The sarco(endo)plasmic reticulum Ca2+−ATPase (SERCA) hydrolyzes ATP to transport Ca2+ from the cytoplasm to the sarcoplasmic reticulum (SR) lumen, thereby inducing muscle relaxation. Dysfunctional ...SERCA has been related to various diseases. The identification of small‐molecule drugs that can activate SERCA may offer a therapeutic approach to treat pathologies connected with SERCA malfunction. Herein, we propose a method to study the mechanism of interaction between SERCA and novel SERCA activators, i. e. CDN1163, using a solid supported membrane (SSM) biosensing approach. Native SR vesicles or reconstituted proteoliposomes containing SERCA were adsorbed on the SSM and activated by ATP concentration jumps. We observed that CDN1163 reversibly interacts with SERCA and enhances ATP‐dependent Ca2+ translocation. The concentration dependence of the CDN1163 effect provided an EC50=6.0±0.3 μM. CDN1163 was shown to act directly on SERCA and to exert its stimulatory effect under physiological Ca2+ concentrations. These results suggest that CDN1163 interaction with SERCA can promote a protein conformational state that favors Ca2+ release into the SR lumen.
Electric insight: The interaction between the small‐molecule drug CDN1163 and sarcoplasmic reticulum Ca2+−ATPase was investigated by electrical current measurements. It was shown that CDN1163 reversibly binds to the ATPase protein and significantly enhances ATP‐dependent calcium translocation. CDN1163 was able to exert its stimulatory effect on Ca2+−ATPase transport activity under physiological Ca2+ concentrations.
Cisplatin, carboplatin, and oxaliplatin are widely used anticancer drugs. Their efficacy is strongly reduced by development of cell resistance. Down‐regulation of CTR1 and up‐regulation of the ...Cu‐ATPases, ATP7A and ATP7B, have been associated to augmented drug resistance. To gain information on translocation of Pt drugs by human Cu‐ATPases, we performed electrical measurements on the COS‐1 cell microsomal fraction, enriched with recombinant ATP7A, ATP7B, and selected mutants, and adsorbed on a solid supported membrane. The experimental results indicate that Pt drugs activate Cu‐ATPases and undergo ATP‐dependent translocation in a fashion similar to that of Cu. We then used NMR spectroscopy and ESI‐MS to determine the binding mode of these drugs to the first N‐terminal metal‐binding domain of ATP7A (Mnk1).
Charge measurements, in the presence of cisplatin or oxaliplatin, on COS‐1 microsomes enriched with Cu‐ATPases, show vectorial displacement of a charged Pt species upon ATP addition. Co‐administration of a Pt drug and Cu inhibits charge translocation. ESI‐MS and NMR experiments demonstrate formation of monodentate Pt drug adducts, that evolve into stable and unreactive chelate adducts with CXXC motifs.
The Ca
2+
transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca
2+
in intracellular membrane bound compartments, thereby ...lowering cytosolic Ca
2+
to induce relaxation. The stored Ca
2+
is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca
2+
, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca
2+
. We review here biochemical and biophysical evidence demonstrating that release of bound Ca
2+
into the lumen of SR requires Ca
2+
/H
+
exchange at the low affinity Ca
2+
sites. Rise of lumenal Ca
2+
above its dissociation constant from low affinity sites, or reduction of the H
+
concentration by high pH, prevent Ca
2+
/H
+
exchange. Under these conditions Ca
2+
release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca
2+
pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.
P-type ATPases are a large family of membrane transporters that are found in all forms of life. These enzymes couple ATP hydrolysis to the transport of various ions or phospholipids across cellular ...membranes, thereby generating and maintaining crucial electrochemical potential gradients. P-type ATPases have been studied by a variety of methods that have provided a wealth of information about the structure, function, and regulation of this class of enzymes. Among the many techniques used to investigate P-type ATPases, the electrical method based on solid supported membranes (SSM) was employed to investigate the transport mechanism of various ion pumps. In particular, the SSM method allows the direct measurement of charge movements generated by the ATPase following adsorption of the membrane-bound enzyme on the SSM surface and chemical activation by a substrate concentration jump. This kind of measurement was useful to identify electrogenic partial reactions and localize ion translocation in the reaction cycle of the membrane transporter. In the present review, we discuss how the SSM method has contributed to investigate some key features of the transport mechanism of P-type ATPases, with a special focus on sarcoplasmic reticulum Ca
-ATPase, mammalian Cu
-ATPases (ATP7A and ATP7B), and phospholipid flippase ATP8A2.
Background and Purpose
Calcium handling is known to be deranged in heart failure. Interventions aimed at improving cell Ca2+ cycling may represent a promising approach to heart failure therapy. ...Istaroxime is a new luso‐inotropic compound that stimulates cardiac contractility and relaxation in healthy and failing animal models and in patients with acute heart failure (AHF) syndrome. Istaroxime is a Na‐K ATPase inhibitor with the unique property of increasing sarcoplasmic reticulum (SR) SERCA2a activity as shown in heart microsomes from humans and guinea pigs. The present study addressed the molecular mechanism by which istaroxime increases SERCA2a activity.
Experimental Approach
To study the effect of istaroxime on SERCA2a‐phospholamban (PLB) complex, we applied different methodologies in native dog healthy and failing heart preparations and heterologous canine SERCA2a/PLB co‐expressed in Spodoptera frugiperda (Sf21) insect cells.
Key Results
We showed that istaroxime enhances SERCA2a activity, Ca2+ uptake and the Ca2+‐dependent charge movements into dog healthy and failing cardiac SR vesicles. Although not directly demonstrated, the most probable explanation of these activities is the displacement of PLB from SERCA2a.E2 conformation, independently from cAMP/PKA. We propose that this displacement may favour the SERCA2a conformational transition from E2 to E1, thus resulting in the acceleration of Ca2+ cycling.
