Intermolecular interactions in the mechanism of skeletal muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA1): Evidence for a triprotomer

James E. Mahaney, David D. Thomas, Iain K. Farrance, Jeffrey P. Froehlich

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Native membrane sarcoplasmic reticulum (SR) Ca2+-ATPase isolated from skeletal muscle (SERCA1) exhibits oligomeric kinetic behavior [Mahaney, J. E., Thomas, D. D., and Froehlich, J. P. (2004) Biochemistry 43, 4400-4416]. In the present study we used quenched-flow mixing, electron paramagnetic resonance (EPR), and chemical cross-linking to probe for intermolecular interactions at physiological (0.1 M) and high (0.4 M) KCl. Exposure of SR membranes to water- and lipid-soluble cross-linking reagents revealed a mixture of SERCA1 oligomeric species consisting mainly of dimers and trimers. Titration of iodoacetamide spin-labeled SERCA1 with AMPPCP in the presence of 10 μM Ca2+ and 0.1 M KCl revealed high-(KD = 45 μM) and low-affinity (KD ) 315 μM) nucleotide binding sites in a 2:1 ratio, respectively. Raising the [KCl] to 0.4 M increased the fraction of weak binding sites and lowered the K D of the high-affinity component (20 μM). Phosphorylation by 10 μM ATP at 21°C and 0.1 M KCl produced an early burst of Pi production without a corresponding decline in the steady-state phosphoenzyme (EP) level. The steady-state EP level was twice as large as the Pi burst and received equal contributions from E1P and E2P. Chasing the phosphoenzyme at 0.4 M KCl and 2°C with ADP revealed a biphasic time course of E1P formation with a slow phase that matched the kinetics of the transient EPR signal from the spin-labeled Ca2+-ATPase. The absence of a fast component in the EPR signal excludes E1P as its source. Instead, it arises from a slow, KCl-dependent transformation at the start of the cycle which controls the formation of downstream intermediates with an increased mole fraction of rotationally restricted probes. We modeled this behavior with a SERCA1 trimer in which the formation of E1P/E2/E2P from E1ATP/E2P/E1P results from concerted transformations in the subunits coupling phosphorylation (E1ATP → E1P + ADP) to dephosphorylation (E2P → E2 + Pi) and the conversion of E1P to E2P.

Original languageEnglish (US)
Pages (from-to)13711-13725
Number of pages15
Issue number51
StatePublished - Dec 23 2008


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