The sarco(endo)plasmic reticulum Ca 2 + À 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.
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 P-type ATPases, electrical measurements were performed on sarcoplasmic reticulum Ca-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 IC value of 1.3 μM) than of Na,K-ATPase (with an IC value 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.
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