and neurotransmitters

The identical increase of Na, K-ATPase activity is caused by oxidated and reduced forms of noradrenaline, serotonin and dopamine through the synaptosomal activating factors. The synaptosomal inhibiting factor, orthovanadate and calcium ions independently inhibit Na, K-ATPase activity. The inhibition constant (Ki) for vanadate does not change in the presence of EDTA, whereas in the presence of synaptosomal factors regulating the Na, K-ATPase factors, noradrenaline causes drastic increase of Ki for vanadate. It has been concluded, that the data point to the existence of special regulating system of brain synaptosomal Na, K-ATPase.     

Regulation of Na,K-ATPase biosynthesis in developing Artemia salina

Regulation of the biosynthesis of the sodium- and potassium-activated adenosine triphosphatase (Na,K-ATPase) (EC 3.6.1.3) was studied in the developing brine shrimp, Artemia salina. Measurement of levels of the subunits of the Na,K-ATPase by radioimmunoassay indicated the presence of both alpha and beta subunits in undeveloped cysts and developing embryos prior to the appearance of enzymatic activity. The quantity of each subunit increased dramatically between 8 and 24 h of development and then reached a plateau at about 32 h. The quantities of translationally active mRNA alpha and mRNA beta were also determined. Undeveloped cysts contained mRNA alpha and mRNA beta, and the amounts increased 9- and 3-fold, respectively, during the first 24 h of development. The data suggest that the increase in Na,K-ATPase activity was at least in part due to increases in protein synthesis related to changes in mRNA levels. The data also suggest involvement of additional regulatory mechanisms. The alpha-subunit has been detected as two molecular weight forms (alpha 1 and alpha 2) which demonstrate changes in relative amounts during development (Peterson, G. L., Churchill, L., Fisher, J. A., and Hokin, L. E. (1982) J. Exp. Zool. 221, 295-308). We show here that this was not due to changes in mRNA alpha 1 and mRNA alpha 2.     

Regulation of Na, K-ATPase activity by monovalent cations]

A deviation from optimal conditions of the Na, K-ATPase reaction results in a drastic change in the plot: enzyme activity versus Na/K ratio. Acidification of the medium and a decrease in Mg2+ concentration and temperature results in two peaks on the curve at Na/K ratio of about 1 and at Na/K ratio greater than 4. The enhancement of pH of the medium and increase in Mg2+ concentration decreases the first peak and increases the second one. A comparison of these curves for hydrolysis of ATP, UTP and p-nitrophenylphosphate and temperature dependence of the hydrolysis of the substrates suggest that the anomalies observed may be accounted for the Na+ effect on the K-sites or K+ effect on the Na-sites under conditions when cation-binding sites are heterogeneous.     

Structural Insights into Ca2+-dependent Regulation of Inositol 1,4,5-Trisphosphate Receptors by CaBP1

