A new approach to detect and study ion channel formation in microBLMs

In this paper, an innovative and versatile approach to detect pore formation in microBLMs and to investigate their ion selectivity is described. For the first time, both electrochemical impedance spectroscopy and conductivity measurements are performed on the same membrane, providing a simple, long-lasting and cheap method which can find applications in various ion channel-involving research fields. We make use of this improved experimental procedure to obtain for the first time a range of values to characterize homogeneous polycarbonate-supported microBLMs. To test the validity of our method, the well-known, channel-forming peptide gramicidin D was first employed. Then, we applied our approach to the study of the still unclear membrane interaction mechanisms of the peptides trichogin GA IV and phospholamban, finding that both can form ion channels in biomimetic models. The phospholamban-generated ion channels could be a promising therapeutic target in heart failures and other cardiac diseases. Our methodology might also be useful for detecting new drugs with ion-channel blocking action. Keywords: Bilayer lipid membranes, Ion channel, Conductivity measurements, Phospholamban, Trichogin GA IV, MicroBLM

Phospholamban generates cation selective ion channels.

Phosholamban (PLN) is involved in the contractility of cardiac muscles by regulating the intracellular calcium concentration (Ca(2+)(cyt)) of cardiac myocytes. This occurs via a modulation of the sarco-/endoplasmic CaATPase (SERCA). In spite of high-resolution structures the molecular mode of PLN action is yet not known. In the present paper we readdress the question whether PLN proteins can generate ion channel activity. Reconstitution of PLN in planar lipid bilayers reveals single channel fluctuations, which are characterized by two conductance levels, long open/closed dwell times, moderate selectivity between monovalent cations and no perceivable Ca(2+) permeability. The PLN generated channel activity could be inhibited by a PLN antibody (abPLN) implying that the channel activity is indeed due to the inherent channel function of the PLN protein

Drug Interactions With the Ca2+-ATPase From Sarco(Endo)Plasmic Reticulum (SERCA)

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is an intracellular membrane transporter that utilizes the free energy provided by ATP hydrolysis for active transport of Ca2+ ions from the cytoplasm to the lumen of sarco(endo)plasmic reticulum. SERCA plays a fundamental role for cell calcium homeostasis and signaling in muscle cells and also in cells of other tissues. Because of its prominent role in many physiological processes, SERCA dysfunction is associated to diseases displaying various degrees of severity. SERCA transport activity can be inhibited by a variety of compounds with different chemical structures. Specific SERCA inhibitors were identified which have been instrumental in studies of the SERCA catalytic and transport mechanism. It has been proposed that SERCA inhibition may represent a novel therapeutic strategy to cure certain diseases by targeting SERCA activity in pathogens, parasites and cancer cells. Recently, novel small molecules have been developed that are able to stimulate SERCA activity. Such SERCA activators may also offer an innovative and promising therapeutic approach to treat diseases, such as heart failure, diabetes and metabolic disorders. In the present review the effects of pharmacologically relevant compounds on SERCA transport activity are presented. In particular, we will discuss the interaction of SERCA with specific inhibitors and activators that are potential therapeutic agents for different diseases

Effect of cisplatin on the transport activity of PII-type ATPases

Cisplatin (cis-diamminedichlorido-Pt(ii)) is extensively used as a chemotherapeutic agent against various types of tumors. However, cisplatin administration causes serious side effects, including nephrotoxicity, ototoxicity and neurotoxicity. It has been shown that cisplatin can interact with P-type ATPases, e.g., Cu+-ATPases (ATP7A and ATP7B) and Na+,K+-ATPase. Cisplatin-induced inhibition of Na+,K+-ATPase has been related to the nephrotoxic effect of the drug. To investigate the inhibitory effects of cisplatin on the pumping activity of PII-type ATPases, electrical measurements were performed on sarcoplasmic reticulum Ca2+-ATPase (SERCA) and Na+,K+-ATPase embedded in vesicles/membrane fragments adsorbed on a solid-supported membrane. We found that cisplatin inhibits SERCA and Na+,K+-ATPase only when administered without a physiological reducing agent (GSH); in contrast, inhibition was also observed in the case of Cu+-ATPases in the presence of 1 mM GSH. Our results indicate that cisplatin is a much stronger inhibitor of SERCA (with an IC50value of 1.3 μM) than of Na+,K+-ATPase (with an IC50value of 11.1 μM); moreover, cisplatin inhibition of Na+,K+-ATPase is reversible, whereas it is irreversible in the case of SERCA. In the absence of a physiological substrate, while Cu+-ATPases are able to translocate cisplatin, SERCA and Na+,K+-ATPase do not perform ATP-dependent cisplatin displacement

Structure-Function Relation of Phospholamban: Modulation of Channel Activity as a Potential Regulator of SERCA Activity.

Phospholamban (PLN) is a small integral membrane protein, which binds and inhibits in a yet unknown fashion the Ca(2+)-ATPase (SERCA) in the sarcoplasmic reticulum. When reconstituted in planar lipid bilayers PLN exhibits ion channel activity with a low unitary conductance. From the effect of non-electrolyte polymers on this unitary conductance we estimate a narrow pore with a diameter of ca. 2.2 Å for this channel. This value is similar to that reported for the central pore in the structure of the PLN pentamer. Hence the PLN pentamer, which is in equilibrium with the monomer, is the most likely channel forming structure. Reconstituted PLN mutants, which either stabilize (K27A and R9C) or destabilize (I47A) the PLN pentamer and also phosphorylated PLN still generate the same unitary conductance of the wt/non-phosphorylated PLN. However the open probability of the phosphorylated PLN and of the R9C mutant is significantly lower than that of the respective wt/non-phosphorylated control. In the context of data on PLN/SERCA interaction and on Ca(2+) accumulation in the sarcoplasmic reticulum the present results are consistent with the view that PLN channel activity could participate in the balancing of charge during Ca(2+) uptake. A reduced total conductance of the K(+) transporting PLN by phosphorylation or by the R9C mutation may stimulate Ca(2+) uptake in the same way as an inhibition of K(+) channels in the SR membrane. The R9C-PLN mutation, a putative cause of dilated cardiomyopathy, might hence affect SERCA activity also via its inherent low open probability