The fourth extracellular loop of the alpha subunit of Na,K-ATPase. Functional evidence for close proximity with the second extracellular loop.

Na,K-ATPase is the main active transport system that maintains the large gradients of Na(+) and K(+) across the plasma membrane of animal cells. The crystal structure of a K(+)-occluding conformation of this protein has been recently published, but the movements of its different domains allowing for the cation pumping mechanism are not yet known. The structure of many more conformations is known for the related calcium ATPase SERCA, but the reliability of homology modeling is poor for several domains with low sequence identity, in particular the extracellular loops. To better define the structure of the large fourth extracellular loop between the seventh and eighth transmembrane segments of the alpha subunit, we have studied the formation of a disulfide bond between pairs of cysteine residues introduced by site-directed mutagenesis in the second and the fourth extracellular loop. We found a specific pair of cysteine positions (Y308C and D884C) for which extracellular treatment with an oxidizing agent inhibited the Na,K pump function, which could be rapidly restored by a reducing agent. The formation of the disulfide bond occurred preferentially under the E2-P conformation of Na,K-ATPase, in the absence of extracellular cations. Using recently published crystal structure and a distance constraint reproducing the existence of disulfide bond, we performed an extensive conformational space search using simulated annealing and showed that the Tyr(308) and Asp(884) residues can be in close proximity, and simultaneously, the SYGQ motif of the fourth extracellular loop, known to interact with the extracellular domain of the beta subunit, can be exposed to the exterior of the protein and can easily interact with the beta subunit.

T-type Ca2+ channels, SK2 channels and SERCAs gate sleep-related oscillations in thalamic dendrites.

T-type Ca2+ channels (T channels) underlie rhythmic burst discharges during neuronal oscillations that are typical during sleep. However, the Ca2+-dependent effectors that are selectively regulated by T currents remain unknown. We found that, in dendrites of nucleus reticularis thalami (nRt), intracellular Ca2+ concentration increases were dominated by Ca2+ influx through T channels and shaped rhythmic bursting via competition between Ca2+-dependent small-conductance (SK)-type K+ channels and Ca2+ uptake pumps. Oscillatory bursting was initiated via selective activation of dendritically located SK2 channels, whereas Ca2+ sequestration by sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) and cumulative T channel inactivation dampened oscillations. Sk2-/- (also known as Kcnn2) mice lacked cellular oscillations, showed a greater than threefold reduction in low-frequency rhythms in the electroencephalogram of non-rapid-eye-movement sleep and had disrupted sleep. Thus, the interplay of T channels, SK2 channels and SERCAs in nRt dendrites comprises a specialized Ca2+ signaling triad to regulate oscillatory dynamics related to sleep.

The fourth transmembrane segment of the Na,K-ATPase alpha subunit: a systematic mutagenesis study.

The Na,K-ATPase is a major ion-motive ATPase of the P-type family responsible for many aspects of cellular homeostasis. To determine the structure of the pathway for cations across the transmembrane portion of the Na,K-ATPase, we mutated 24 residues of the fourth transmembrane segment into cysteine and studied their function and accessibility by exposure to the sulfhydryl reagent 2-aminoethyl-methanethiosulfonate. Accessibility was also examined after treatment with palytoxin, which transforms the Na,K-pump into a cation channel. Of the 24 tested cysteine mutants, seven had no or a much reduced transport function. In particular cysteine mutants of the highly conserved "PEG" motif had a strongly reduced activity. However, most of the non-functional mutants could still be transformed by palytoxin as well as all of the functional mutants. Accessibility, determined as a 2-aminoethyl-methanethiosulfonate-induced reduction of the transport activity or as inhibition of the membrane conductance after palytoxin treatment, was observed for the following positions: Phe(323), Ile(322), Gly(326), Ala(330), Pro(333), Glu(334), and Gly(335). In accordance with a structural model of the Na,K-ATPase obtained by homology modeling with the two published structures of sarcoplasmic and endoplasmic reticulum calcium ATPase (Protein Data Bank codes 1EUL and 1IWO), the results suggest the presence of a cation pathway along the side of the fourth transmembrane segment that faces the space between transmembrane segments 5 and 6. The phenylalanine residue in position 323 has a critical position at the outer mouth of the cation pathway. The residues thought to form the cation binding site II ((333)PEGL) are also part of the accessible wall of the cation pathway opened by palytoxin through the Na,K-pump.

