Francesco Tadini-Buoninsegni

Pre-steady State Electrogenic Events of Ca2+/H+ Exchange and Transport by the Ca2+-ATPase

Native or recombinant SERCA (sarco(endo)plasmic reticulum Ca2+ ATPase) was adsorbed on a solid supported membrane and then activated with Ca2+ and ATP concentration jumps through rapid solution exchange. The resulting electrogenic events were recorded as electrical currents flowing along the external circuit. Current transients were observed following Ca2+ jumps in the absence of ATP and following ATP jumps in the presence of Ca2+. The related charge movements are attributed to Ca2+ reaching its binding sites in the ground state of the enzyme (E1) and to its vectorial release from the enzyme phosphorylated by ATP (E2P). The Ca2+ concentration and pH dependence as well as the time frames of the observed current transients are consistent with equilibrium and pre-steady state biochemical measurements of sequential steps within a single enzymatic cycle. Numerical integration of the current transients recorded at various pH values reveal partial charge compensation by H+ in exchange for Ca2+ at acidic (but not at alkaline) pH. Most interestingly, charge movements induced by Ca2+ and ATP vary over different pH ranges, as the protonation probability of residues involved in Ca2+/H+ exchange is lower in the E1 than in the E2P state. Our single cycle measurements demonstrate that this difference contributes directly to the reduction of Ca2+ affinity produced by ATP utilization and results in the countertransport of two Ca2+ and two H+ within each ATPase cycle at pH 7.0. The effects of site-directed mutations indicate that Glu-771 and Asp-800, within the Ca2+ binding domain, are involved in the observed Ca2+/H+ exchange.

Translocation of Platinum Anticancer Drugs by Human Copper ATPases ATP7A and ATP7B

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).

A voltammetric study of monolayers and bilayers self-assembled on metal electrodes

Abstract Three types of aliphatic thiols, n-dodecanethiol, n-hexadecanethiol and n-octadecanethiol, differing in their physical state in self-assembled monolayers at ambient temperature were tested from the point of view of their integrity and usefulness as the sub-layer for the second, adjacent layer of phospholipids. These self-assembled monolayers were formed on mercury and polycrystalline gold electrodes in order to assess their electrochemical behavior as monolayers passivating the electrodes against various redox probes present in aqueous phase, such as hexaamineruthenium(III) chloride and benzoquinone, differing in their heterogeneous rate constants and hydrophobic–hydrophilic character. Subsequently, as such a type of hydrophobic alkanethiol monolayer is frequently used as a part of alkanethiol|phospholipid asymmetric bilayer membranes, chemically bound to the metallic surface, the monolayer was covered with the adjacent phosphatidylcholine monolayer, and the electrochemical behavior of such an asymmetric system was again tested. The results obtained show that the liquid-like monolayers, particularly those formed on mercury, possess better passivating properties (much smaller number of defects) as compared to crystalline thiols, also providing a better driving force for the attachment of the second liquid-crystalline phosphatidylcholine monolayer. An interesting observation was also noted that the outer phospholipid monolayer imposes a larger energy barrier to the penetration of benzoquinone that hexaamineruthenium(III) cations.

Mimicking the intramolecular hydrogen bond: synthesis, biological evaluation, and molecular modeling of benzoxazines and quinazolines as potential antimalarial agents.

The intramolecular hydrogen bond formed between a protonated amine and a neighboring H-bond acceptor group in the side chain of amodiaquine and isoquine is thought to play an important role in their antimalarial activities. Here we describe isoquine-based compounds in which the intramolecular H-bond is mimicked by a methylene linker. The antimalarial activities of the resulting benzoxazines, their isosteric tetrahydroquinazoline derivatives, and febrifugine-based 1,3-quinazolin-4-ones were examined in vitro (against Plasmodium falciparum ) and in vivo (against Plasmodium berghei ). Compounds 6b,c caused modest inhibition of chloroquine transport via the parasites chloroquine resistance transporter (PfCRT) in a Xenopus laevis oocyte expression system. In silico predictions and experimental evaluation of selected drug-like properties were also performed on compounds 6b,c. Compound 6c emerged from this work as the most promising analogue of the series; it possessed low toxicity and good antimalarial activity when administered orally to P. berghei -infected mice.

Clotrimazole Inhibits the Ca2+-ATPase (SERCA) by Interfering with Ca2+ Binding and Favoring the E2 Conformation

Clotrimazole (CLT) is an antimycotic imidazole derivative that is known to inhibit cytochrome P-450, ergosterol biosynthesis and proliferation of cells in culture, and to interfere with cellular Ca2+ homeostasis. We found that CLT inhibits the Ca2+-ATPase of rabbit fast-twitch skeletal muscle (SERCA1), and we characterized in detail the effect of CLT on this calcium transport ATPase. We used biochemical methods for characterization of the ATPase and its partial reactions, and we also performed measurements of charge movements following adsorption of sarcoplasmic reticulum vesicles containing the ATPase onto a gold-supported biomimetic membrane. CLT inhibits Ca2+-ATPase and Ca2+ transport with a KI of 35 μm. Ca2+ binding in the absence of ATP and phosphoenzyme formation by the utilization of ATP in the presence of Ca2+ are also inhibited within the same CLT concentration range. On the other hand, phosphoenzyme formation by utilization of Pi in the absence of Ca2+ is only minimally inhibited. It is concluded that CLT inhibits primarily Ca2+ binding and, consequently, the Ca2+-dependent reactions of the SERCA cycle. It is suggested that CLT resides within the membrane-bound region of the transport ATPase, thereby interfering with binding and the conformational effects of the activating cation.