Calcium-binding protein 1 (CaBP1), a neuron-specific member of the calmodulin (CaM) superfamily, modulates Ca²⁺-dependent activity of inositol 1,4,5-trisphosphate receptors (InsP₃Rs). Here we present NMR structures of CaBP1 in both Mg²⁺-bound and Ca²⁺-bound states and their structural interaction with InsP₃Rs. CaBP1 contains four EF-hands in two separate domains. The N-domain consists of EF1 and EF2 in a closed conformation with Mg²⁺ bound at EF1. The C-domain binds Ca²⁺ at EF3 and EF4, and exhibits a Ca²⁺-induced closed to open transition like that of CaM. The Ca²⁺-bound C-domain contains exposed hydrophobic residues (Leu¹³², His¹³⁴, Ile¹⁴¹, Ile¹⁴⁴, and Val¹⁴⁸) that may account for selective binding to InsP₃Rs. Isothermal titration calorimetry analysis reveals a Ca²⁺-induced binding of the CaBP1 C-domain to the N-terminal region of InsP₃R (residues 1-587), whereas CaM and the CaBP1 N-domain did not show appreciable binding. CaBP1 binding to InsP₃Rs requires both the suppressor and ligand-binding core domains, but has no effect on InsP₃ binding to the receptor. We propose that CaBP1 may regulate Ca²⁺-dependent activity of InsP₃Rs by promoting structural contacts between the suppressor and core domains.Calcium ion (Ca²⁺) in the cell functions as an important messenger that controls neurotransmitter release, gene expression, muscle contraction, apoptosis, and disease processes (1). Receptor stimulation in neurons promotes large increases in intracellular Ca²⁺ levels controlled by Ca²⁺ release from intracellular stores through InsP₃Rs (2). The neuronal type-1 receptor (InsP₃R1)² is positively and negatively regulated by cytosolic Ca²⁺ (3-6), important for the generation of repetitive Ca²⁺ transients known as Ca²⁺ spikes and waves (1). Ca²⁺-dependent activation of InsP₃R1 contributes to the fast rising phase of Ca²⁺ signaling known as Ca²⁺-induced Ca²⁺ release (7). Ca²⁺-induced inhibition of InsP₃R1, triggered at higher cytosolic Ca²⁺ levels, coordinates the temporal decay of Ca²⁺ transients (6). The mechanism of Ca²⁺-dependent regulation of InsP₃Rs is complex (8, 9), and involves direct Ca²⁺ binding sites (5, 10) as well as remote sensing by extrinsic Ca²⁺-binding proteins such as CaM (11, 12), CaBP1 (13, 14), CIB1 (15), and NCS-1 (16).Neuronal Ca²⁺-binding proteins (CaBP1-5 (17)) represent a new sub-branch of the CaM superfamily (18) that regulate various Ca²⁺ channel targets. Multiple splice variants and isoforms of CaBPs are localized in different neuronal cell types (19-21) and perform specialized roles in signal transduction. CaBP1, also termed caldendrin (22), has been shown to modulate the Ca²⁺-sensitive activity of InsP₃Rs (13, 14). CaBP1 also regulates P/Q-type voltage-gated Ca²⁺ channels (23), L-type channels (24), and the transient receptor potential channel, TRPC5 (25). CaBP4 regulates Ca²⁺-dependent inhibition of L-type channels in the retina and may be genetically linked to retinal degeneration (26). Thus, the CaBP proteins are receiving increased attention as a family of Ca²⁺ sensors that control a variety of Ca²⁺ channel targets implicated in neuronal degenerative diseases.CaBP proteins contain four EF-hands, similar in sequence to those found in CaM and troponin C (18) (Fig. 1). By analogy to CaM (27), the four EF-hands are grouped into two domains connected by a central linker that is four residues longer in CaBPs than in CaM. In contrast to CaM, the CaBPs contain non-conserved amino acids within the N-terminal region that may confer target specificity. Another distinguishing property of CaBPs is that the second EF-hand lacks critical residues required for high affinity Ca²⁺ binding (17). CaBP1 binds Ca²⁺ only at EF3 and EF4, whereas it binds Mg²⁺ at EF1 that may serve a functional role (28). Indeed, changes in cytosolic Mg²⁺ levels have been detected in cortical neurons after treatment with neurotransmitter (29). Other neuronal Ca²⁺-binding proteins such as DREAM (30), CIB1 (31), and NCS-1 (32) also bind Mg²⁺ and exhibit Mg²⁺-induced physiological effects. Mg²⁺ binding in each of these proteins helps stabilize their Ca²⁺-free state to interact with signaling targets.Open in a separate windowFIGURE 1.Amino acid sequence alignment of human CaBP1 with CaM. Secondary structural elements (α-helices and β-strands) were derived from NMR analysis. The four EF-hands (EF1, EF2, EF3, and EF4) are highlighted green, red, cyan, and yellow. Residues in the 12-residue Ca²⁺-binding loops are underlined and chelating residues are highlighted bold. Non-conserved residues in the hydrophobic patch are colored red.Despite extensive studies on CaBP1, little is known about its structure and target binding properties, and regulation of InsP₃Rs by CaBP1 is somewhat controversial and not well understood. Here, we present the NMR solution structures of both Mg²⁺-bound and Ca²⁺-bound conformational states of CaBP1 and their structural interactions with InsP₃R1. These CaBP1 structures reveal important Ca²⁺-induced structural changes that control its binding to InsP₃R1. Our target binding analysis demonstrates that the C-domain of CaBP1 exhibits Ca²⁺-induced binding to the N-terminal cytosolic region of InsP₃R1. We propose that CaBP1 may regulate Ca²⁺-dependent channel activity in InsP₃Rs by promoting a structural interaction between the N-terminal suppressor and ligand-binding core domains that modulates Ca²⁺-dependent channel gating (8, 33, 34).     

Molecular Mechanisms of the Redox Regulation of the Na,K-ATPase

Biophysics - This review considers the molecular mechanisms involved in the redox regulation of the Na,K-ATPase. The enzyme creates a transmembrane gradient of sodium and potassium ions, which is...     

Terahertz vibration modes in Na/K-ATPase

Mechanical vibration in the Terahertz range is believed to be connected with protein functions. In this paper, we present the results of a normal-mode analysis (modal analysis) of a Na/K-ATPase all-atom model, focusing the attention on low-frequency vibration modes. The numerical model helps in the interpretation of experimental results previously obtained by the authors via Raman spectroscopy of Na/K-ATPase samples, where several unassigned peaks were found in the sub-500 cm^?1 range. In particular, vibration modes corresponding to peaks at 27, 190 and 300 cm^?1, found experimentally, are confirmed here numerically, together with some other modes at lower frequencies (wavenumbers) that were not possible to observe in the experimental test. All the aforementioned modes correspond to vibrations involving the protein ends, i.e. portions directly related to the operating mechanism of the sodium-potassium pump.