Trypanosoma cruzi-cardiomyocytes: new contributions regarding a better understanding of this interaction

The present paper summarizes new approaches regarding the progress done to the understanding of the interaction of Trypanosoma cruzi-cardiomyocytes. Mannose receptors localized at the surface of heart muscle cell are involved in binding and uptake of the parasite. One of the most striking events in the parasite-heart muscle cells interaction is the disruption of the actin cytoskeleton. We have investigated the regulation of the actin mRNA during the cytopathology induced in myocardial cells by the parasite. T. cruzi invasion increases calcium resting levels in cardiomyocytes. We have previously shown that Ca2+ ATPase of the sarcoplasmic reticulum (SERCA) is involved in the invasion of T. cruzi in cardiomyocytes. Treating the cells with thapsigargin, a drug that binds to all SERCA ATPases and causes depletion of intracellular calcium stores, we found a 75% inhibition in the T. cruzi-cardiomyocytes invasion.

Control of calcium homeostasis in Schistosoma mansoni

Calcium signalling is fundamental for muscular contractility of Schistosoma mansoni. We have previously described the presence of transport ATPases (Na+,K+-ATPase and (Ca2+-Mg2+)-ATPase) and calcium channels (ryanodine receptors - RyR) involved in control of calcium homeostasis in this worm. Here we briefly review the main technics (ATPase activity, binding with specific radioligands, fluxes of 45Ca2+ and whole worm contractions) and results obtained in order to compare the distribution patterns of these proteins: thapsigargin-sensitive (Ca2+-Mg2+)-ATPase activity and RyR co-purified in P1 and P4 fractions mainly, which is compatible with a sarcoplasmic reticulum localization, while basal ATPase (along with Na+,K+-ATPase) and thapsigargin-resistant (Ca2+-Mg2+)-ATPase have a distinct distribution, indicative of their plasma membrane localization. Finally we attempt to integrate these contributions with data from other groups in order to propose the first synoptic model for control of calcium homeostasis in S. mansoni.

Tonoplast localization of a calmodulin-stimulated Ca2+-pump from maize roots.

The subcellular localization of a calmodulin-stimulated calcium (Ca2+)-ATPase activity from maize roots (Zea mays L., cv LG 11) was studied. For this purpose, an efficient procedure was developed to prepare sealed plasma membrane vesicles allowing the measurement of proton and Ca2+ transport activities. Two-day-old root membranes were fractionated by sucrose and dextran density gradient centrifugation. Marker enzymes were used to study the distribution of the different membranes in the gradients and a filtration technique was developed to measure Ca-45(2+) transport in sealed vesicles. Most of the ATP-dependent Ca2+ transport activity was associated with the ER. However, a small part of this activity was associated with the tonoplast (corresponding to the activity of the H+/Ca2+ antiport) and the plasma membrane. When the Ca2+ transport was measured in the presence of exogenous calmodulin (1 muM), a 3-5-fold increase of uptake was measured. The calmodulin-stimulated activity was associated with the tonoplast vesicles only. This activity was insensitive to monensin, a proton ionophore, ruling out a direct effect of calmodulin on the H+/Ca2+ antiport. In conclusion, four different Ca2+ transporters are present in young maize root cells. A Ca2+/H+ antiport system is present on the tonoplast, whereas, the plasma membrane and the ER possess each a calmodulinin-sensitive Ca2+-ATPase. Finally, a calmodulin-stimulated Ca2+-ATPase is associated with the tonoplast.

Activation of Transient Receptor Potential Canonical 3 (TRPC3)-mediated Ca2+ Entry by A1 Adenosine Receptor in Cardiomyocytes Disturbs Atrioventricular Conduction.

Although the activation of the A(1)-subtype of the adenosine receptors (A(1)AR) is arrhythmogenic in the developing heart, little is known about the underlying downstream mechanisms. The aim of this study was to determine to what extent the transient receptor potential canonical (TRPC) channel 3, functioning as receptor-operated channel (ROC), contributes to the A(1)AR-induced conduction disturbances. Using embryonic atrial and ventricular myocytes obtained from 4-day-old chick embryos, we found that the specific activation of A(1)AR by CCPA induced sarcolemmal Ca(2+) entry. However, A(1)AR stimulation did not induce Ca(2+) release from the sarcoplasmic reticulum. Specific blockade of TRPC3 activity by Pyr3, by a dominant negative of TRPC3 construct, or inhibition of phospholipase Cs and PKCs strongly inhibited the A(1)AR-enhanced Ca(2+) entry. Ca(2+) entry through TRPC3 was activated by the 1,2-diacylglycerol (DAG) analog OAG via PKC-independent and -dependent mechanisms in atrial and ventricular myocytes, respectively. In parallel, inhibition of the atypical PKCζ by myristoylated PKCζ pseudosubstrate inhibitor significantly decreased the A(1)AR-enhanced Ca(2+) entry in both types of myocytes. Additionally, electrocardiography showed that inhibition of TRPC3 channel suppressed transient A(1)AR-induced conduction disturbances in the embryonic heart. Our data showing that A(1)AR activation subtly mediates a proarrhythmic Ca(2+) entry through TRPC3-encoded ROC by stimulating the phospholipase C/DAG/PKC cascade provide evidence for a novel pathway whereby Ca(2+) entry and cardiac function are altered. Thus, the A(1)AR-TRPC3 axis may represent a potential therapeutic target.