Istaroxime stimulates SERCA2a and accelerates calcium cycling in heart failure by relieving phospholamban inhibition

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.

Ca2+/H+ exchange, lumenal Ca2+ release and Ca2+/ATP coupling ratios in the sarcoplasmic reticulum ATPase

The Ca2+ transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca2+ in intracellular membrane bound compartments, thereby lowering cytosolic Ca2+ to induce relaxation. The stored Ca2+ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca2+, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca2+. We review here biochemical and biophysical evidence demonstrating that release of bound Ca2+ into the lumen of SR requires Ca2+/H+ exchange at the low affinity Ca2+ sites. Rise of lumenal Ca2+ above its dissociation constant from low affinity sites, or reduction of the H+ concentration by high pH, prevent Ca2+/H+ exchange. Under these conditions Ca2+ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca2+pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.

Charge transfer in P-type ATPases investigated on planar membranes

Planar lipid bilayers, e.g., black lipid membranes (BLM) and solid supported membranes (SSM), have been employed to investigate charge movements during the reaction cycle of P-type ATPases. The BLM/SSM method allows a direct measurement of the electrical currents generated by the cation transporter following chemical activation by a substrate concentration jump. The electrical current transients provides information about the reaction mechanism of the enzyme. In particular, the BLM/SSM technique allows identification of electrogenic steps which in turn may be used to localize ion translocation during the reaction cycle of the pump. In addition, using the high time resolution of the technique, especially when rapid activation via caged ATP is employed, rate constants of electrogenic and electroneutral steps can be determined. In the present review, we will discuss the main results obtained by the BLM and SSM methods and how they have contributed to unravel the transport mechanism of P-type ATPases.

Distinctive Features of Catalytic and Transport Mechanisms in Mammalian Sarco-endoplasmic Reticulum Ca2+ ATPase (SERCA) and Cu+ (ATP7A/B) ATPases

Background: SERCA and ATP7A/B are P-type ATPases entailing phosphoenzyme intermediate (E-P) formation. Results: Biochemical characterization, however, reveals distinctive features. Conclusion: A H+-gated Ca2+ pathway coupled to E1-P to E2-P transition is SERCA-specific. Headpiece interactions with an N-metal binding extension (NMBD) and related conformational constraints are ATP7A/B-specific. Significance: SERCA and ATP7A/B mechanisms present significant differences even though they are both P-type. Ca2+ (sarco-endoplasmic reticulum Ca2+ ATPase (SERCA)) and Cu+ (ATP7A/B) ATPases utilize ATP through formation of a phosphoenzyme intermediate (E-P) whereby phosphorylation potential affects affinity and orientation of bound cation. SERCA E-P formation is rate-limited by enzyme activation by Ca2+, demonstrated by the addition of ATP and Ca2+ to SERCA deprived of Ca2+ (E2) as compared with ATP to Ca2+-activated enzyme (E1·2Ca2+). Activation by Ca2+ is slower at low pH (2H+·E2 to E1·2Ca2+) and little sensitive to temperature-dependent activation energy. On the other hand, subsequent (forward or reverse) phosphoenzyme processing is sensitive to activation energy, which relieves conformational constraints limiting Ca2+ translocation. A “H+-gated pathway,” demonstrated by experiments on pH variations, charge transfer, and Glu-309 mutation allows luminal Ca2+ release by H+/Ca2+ exchange. As compared with SERCA, initial utilization of ATP by ATP7A/B is much slower and highly sensitive to temperature-dependent activation energy, suggesting conformational constraints of the headpiece domains. Contrary to SERCA, ATP7B phosphoenzyme cleavage shows much lower temperature dependence than EP formation. ATP-dependent charge transfer in ATP7A and -B is observed, with no variation of net charge upon pH changes and no evidence of Cu+/H+ exchange. As opposed to SERCA after Ca2+ chelation, ATP7A/B does not undergo reverse phosphorylation with Pi after copper chelation unless a large N-metal binding extension segment is deleted. This is attributed to the inactivating interaction of the copper-deprived N-metal binding extension with the headpiece domains. We conclude that in addition to common (P-type) phosphoenzyme intermediate formation, SERCA and ATP7A/B possess distinctive features of catalytic and transport mechanisms.

ATP dependent charge movement in ATP7B Cu+-ATPase is demonstrated by pre-steady state electrical measurements.

ATP7B is a copper dependent P‐type ATPase, required for copper homeostasis. Taking advantage of high yield heterologous expression of recombinant protein, we investigated charge transfer in ATP7B. We detected charge displacement within a single catalytic cycle upon ATP addition and formation of phosphoenzyme intermediate. We attribute this charge displacement to movement of bound copper within ATP7B. Based on specific mutations, we demonstrate that enzyme activation by copper requires occupancy of a site in the N‐terminus extension which is not present in other transport ATPases, as well as of a transmembrane site corresponding to the cation binding site of other ATPases.