Investigation of Na+,K+-ATPase on a solid supported membrane: the role of acylphosphatase on the ion transport mechanism

AbstractCharge translocation by Na+,K+-ATPase was investigated by adsorbing membrane fragments containing Na+,K+-ATPase from pig kidney on a solid supported membrane (SSM). Upon adsorption, the ion pumps were activated by performing ATP concentration jumps at the surface of the SSM, and the capacitive current transients generated by Na+,K+-ATPase were measured under potentiostatic conditions. To study the behavior of the ion pump under multiple turnover conditions, ATP concentration jump experiments were carried out in the presence of Na+ and K+ ions. Current transients induced by ATP concentration jumps were also recorded in the presence of the enzyme α-chymotrypsin. The effect of acylphosphatase (AcP), a cytosolic enzyme that may affect the functioning of Na+,K+-ATPase by hydrolyzing its acylphosphorylated intermediate, was investigated by performing ATP concentration jumps both in the presence and in the absence of AcP. In the presence of Na+ but not of K+, the addition of AcP causes the charge translocated as a consequence of ATP concentration jumps to decrease by about 50% over the pH range from 6 to 7, and to increase by about 20% at pH 8. Conversely, no appreciable effect of pH upon the translocated charge is observed in the absence of AcP. The above behavior suggests that protons are involved in the AcP-catalyzed dephosphorylation of the acylphosphorylated intermediate of Na+,K+-ATPase

Stimulation of Ca2+-ATPase Transport Activity by a Small-Molecule Drug

The sarco(endo)plasmic reticulum Ca(2+)−ATPase (SERCA) hydrolyzes ATP to transport Ca(2+) 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 Ca(2+) translocation. The concentration dependence of the CDN1163 effect provided an EC(50)=6.0±0.3 μM. CDN1163 was shown to act directly on SERCA and to exert its stimulatory effect under physiological Ca(2+) concentrations. These results suggest that CDN1163 interaction with SERCA can promote a protein conformational state that favors Ca(2+) release into the SR lumen

Pre-steady state electrogenic events of Ca2+/H+ exchange and transport by the Ca2+-ATPase.

Native or recombinant SERCA (sarco(endo)plasmic reticulum Ca(2+) ATPase) was adsorbed on a solid supported membrane and then activated with Ca(2+) 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 Ca(2+) jumps in the absence of ATP and following ATP jumps in the presence of Ca(2+). The related charge movements are attributed to Ca(2+) reaching its binding sites in the ground state of the enzyme (E(1)) and to its vectorial release from the enzyme phosphorylated by ATP (E(2)P). The Ca(2+) 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 Ca(2+) at acidic (but not at alkaline) pH. Most interestingly, charge movements induced by Ca(2+) and ATP vary over different pH ranges, as the protonation probability of residues involved in Ca(2+)/H(+) exchange is lower in the E(1) than in the E(2)P state. Our single cycle measurements demonstrate that this difference contributes directly to the reduction of Ca(2+) affinity produced by ATP utilization and results in the countertransport of two Ca(2+) 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 Ca(2+) binding domain, are involved in the observed Ca(2+)/H(+) exchange

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 Ca(2+) homeostasis. We found that CLT inhibits the Ca(2+)-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 Ca(2+)-ATPase and Ca(2+) transport with a K(I) of 35 mum. Ca(2+) binding in the absence of ATP and phosphoenzyme formation by the utilization of ATP in the presence of Ca(2+) are also inhibited within the same CLT concentration range. On the other hand, phosphoenzyme formation by utilization of P(i) in the absence of Ca(2+) is only minimally inhibited. It is concluded that CLT inhibits primarily Ca(2+) binding and, consequently, the Ca(2+)-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

A case of insect colonization before the death

Forensic entomology is a branch of forensic science in which insects are used as evidence in legal investigations relating to humans, domestic animals and wildlife. One of the theoretical pillars on which the discipline is based concerns the fact that flies colonize a body after death. However in cases of myiasis, maggots are present before death, with consequences in the correct estimation of the minimum postmortem interval (mPMI). We report here the case of a woman, largely colonized by fly larvae, who has lain alive in her garden for four days prior to being rescued. Larvae were found on the conjunctivae, the bronchi, the rectum and vagina. The woman's death, two months later, was caused by tetanus. The consequences of myiasis on mPMI estimation are here discussed. In fact, despite she was still alive larvae, indicated and estimated age of 1.5–2.5 days, based on environmental and body temperature

Hofmeister effect of anions on calcium translocation by sarcoplasmic reticulum Ca2+-ATPase

The occurrence of Hofmeister (specific ion) effects in various membrane-related physiological processes is well documented. For example the effect of anions on the transport activity of the ion pump Na(+), K(+)-ATPase has been investigated. Here we report on specific anion effects on the ATP-dependent Ca(2+) translocation by the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA). Current measurements following ATP concentration jumps on SERCA-containing vesicles adsorbed on solid supported membranes were carried out in the presence of different potassium salts. We found that monovalent anions strongly interfere with ATP-induced Ca(2+) translocation by SERCA, according to their increasing chaotropicity in the Hofmeister series. On the contrary, a significant increase in Ca(2+) translocation was observed in the presence of sulphate. We suggest that the anions can affect the conformational transition between the phosphorylated intermediates E(1)P and E(2)P of the SERCA cycle. In particular, the stabilization of the E(1)P conformation by chaotropic anions seems to be related to their adsorption at the enzyme/water and/or at the membrane/water interface, while the more kosmotropic species affect SERCA conformation and functionality by modifying the hydration layers of the enzyme

A sulfur-based transport pathway in Cu^+-ATPases

Cells regulate copper levels tightly to balance the biogenesis and integrity of copper centers in vital enzymes against toxic levels of copper. P_(IB)‐type Cu^+‐ATPases play a central role in copper homeostasis by catalyzing the selective translocation of Cu^+ across cellular membranes. Crystal structures of a copper‐free Cu^+‐ATPase are available, but the mechanism of Cu^+ recognition, binding, and translocation remains elusive. Through X‐ray absorption spectroscopy, ATPase activity assays, and charge transfer measurements on solid‐supported membranes using wild‐type and mutant forms of the Legionella pneumophila Cu^+‐ATPase (LpCopA), we identify a sulfur‐lined metal transport pathway. Structural analysis indicates that Cu^+ is bound at a high‐affinity transmembrane‐binding site in a trigonal‐planar coordination with the Cys residues of the conserved CPC motif of transmembrane segment 4 (C382 and C384) and the conserved Met residue of transmembrane segment 6 (M717 of the MXXXS motif). These residues are also essential for transport. Additionally, the studies indicate essential roles of other conserved intramembranous polar residues in facilitating copper binding to the high‐affinity site and subsequent release through the exit pathway