Cation Transport Coupled to ATP Hydrolysis By the (Na, K)-ATPase : an integrated, animated model

An Adobe (R) animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na+ and K+ translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P-2c-type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also known as an E-1/E-2-ATPase as it undergoes conformational changes between the E-1 and E-2 forms during the pumping cycle, altering the affinity and accessibility of the transmembrane ion-binding sites. The animation is based on Horisberger's scheme that incorporates the most recent significant findings to have improved our understanding of the (Na, K)-ATPase structure function relationship. The movements of the various domains within the (Na, K)-ATPase alpha-subunit illustrate the conformational changes that occur during Na+ and K+ translocation across the membrane and emphasize involvement of the actuator, nucleotide, and phosphorylation domains, that is, the "core engine" of the pump, with respect to ATP binding, cation transport, and ADP and P-i release.

Eucalyptol, an essential oil, reduces contractile activity in rat cardiac muscle

Eucalyptol is an essential oil that relaxes bronchial and vascular smooth muscle although its direct actions on isolated myocardium have not been reported. We investigated a putative negative inotropic effect of the oil on left ventricular papillary muscles from male Wistar rats weighing 250 to 300 g, as well as its effects on isometric force, rate of force development, time parameters, post-rest potentiation, positive inotropic interventions produced by Ca2+ and isoproterenol, and on tetanic tension. The effects of 0.3 mM eucalyptol on myosin ATPase activity were also investigated. Eucalyptol (0.003 to 0.3 mM) reduced isometric tension, the rate of force development and time parameters. The oil reduced the force developed by steady-state contractions (50% at 0.3 mM) but did not alter sarcoplasmic reticulum function or post-rest contractions and produced a progressive increase in relative potentiation. Increased extracellular Ca2+ concentration (0.62 to 5 mM) and isoproterenol (20 nM) administration counteracted the negative inotropic effects of the oil. The activity of the contractile machinery evaluated by tetanic force development was reduced by 30 to 50% but myosin ATPase activity was not affected by eucalyptol (0.3 mM), supporting the idea of a reduction of sarcolemmal Ca2+ influx. The present results suggest that eucalyptol depresses force development, probably acting as a calcium channel blocker.

The importance of the Thr17 residue of phospholamban as a phosphorylation site under physiological and pathological conditions

The sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) is under the control of an SR protein named phospholamban (PLN). Dephosphorylated PLN inhibits SERCA2a, whereas phosphorylation of PLN at either the Ser16 site by PKA or the Thr17 site by CaMKII reverses this inhibition, thus increasing SERCA2a activity and the rate of Ca2+ uptake by the SR. This leads to an increase in the velocity of relaxation, SR Ca2+ load and myocardial contractility. In the intact heart, ß-adrenoceptor stimulation results in phosphorylation of PLN at both Ser16 and Thr17 residues. Phosphorylation of the Thr17 residue requires both stimulation of the CaMKII signaling pathways and inhibition of PP1, the major phosphatase that dephosphorylates PLN. These two prerequisites appear to be fulfilled by ß-adrenoceptor stimulation, which as a result of PKA activation, triggers the activation of CaMKII by increasing intracellular Ca2+, and inhibits PP1. Several pathological situations such as ischemia-reperfusion injury or hypercapnic acidosis provide the required conditions for the phosphorylation of the Thr17 residue of PLN, independently of the increase in PKA activity, i.e., increased intracellular Ca2+ and acidosis-induced phosphatase inhibition. Our results indicated that PLN was phosphorylated at Thr17 at the onset of reflow and immediately after hypercapnia was established, and that this phosphorylation contributes to the mechanical recovery after both the ischemic and acidic insults. Studies on transgenic mice with Thr17 mutated to Ala (PLN-T17A) are consistent with these results. Thus, phosphorylation of the Thr17 residue of PLN probably participates in a protective mechanism that favors Ca2+ handling and limits intracellular Ca2+ overload in pathological situations.

