Expanding the range of free calcium regulation in biological solutions

Dweck, David; Reyes-Alfonso, Avelino; Potter, James D.

doi:10.1016/j.ab.2005.09.025

Cited by 80 publications

(100 citation statements)

References 5 publications

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“…The fiber slack was removed until tension developed, and then the fiber was stretched 1.2ϫ its length. This results in estimated sarcomere lengths between 2.2 and 2.3 m. The various pCa solutions were prepared using the binding constants at 20°C (18). The contraction solution (pCa 4.0) was the same composition as the pCa 8.0 solution, except that the free Ca 2ϩ concentration was 10 Ϫ4 mol/liter and was used to measure the initial force.…”

Section: Methodsmentioning

confidence: 99%

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A Dilated Cardiomyopathy Troponin C Mutation Lowers Contractile Force by Reducing Strong Myosin-Actin Binding

Dweck¹,

Reynaldo

Pinto³

et al. 2010

Journal of Biological Chemistry

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In this study we explore the mechanisms by which a double mutation (E59D/D75Y) in cardiac troponin C (CTnC) associated with dilated cardiomyopathy reduces the Ca 2؉ -activated maximal tension of cardiac muscle. Studying the single mutants (i.e. E59D or D75Y) indicates that D75Y, but not E59D, causes a reduction in the calcium affinity of CTnC in troponin complex, regulated thin filaments (RTF), and the Ca 2؉ sensitivity of contraction and ATPase in cardiac muscle preparations. However, both D75Y and E59D are required to reduce the actomyosin ATPase activity and maximal force in muscle fibers, indicating that E59D enhances the effects of D75Y. Part of the reduction in force/ATPase may be due to a defect in the interactions between CTnC and cardiac troponin T, which are known to be necessary for ATPase activation. An additional mechanism for the reduction in force/ATPase comes from measurements of the binding stoichiometry of myosin subfragment-1 (S-1) to the RTF. Using wild type RTFs, 4.8 mol S-1 was bound per mol filament (seven actins), whereas with E59D/D75Y RTFs, the number of binding sites was reduced by ϳ23% to 3.7. Altogether, these results suggest that the reduction in force and ATPase activation is possibly due to a thin filament conformation that promotes fewer accessible S-1-binding sites. In the absence of any family segregation data, the functional results presented here support the concept that this is likely a dilated cardiomyopathy-causing mutation.

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Section: Methodsmentioning

confidence: 99%

“…Preparation of Buffered Calcium Solutions-Methods for solving the free and bound metal ion equilibria in our solutions were provided for by the computer program pCa Calculator (18).…”

Section: Methodsmentioning

confidence: 99%

A Dilated Cardiomyopathy Troponin C Mutation Lowers Contractile Force by Reducing Strong Myosin-Actin Binding

Dweck¹,

Reynaldo

Pinto³

et al. 2010

Journal of Biological Chemistry

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“…Each duplicate control well received an equal volume of KCl, reflecting the volume of CaCl 2 added to the experimental well. The volume of CaCl 2 to be added was calculated using a pCa calculator (43). ANS fluorescence was measured in top reading mode using a Tecan Infinite M200Pro with excitation of 325 Ϯ 4.5 nm and emission at 450 Ϯ 10 nm, with an integration time of 2 ms and a gain of 136.…”

Section: With Nbd a 2 MM Stock Solution Of N-((2-(iodoacetoxy)ethyl)mentioning

confidence: 99%

“…The 10ϫ pCa buffers were prepared with 20 mM EGTA, 40 mM nitrilotriacetic acid, and varying concentrations of CaCl 2 . Free Ca 2ϩ concentrations were calculated using the pCa calculator developed by Dweck et al (43). Assays contained 7 M actin, 2 M Tm, 3 M Tn, 0.1 M human ␤-cardiac S1 and the appropriate 1ϫ pCa buffer in a 96-well plate.…”

Section: With Nbd a 2 MM Stock Solution Of N-((2-(iodoacetoxy)ethyl)mentioning

confidence: 99%

Mechanistic Heterogeneity in Contractile Properties of α-Tropomyosin (TPM1) Mutants Associated with Inherited Cardiomyopathies

Gupte

Haque

Gangadharan

et al. 2015

Journal of Biological Chemistry

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Background: Single residue substitutions in sarcomeric proteins cause most inherited cardiomyopathies. Results: Mutant ␣-tropomyosins cause multiple functional alterations in actin affinity and Ca 2ϩ sensitivity. Conclusion: Mutants follow distinct mechanisms to change Ca 2ϩ sensitivity. Significance: Fluorescence assays to measure changes in troponin C conformation may provide a simple platform for preliminary high throughput screening of modulatory small molecules to treat inherited cardiomyopathies.

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“…Once relaxed, the fibers were exposed to solutions of increasing [Ca 2ϩ ] (from pCa 8.0 to 4.0). The various pCa solutions were calculated using a computer program (pCa calculator) developed in our laboratory (26). Data were analyzed using the following equation: Percent change in force ϭ 100…”

Section: Skinned Fiber Studiesmentioning

confidence: 99%

The Functional Properties of Human Slow Skeletal Troponin T Isoforms in Cardiac Muscle Regulation

Pinto

Gomes

Jones

et al. 2012

Journal of Biological Chemistry

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Expanding the range of free calcium regulation in biological solutions

Cited by 80 publications

References 5 publications

A Dilated Cardiomyopathy Troponin C Mutation Lowers Contractile Force by Reducing Strong Myosin-Actin Binding

A Dilated Cardiomyopathy Troponin C Mutation Lowers Contractile Force by Reducing Strong Myosin-Actin Binding

Mechanistic Heterogeneity in Contractile Properties of α-Tropomyosin (TPM1) Mutants Associated with Inherited Cardiomyopathies

The Functional Properties of Human Slow Skeletal Troponin T Isoforms in Cardiac Muscle Regulation

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