Abstract
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.
Highlights
The sarco(endo)plasmic reticulum Ca2+À ATPase (SERCA), belonging to the superfamily of membrane transporters known as P-type ATPases, is an intracellular membrane-associated enzyme of approximately 110 kDa.[1,2,3] In muscle cells SERCA couples the hydrolysis of one ATP molecule to the transport of two Ca2+ ions against their electrochemical potential gradient from the cytoplasm into the lumen of the sarcoplasmic reticulum (SR)
We employed the solid supported membrane (SSM) method to characterize the interaction of CDN1163 with SERCA with the aim of elucidating at a molecular level the ability of CDN1163 to enhance ATPdependent translocation of Ca2+ ions by SERCA
This result indicates that CDN1163 directly interacts with SERCA, and its stimulatory effect does not depend on the presence of SERCA regulatory partners such as sarcolipin that is found in the native SR vesicles
Summary
The sarco(endo)plasmic reticulum Ca2+À ATPase (SERCA), belonging to the superfamily of membrane transporters known as P-type ATPases, is an intracellular membrane-associated enzyme of approximately 110 kDa.[1,2,3] In muscle cells SERCA couples the hydrolysis of one ATP molecule to the transport of two Ca2+ ions against their electrochemical potential gradient from the cytoplasm into the lumen of the sarcoplasmic reticulum (SR). The SERCA transport cycle includes initial enzyme activation triggered by high affinity binding of two Ca2+ ions from the cytoplasmic side. This initial step is followed by enzyme phosphorylation by ATP and formation of a phosphorylated intermediate. In this study we have employed a biosensing approach based on a solid supported membrane (SSM) to characterize the mechanism of interaction between SERCA and CDN1163. This method has been used to investigate in detail the ion translocation mechanism of the SERCA enzyme.[25] The SSM. Electrical measurements were performed to evaluate the concentration dependence of the CDN1163 stimulatory effect on SERCA transport activity
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