STIM1/Orai1-mediated store-operated Ca2+ entry: the tip of the iceberg

Highly efficient mechanisms regulate intracellular calcium (Ca2+) levels. The recent discovery of new components linking intracellular Ca2+ stores to plasma membrane Ca2+ entry channels has brought new insight into the understanding of Ca2+ homeostasis. Stromal interaction molecule 1 (STIM1) was identified as a Ca2+ sensor essential for Ca2+ store depletion-triggered Ca2+ influx. Orai1 was recognized as being an essential component for the Ca2+ release-activated Ca2+ (CRAC) channel. Together, these proteins participate in store-operated Ca2+ channel function. Defective regulation of intracellular Ca2+ is a hallmark of several diseases. In this review, we focus on Ca2+ regulation by the STIM1/Orai1 pathway and review evidence that implicates STIM1/Orai1 in several pathological conditions including cardiovascular and pulmonary diseases, among others.

Acute exposure to lead increases myocardial contractility independent of hypertension development

We studied the effects of the acute administration of small doses of lead over time on hemodynamic parameters in anesthetized rats to determine if myocardial contractility changes are dependent or not on the development of hypertension. Male Wistar rats received 320 µg/kg lead acetateiv once, and their hemodynamic parameters were measured for 2 h. Cardiac contractility was evaluated in vitro using left ventricular papillary muscles as were Na+,K+-ATPase and myosin Ca2+-ATPase activities. Lead increased left- (control: 112 ± 3.7 vs lead: 129 ± 3.2 mmHg) and right-ventricular systolic pressures (control: 28 ± 1.2vs lead: 34 ± 1.2 mmHg) significantly without modifying heart rate. Papillary muscles were exposed to 8 µM lead acetate and evaluated 60 min later. Isometric contractions increased (control: 0.546 ± 0.07 vs lead: 0.608 ± 0.06 g/mg) and time to peak tension decreased (control: 268 ± 13vs lead: 227 ± 5.58 ms), but relaxation time was unchanged. Post-pause potentiation was similar between groups (n = 6 per group), suggesting no change in sarcoplasmic reticulum activity, evaluated indirectly by this protocol. After 1-h exposure to lead acetate, the papillary muscles became hyperactive in response to a β-adrenergic agonist (10 µM isoproterenol). In addition, post-rest contractions decreased, suggesting a reduction in sarcolemmal calcium influx. The heart samples treated with 8 µM lead acetate presented increased Na+,K+-ATPase (approximately 140%, P < 0.05 for control vs lead) and myosin ATPase (approximately 30%, P < 0.05 for control vs lead) activity. Our results indicated that acute exposure to low lead concentrations produces direct positive inotropic and lusitropic effects on myocardial contractility and increases the right and left ventricular systolic pressure, thus potentially contributing to the early development of hypertension.

Effect of exercise training on Ca2+ release units of left ventricular myocytes of spontaneously hypertensive rats

In cardiomyocytes, calcium (Ca2+) release units comprise clusters of intracellular Ca2+ release channels located on the sarcoplasmic reticulum, and hypertension is well established as a cause of defects in calcium release unit function. Our objective was to determine whether endurance exercise training could attenuate the deleterious effects of hypertension on calcium release unit components and Ca2+ sparks in left ventricular myocytes of spontaneously hypertensive rats. Male Wistar and spontaneously hypertensive rats (4 months of age) were divided into 4 groups: normotensive (NC) and hypertensive control (HC), and normotensive (NT) and hypertensive trained (HT) animals (7 rats per group). NC and HC rats were submitted to a low-intensity treadmill running protocol (5 days/week, 1 h/day, 0% grade, and 50-60% of maximal running speed) for 8 weeks. Gene expression of the ryanodine receptor type 2 (RyR2) and FK506 binding protein (FKBP12.6) increased (270%) and decreased (88%), respectively, in HC compared to NC rats. Endurance exercise training reversed these changes by reducing RyR2 (230%) and normalizing FKBP12.6 gene expression (112%). Hypertension also increased the frequency of Ca2+ sparks (HC=7.61±0.26 vs NC=4.79±0.19 per 100 µm/s) and decreased its amplitude (HC=0.260±0.08 vs NC=0.324±0.10 ΔF/F0), full width at half-maximum amplitude (HC=1.05±0.08 vs NC=1.26±0.01 µm), total duration (HC=11.51±0.12 vs NC=14.97±0.24 ms), time to peak (HC=4.84±0.06 vs NC=6.31±0.14 ms), and time constant of decay (HC=8.68±0.12 vs NC=10.21±0.22 ms). These changes were partially reversed in HT rats (frequency of Ca2+ sparks=6.26±0.19 µm/s, amplitude=0.282±0.10 ΔF/F0, full width at half-maximum amplitude=1.14±0.01 µm, total duration=13.34±0.17 ms, time to peak=5.43±0.08 ms, and time constant of decay=9.43±0.15 ms). Endurance exercise training attenuated the deleterious effects of hypertension on calcium release units of left ventricular myocytes.