Conclusions and Implications
Istaroxime represents the first example of a small molecule that exerts a luso‐inotropic effect in the failing human heart through the stimulation of SERCA2a ATPase activity and the enhancement of Ca2+ uptake into the SR by relieving the PLB inhibitory effect on SERCA2a in a cAMP/PKA independent way.
Linked Articles
This article is commented on by Huang, pp. 486–488 of volume 170 issue 3. To view this commentary visit http://dx.doi.org/10.1111/bph.12288.
Cancer is a multifactorial family of diseases that is still a leading cause of death worldwide. More than 100 different types of cancer affecting over 60 human organs are known. Chemotherapy plays a ...central role for treating cancer. The development of new anticancer drugs or new uses for existing drugs is an exciting and increasing research area. This is particularly important since drug resistance and side effects can limit the efficacy of the chemotherapy. Thus, there is a need for multiplexed, cost-effective, rapid, and novel screening methods that can help to elucidate the mechanism of the action of anticancer drugs and the identification of novel drug candidates. This review focuses on different label-free bioelectrochemical approaches, in particular, impedance-based methods, the solid supported membranes technique, and the DNA-based electrochemical sensor, that can be used to evaluate the effects of anticancer drugs on nucleic acids, membrane transporters, and living cells. Some relevant examples of anticancer drug interactions are presented which demonstrate the usefulness of such methods for the characterization of the mechanism of action of anticancer drugs that are targeted against various biomolecules.
Copper ATPases, in analogy with other members of the P-ATPase superfamily, contain a catalytic headpiece including an aspartate residue reacting with ATP to form a phosphoenzyme intermediate, and ...transmembrane helices containing cation-binding sites TMBS (transmembrane metal-binding sites) for catalytic activation and cation translocation. Following phosphoenzyme formation by utilization of ATP, bound copper undergoes displacement from the TMBS to the lumenal membrane surface, with no H+ exchange. Although PII-type ATPases sustain active transport of alkali/alkali-earth ions (i.e. Na+, Ca2+) against electrochemical gradients across defined membranes, PIB-type ATPases transfer transition metal ions (i.e. Cu+) from delivery to acceptor proteins and, prominently in mammalian cells, undergo trafficking from/to various membrane compartments. A specific component of copper ATPases is the NMBD (N-terminal metal-binding domain), containing up to six copper-binding sites in mammalian (ATP7A and ATP7B) enzymes. Copper occupancy of NMBD sites and interaction with the ATPase headpiece are required for catalytic activation. Furthermore, in the presence of copper, the NMBD allows interaction with protein kinase D, yielding phosphorylation of serine residues, ATP7B trafficking and protection from proteasome degradation. A specific feature of ATP7A is glycosylation and stabilization on plasma membranes. Cisplatin, a platinum-containing anti-cancer drug, binds to copper sites of ATP7A and ATP7B, and undergoes vectorial displacement in analogy with copper.
ATP8A2 is a mammalian P4‐ATPase (flippase) that translocates the negatively charged lipid substrate phosphatidylserine from the exoplasmic leaflet to the cytoplasmic leaflet of cellular membranes. ...Using an electrophysiological method based on solid supported membranes, we investigated the electrogenicity of specific reaction steps of ATP8A2 and explored a potential phospholipid translocation pathway involving residues with positively charged side chains. Changes to the current signals caused by mutations show that the main electrogenic event occurs in connection with the release of the bound phosphatidylserine to the cytoplasmic leaflet and support the hypothesis that the phospholipid interacts with specific lysine and arginine residues near the cytoplasmic border of the lipid bilayer during the translocation and reorientation required for insertion into the cytoplasmic leaflet.
We used an electrophysiological method based on solid supported membranes to investigate the electrogenicity of mammalian flippase ATP8A2. Mutations of specific positively charged residues strongly affected the current signal generated by ATP8A2 in the presence of the substrate phosphatidylserine (PS). We propose that PS interacts with these residues during translocation and reorientation required for insertion into the cytoplasmic leaflet.
Mexiletine is a sodium channel blocker, primarily used in the treatment of ventricular arrhythmias. Moreover, recent studies have demonstrated its therapeutic value to treat myotonic syndromes and to ...relieve neuropathic pain. The present study aims at investigating the direct blockade of hERG potassium channel by mexiletine and its metabolite m‐hydroxymexiletine (MHM). Our data show that mexiletine inhibits hERG in a time‐ and voltage‐dependent manner, with an IC50 of 3.7 ± 0.7 μmol/L. Analysis of the initial onset of current inhibition during a depolarizing test pulse indicates mexiletine binds preferentially to the open state of the hERG channel. Looking for a possible mexiletine alternative, we show that m‐hydroxymexiletine (MHM), a minor mexiletine metabolite recently reported to be as active as the parent compound in an arrhythmia animal model, is a weaker hERG channel blocker, compared to mexiletine (IC50 = 22.4 ± 1.2 μmol/L). The hERG aromatic residues located in the S6 helix (Tyr652 and Phe656) are crucial in the binding of mexiletine and the different affinities of mexiletine and MHM with hERG channel are interpreted by modeling their corresponding binding interactions through ab initio calculations. The simulations demonstrate that the introduction of a hydroxyl group on the meta‐position of the aromatic portion of mexiletine weakens the interaction of the drug xylyloxy moiety with Tyr652. These results provide further insights into the molecular basis of drug/hERG interactions and, in agreement with previously reported results on clofilium and ibutilide analogs, support the possibility of reducing hERG potency and related toxicity by modifying the aromatic pattern of substitution of clinically relevant compounds.
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