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Interchange of autoinhibitory domain conformations in plasma-membrane Ca²⁺-ATPase–calmodulin complexes

¹ Department of Chemistry, University of Kansas, Lawrence, Kansas 66045, USA
² Department of Pharmacology and Toxicology, University of Kansas, Lawrence, Kansas 66045, USA

(RECEIVED September 13, 2007; FINAL REVISION November 19, 2007; ACCEPTED November 27, 2007)

The Ca²⁺ signaling protein calmodulin (CaM) stimulates Ca²⁺ pumping in the plasma-membrane Ca²⁺-ATPase (PMCA) by binding to an autoinhibitory domain, which then dissociates from the catalytic domain of PMCA to allow full activation of the enzyme. We measured single-molecule fluorescence trajectories with polarization modulation to track the conformation of the autoinhibitory domain of PMCA pump bound to fluorescently labeled CaM. Interchange of the autoinhibitory domain between associated and dissociated conformations was detected at a physiological Ca²⁺ concentration of 0.15 µM, where the enzyme is only partially active, but not at 25 µM, where the enzyme is fully activated. In previous work we showed that the conformation of the autoinhibitory domain in PMCA-CaM complexes could be monitored by the extent of modulation of single-molecule fluorescence generated with rotating excitation polarization. In the present work, we determined the timescale of association and dissociation of the autoinhibitory domain with the catalytic regions of the PMCA. Association of the autoinhibitory domain was rare at a high Ca²⁺ concentration (25 µM). At a lower Ca²⁺ concentration (0.15 µM), conformations of the autoinhibitory domain interchanged with a dissociation rate of 0.042 ± 0.011 sec^–1 and an association rate of 0.023 ± 0.006 sec^–1. The results indicate that the response time of PMCA upon a reduction in Ca²⁺ is limited to tens of seconds by autoinhibitory dynamics. This property may reduce the sensitivity of PMCA to transient reductions in intracellular Ca²⁺. We suggest that the dynamics of the autoinhibitory domain may play a novel role in regulating PMCA activity.

Keywords: calmodulin; plasma-membrane Ca²⁺-ATPase; autoinhibitory domain, single-molecule fluorescence; polarization modulation

Reprint requests to: Carey K. Johnson, Department of Chemistry, 1251 Wescoe Drive, University of Kansas, Lawrence, KS 66045, USA; e-mail: ckjohnson{at}ku.edu; fax: (785) 864-5396.

Abbreviations: CaM, calmodulin; CaM-TMR, calmodulin fluorescently labeled with tetramethylrhodamine; EDTA, ethylene diamine tetraacetic acid; EGTA, ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid; HEPES, N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid); MLE, maximum-likelihood estimator; PMCA, plasma-membrane Ca²⁺-ATPase; TMR, tetramethylrhodamine-5-maleimide

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.073239108.

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