Biological Function and Site II Ca2+-induced Opening of the Regulatory Domain of Skeletal Troponin C Are Impaired by Invariant Site I or II Glu Mutations

Pearlstone, Joyce R.; Chandra, Murali; Sorenson, Martha M.; Smillie, Lawrence B.

doi:10.1074/jbc.m001000200

Cited by 23 publications

(22 citation statements)

References 75 publications

(117 reference statements)

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“…K d s for magnesium binding were 2.2 mM and 0.5 mM for TN-L15 and TN-humTnC, respectively. Site-directed mutagenesis has been used extensively to study ligand-binding properties and conformational change within troponin C. Therefore, we inactivated individual EF-hands systematically by exchanging crucial aspartate or glutamate residues within the binding loops with either alanine or glutamine (20,21). The mutation D107A (20), by which the third, Cterminal EF-hand was inactivated within TN-L15, resulted in an indicator with reduced calcium affinity, in agreement with published results.…”

Section: Ratiometric Indicators With New Calcium-binding Moieties-itsupporting

confidence: 74%

Genetically Encoded Indicators of Cellular Calcium Dynamics Based on Troponin C and Green Fluorescent Protein

Heim

Griesbeck

2004

Journal of Biological Chemistry

237

236

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Genetic calcium probes offer tremendous potential in the fields of neuroscience, cell biology, and pharmaceutical screening. Previously, ratiometric and non-ratiometric indicators of cellular calcium dynamics have been described that consist of mutants of the green fluorescent protein (GFP) as fluorophores and calmodulin as calcium-binding moiety in several configurations. However, these calmodulin-based types of probes have a series of deficiencies, such as reduced dynamic ranges, when expressed within transgenic organisms and lack of calcium sensitivity in certain targetings. We developed novel types of calcium probes based on troponin C variants from skeletal and cardiac muscle. These indicators have ratio changes up to 140%, K d s ranging from 470 nM to 29 M, and improved subcellular targeting properties. We targeted the indicators to the plasma membrane of HEK293 cells and primary hippocampal neurons. Upon long lasting depolarization, submembrane calcium levels in hippocampal neurons were found to be in equilibrium with bulk cytosolic calcium levels, suggesting no standing gradient persists from the membrane toward the cytosol. We expect that such novel indicators using specialized calcium sensing proteins will be minimally interacting with the cellular biochemical machinery.Genetically encoded fluorescent indicators used to visualize cellular calcium levels have many advantages over other fluorescent dyes that have to be applied externally. They are generated in situ inside cells after transfection, do not require cofactors, can be specifically targeted to cell organelles and cellular microenvironments, and do not leak out of cells during longer recording sessions. Furthermore, they can be expressed within intact tissues of transgenic organisms and thus solve the problem of loading an indicator dye into tissue, while allowing the labeling of specific subsets of cells of interest (for review, see Ref. 1). Two classes of green fluorescent protein (GFP) 1 -based calcium indicators have been described so far: (i) ratiometric indicators (termed "cameleons") consisting of a pair of fluorescent proteins engineered for fluorescence resonance energy transfer (FRET) which carry the calcium-binding protein calmodulin as well as a calmodulin target peptide sandwiched between the GFPs (2-4); and (ii) various non-ratiometric indicators with calmodulin directly inserted into a single fluorescent protein (5-8). However, calmodulin-based indicators show deficiencies in certain applications, e.g. they display only a reduced dynamic range in transgenic invertebrates compared with in vitro data of the purified indicator proteins and acute transfections (9 -11) and fail to show calcium responses when targeted to certain sites within cells. No successful transgenic expression in mammals has been reported yet. Calmodulin is a ubiquitous signal protein in cell metabolism and thus under stringent regulation involving a plethora of calmodulinbinding proteins (12). It activates numerous kinases and phosphatases, modulates ion channe...

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Section: Ratiometric Indicators With New Calcium-binding Moieties-itsupporting

confidence: 74%

Genetically Encoded Indicators of Cellular Calcium Dynamics Based on Troponin C and Green Fluorescent Protein

Heim

Griesbeck

2004

Journal of Biological Chemistry

237

236

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“…However, Pearlstone et al (57) highlighted the difficulty in conferring biological significance to cooperativity inferred from Ca 2ϩ titrations using indirect spectroscopic methods. This is mainly because of Hill coefficients greater than unity which were predicted for processes known to be related to the filling of only one site (57). Although our fluorescence data led in some cases to predicted Hill coefficients different from unity (Table II), we consider that there is no secure reason to assume all those values as real indications of positive or negative cooperativity.…”

Section: Ca 2ϩ Binding To the Regulatory Sites Of Tncmentioning

confidence: 42%

“…Pearlstone et al (57) suggested that this decrease is associated with opening of the N-domain, based on the characteristics of cardiac TnC (cTnC) and the E41A mutant of TnC. These proteins show neither the CD band decrease nor the opening of N-domain upon calcium binding (53,55).…”

Section: Ca 2ϩ Binding To the Regulatory Sites Of Tncmentioning

confidence: 99%

Parallel Measurement of Ca2+ Binding and Fluorescence Emission upon Ca2+ Titration of Recombinant Skeletal Muscle Troponin C

Valência

Paulucci²,

Quaggio

et al. 2003

Journal of Biological Chemistry

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“…Preparation of cTnC"WT" and fsTnC-The pET3a⅐cTnC and pET3a⅐fsTnC constructs have been described previously (15,16). For the present study, the Cys-34 and Cys-83 codons (TGC) of pET3a⅐cTnC were mutated to Ser codons (AGC) using paired 31-and 37-mer oligonucleotides, respectively (Stratagene).…”

Section: Methodsmentioning

confidence: 99%

Single Mutation (A162H) in Human Cardiac Troponin I Corrects Acid pH Sensitivity of Ca2+-regulated Actomyosin S1 ATPase

Dargis

Pearlstone

Barrette-Ng³

et al. 2002

Journal of Biological Chemistry

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Biological Function and Site II Ca2+-induced Opening of the Regulatory Domain of Skeletal Troponin C Are Impaired by Invariant Site I or II Glu Mutations

Cited by 23 publications

References 75 publications

Genetically Encoded Indicators of Cellular Calcium Dynamics Based on Troponin C and Green Fluorescent Protein

Genetically Encoded Indicators of Cellular Calcium Dynamics Based on Troponin C and Green Fluorescent Protein

Parallel Measurement of Ca2+ Binding and Fluorescence Emission upon Ca2+ Titration of Recombinant Skeletal Muscle Troponin C

Single Mutation (A162H) in Human Cardiac Troponin I Corrects Acid pH Sensitivity of Ca2+-regulated Actomyosin S1 ATPase

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