Capsaicin Stimulates Uncoupled ATP Hydrolysis by the Sarcoplasmic Reticulum Calcium Pump

Abstract

In muscle cells the sarcoplasmic reticulum (SR) Ca2؉ -ATPase (SERCA) couples the free energy of ATP hydrolysis to pump Ca 2؉ ions from the cytoplasm to the SR lumen. In addition, SERCA plays a key role in non-shivering thermogenesis through uncoupled reactions, where ATP hydrolysis takes place without active Ca 2؉ translocation. Capsaicin (CPS) is a naturally occurring vanilloid, the consumption of which is linked with increased metabolic rate and core body temperature. Here we document the stimulation by CPS of the Ca 2؉ -dependent ATP hydrolysis by SERCA without effects on Ca 2؉ accumulation. The stimulation by CPS was significantly dependent on the presence of a Ca 2؉ gradient across the SR membrane. ATP activation assays showed that the drug reduced the nucleotide affinity at the catalytic site, whereas the affinity at the regulatory site increased. Several biochemical analyses indicated that CPS stabilizes an ADP-insensitive E 2 P-related conformation that dephosphorylates at a higher rate than the control enzyme. Under conditions where uncoupled SERCA was specifically inhibited by the treatment with fluoride, low temperatures, or dimethyl sulfoxide, CPS had no stimulatory effect on ATP hydrolysis by SERCA. It is concluded that CPS stabilizes a SERCA sub-conformation where Ca 2؉ is released from the phosphorylated intermediate to the cytoplasm instead of the SR lumen, increasing ATP hydrolysis not coupled with Ca 2؉ transport. To the best of our knowledge CPS is the first natural drug that augments uncoupled SERCA, presumably resulting in thermogenesis. The role of CPS as a SERCA modulator is discussed. Transient increase in cytoplasmic Ca2ϩ concentration ([Ca 2ϩ ] cyt ) is a common mechanism in cellular signaling. In the working muscle, the sarcoplasmic reticulum (SR) 2 Ca 2ϩ -ATPase (SERCA) rapidly clears [Ca 2ϩ ] cyt to ensure muscle relaxation. SERCA uses energy from ATP hydrolysis to build up a Ca 2ϩ gradient across the SR membrane that can reach up to 4 orders of magnitude. SERCA is the best-characterized member of the P-type ATPase family (1). Our understanding of this ion pump was initially based on extensive biochemical and molecular biological studies (e.g. for review, see Refs. 2 and 3). Crystal structures of several SERCA isomers have provided essential structural information and largely exposed the molecular mechanism of this pump (Refs. 4 -8; for review, see Refs. 9 and 10).An interesting feature in the crystal structures of SERCA is that the cytoplasmic and the transmembrane (TM) domains, which are rigid structures, are interconnected with flexible links (4 -10). Because of their importance in energy transduction, the functional importance of the flexible links in SERCA has been intensively investigated by mutational analyses (e.g. 11-13). As discussed previously (14), one of the functional roles of the flexible links could be to generate complex domain movements, possibly allowing the pump to isomerate among several distinct sub-conformations. Indeed, several SERCA isomerization steps ha...

The role of skeletal‐muscle‐based thermogenic mechanisms in vertebrate endothermy

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Uncoupling of sarcoplasmic reticulum Ca2+‐ATPase by N‐arachidonoyl dopamine. Members of the endocannabinoid family as thermogenic drugs

“…We have studied how dissipation of the Ca 2+ gradient using a Ca 2+ ionophore would affect the stimulation of SERCA by NADA. Results in Figure 2B point to a fundamental difference between NADA and capsaicin (Mahmmoud, 2008); although NADA stimulated ATP hydrolysis by SERCA in the presence of a Ca 2+ gradient, i.e., at concentrations of A23187 < 0.2 µg·mL −1 , no stimulation was observed in the absence of the gradient, at concentrations of A23187 > 0.2 µg·mL −1 , implying that uncoupled ATP hydrolysis was not stimulated in the absence of a Ca 2+ gradient across the membrane.…”

“…SDS‐PAGE was performed as described previously (Mahmmoud, 2008). Phosphorylation was performed in 44 mM MOPS buffer, 50 mM KCl, 2 mM EGTA, 2 mM MgCl 2 , 0.72 mM ATP and 50 µg SR protein.…”

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