Kinetic and Equilibrium Properties of Regulatory Calcium Sensors of NCX1 Protein

Boyman, Liron; Mikhasenko, Helen; Hiller, Reuben; Khananshvili, Daniel

doi:10.1074/jbc.m809012200

Cited by 63 publications

(122 citation statements)

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“…4). A recent study measured Ca 2ϩ binding to purified CBD1 or CBD2 using a fluorescence-based assay and by 45 Ca 2ϩ binding (17). This study concluded that between 1.8 and 2.4 Ca 2ϩ ions bind to CBD1, whereas between 1.6 and 2.0 bind to CBD2, suggesting that the binding sites of the CBDs were only partially occupied.…”

Section: Discussionmentioning

confidence: 97%

Structure and Functional Analysis of a Ca2+ Sensor Mutant of the Na+/Ca2+ Exchanger

Chaptal¹,

Ottolia²,

Mercado-Besserer³

et al. 2009

Journal of Biological Chemistry

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The mammalian Na ؉ /Ca 2؉ exchanger, NCX1.1, serves as the main mechanism for Ca 2؉ efflux across the sarcolemma following cardiac contraction. In addition to transporting Ca 2؉ , NCX1.1 activity is also strongly regulated by Ca 2؉ binding to two intracellular regulatory domains, CBD1 and CBD2. The structures of both of these domains have been solved by NMR spectroscopy and x-ray crystallography, greatly enhancing our understanding of Ca 2؉ regulation. Nevertheless, the mechanisms by which Ca 2؉ regulates the exchanger remain incompletely understood. -ATPase) and expulsion from the cell (primarily through the Na ϩ /Ca 2ϩ exchanger, NCX1.1) results in relaxation (1). Altered Ca 2ϩ cycling is observed in a number of pathophysiological situations including ischemia, hypertrophy, and heart failure (2). Understanding the function and regulation of NCX1.1 is thus of fundamental importance to understand cardiac physiology.NCX1.1 utilizes the electrochemical potential of the Na ϩ gradient to extrude Ca 2ϩ in a ratio of three Na ϩ ions to one Ca 2ϩ ion (3). In addition to transporting both Na ϩ and Ca 2ϩ , NCX1.1 is also strongly regulated by these two ions. Intracellular Na ϩ can induce NCX1.1 to enter an inactivated state, whereas Ca 2ϩ bound to regulatory sites removes Na ϩ -dependent inactivation and also activates Na ϩ /Ca 2ϩ exchange (3). These regulatory sites are located on a large cytoplasmic loop (ϳ500 residues located between transmembrane helices V and VI) containing two calcium binding domains (CBD1 and CBD2), which sense cytosolic Ca 2ϩ levels. We have previously shown that Ca 2ϩ binding to the primary site in CBD2 is required for full exchange regulation (4); CBD1, however, is a site of higher affinity and appears to dominate the activation of exchange activity by Ca 2ϩ . Both CBDs have an immunoglobulin fold formed from two antiparallel ␤ sheets generating a ␤ sandwich with a differing number of Ca 2ϩ ions coordinated at the tip of the domain (4, 5). CBD1 binds four Ca 2ϩ ions, whereas CBD2 binds only two Ca 2ϩ ions. An initial NMR study revealed a local unfolding of the upper portion of CBD1 upon release of Ca 2ϩ (6). In contrast, CBD2 did not display an unfolding response upon Ca 2ϩ removal. A comparative analysis between CBDs revealed a difference in charge at residues in equivalent positions near the Ca 2ϩ coordination site; Glu-454 in CBD1 is replaced by Lys-585 in CBD2. The unstructuring of CBD1 upon Ca 2ϩ removal was alleviated by reversing the charge of the acidic residue (E454K) involved in Ca 2ϩ coordination (6). Previously, we solved the structures of the Ca 2ϩ -bound and -free conformations of CBD2 and revealed a charge compensation mechanism involving Lys-585 (4). The positively charged lysine residue assumes the position of one of the Ca 2ϩ ions upon Ca 2ϩ depletion, permitting CBD2 to retain its overall fold (4). A similar phenomenon is predicted to take place in E454K-CBD1 mutant. In addition, Hilge et al. (6) showed that the E454K mutation of CBD1 decreases Ca 2ϩ affinity to a level simi...

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Section: Discussionmentioning

confidence: 97%

Structure and Functional Analysis of a Ca2+ Sensor Mutant of the Na+/Ca2+ Exchanger

Chaptal¹,

Ottolia²,

Mercado-Besserer³

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Stopped-flow Experiments-Quin-2 was used in the stoppedflow experiments to monitor Ca 2ϩ off rates (16,17). In the stopped-flow machine SFM-3 (BioLogic), 150 l (syringe A) of proteins in buffer (100 mM KCl and 10 mM Bistris propane) were mixed with 150 l of buffer plus 200 -600 M Quin-2 (syringe B).…”

Section: Overexpression and Purification Of Cbd1 Cbd2 And Cbd12mentioning

confidence: 99%

“…Proteins-The DNA constructs of CBD1, CBD2, and CBD12 (AD-splice variant) were cloned into a pET23b vector and expressed in Escherichia coli Rosetta2 (DE3) competent cells (Novagen) as described (16,17). Overexpressed proteins were purified on nickel beads (Ͻ95% purity, judged by SDS-PAGE).…”

Section: Overexpression and Purification Of Cbd1 Cbd2 And Cbd12mentioning

confidence: 99%

“…All Ca 2ϩ binding assays were done at 22-23°C. The 45 Ca 2ϩ titration curves were fitted to a Hill or Adair equation (16,17).…”

Section: Overexpression and Purification Of Cbd1 Cbd2 And Cbd12mentioning

confidence: 99%

“…[Ca 2ϩ ] i , pH i , and I NCX were measured in single ventricular myocytes (13)(14)(15). 45 Ca 2ϩ binding to purified preparations of CBD1 and CBD2 was assayed by ultrafiltration and Ca 2ϩ off rates were measured by stopped-flow assay (16,17). We have identified and characterized the Ca 2ϩ sensitivity of NCX at CBD1 and CBD2 and discovered a process that is profoundly modulated by pH.…”

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confidence: 99%

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