Increased Activation of Stromal Interaction Molecule-1/Orai-1 in Aorta From Hypertensive Rats A Novel Insight Into Vascular Dysfunction

Disturbances in the regulation of cytosolic calcium (Ca(2+)) concentration play a key role in the vascular dysfunction associated with arterial hypertension. Stromal interaction molecules (STIMs) and Orai proteins represent a novel mechanism to control store-operated Ca(2+) entry. Although STIMs act as Ca(2+) sensors for the intracellular Ca(2+) stores, Orai is the putative pore-forming component of Ca(2+) release-activated Ca(2+) channels at the plasma membrane. We hypothesized that augmented activation of Ca(2+) release-activated Ca(2+)/Orai-1, through enhanced activity of STIM-1, plays a role in increased basal tonus and vascular reactivity in hypertensive animals. Endothelium-denuded aortic rings from Wistar-Kyoto and stroke-prone spontaneously hypertensive rats were used to evaluate contractions because of Ca(2+) influx. Depletion of intracellular Ca(2+) stores, which induces Ca(2+) release-activated Ca(2+) activation, was performed by placing arteries in Ca(2+) free-EGTA buffer. The addition of the Ca(2+) regular buffer produced greater contractions in aortas from stroke-prone spontaneously hypertensive rats versus Wistar-Kyoto rats. Thapsigargin (10 mu mol/L), an inhibitor of the sarcoplasmic reticulum Ca(2+) ATPase, further increased these contractions, especially in stroke-prone spontaneously hypertensive rat aorta. Addition of the Ca(2+) release-activated Ca(2+) channel inhibitors 2-aminoethoxydiphenyl borate (100 mu mol/L) or gadolinium (100 mu mol/L), as well as neutralizing antibodies to STIM-1 or Orai-1, abolished thapsigargin-increased contraction and the differences in spontaneous tone between the groups. Expression of Orai-1 and STIM-1 proteins was increased in aorta from stroke-prone spontaneously hypertensive rats when compared with Wistar-Kyoto rats. These results support the hypothesis that both Orai-1 and STIM-1 contribute to abnormal vascular function in hypertension. Augmented activation of STIM-1/Orai-1 may represent the mechanism that leads to impaired control of intracellular Ca(2+) levels in hypertension. (Hypertension. 2009; 53[part 2]: 409-416.)

Involvement of L-Type Calcium Channel and SERCA2a in Myocardial Dysfunction Induced by Obesity

Obesity has been shown to impair myocardial performance. Nevertheless, the mechanisms underlying the participation of calcium (Ca2+) handling on cardiac dysfunction in obesity models remain unknown. L-type Ca2+ channels and sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a), may contribute to the cardiac dysfunction induced by obesity. The purpose of this study was to investigate whether myocardial dysfunction in obese rats is related to decreased activity and/or expression of L-type Ca2+ channels and SERCA2a. Male 30-day-old Wistar rats were fed standard (C) and alternately four palatable high-fat diets (Ob) for 15 weeks. Obesity was determined by adiposity index and comorbidities were evaluated. Myocardial function was evaluated in isolated left ventricle papillary muscles under basal conditions and after inotropic and lusitropic maneuvers. L-type Ca2+ channels and SERCA2a activity were determined using specific blockers, while changes in the amount of channels were evaluated by Western blot analysis. Phospholamban (PLB) protein expression and the SERCA2a/PLB ratio were also determined. Compared with C rats, the Ob rats had increased body fat, adiposity index and several comorbidities. The Ob muscles developed similar baseline data, but myocardial responsiveness to post-rest contraction stimulus and increased extracellular Ca2+ was compromised. The diltiazem promoted higher inhibition on developed tension in obese rats. In addition, there were no changes in the L-type Ca2+ channel protein content and SERCA2a behavior (activity and expression). In conclusion, the myocardial dysfunction caused by obesity is related to L-type Ca2+ channel activity impairment without significant changes in SERCA2a expression and function as well as L-type Ca2+ protein levels. J. Cell. Physiol. 226: 2934-2942, 2011. (C) 2011 Wiley-Liss, Inc.