Ca(2+) binding to cardiac troponin C: effects of temperature and pH on mammalian and salmonid isoforms.
A reduction in temperature lowers the Ca(2+) sensitivity of skinned cardiac myofilaments but this effect is attenuated when native cardiac troponin C (cTnC) is replaced with skeletal TnC. This suggests that conformational differences between the two isoforms mediate the influence of temperature on contractility. To investigate this phenomenon, the functional characteristics of bovine cTnC (BcTnC) and that from rainbow trout, Oncorhynchus mykiss, a cold water salmonid (ScTnC), have been compared. Rainbow trout maintain cardiac function at temperatures cardioplegic to mammals. To determine whether ScTnC is more sensitive to Ca(2+) than BcTnC, F27W mutants were used to measure changes in fluorescence with in vitro Ca(2+) titrations of site II, the activation site. When measured under identical conditions, ScTnC was more sensitive to Ca(2+) than BcTnC. At 21 degrees C, pH 7.0, as indicated by K(1/2) (-log[Ca] at half-maximal fluorescence, where [Ca] is calcium concentration), ScTnC was 2.29-fold more sensitive to Ca(2+) than BcTnC. When pH was kept constant (7.0) and temperature was lowered from 37.0 to 21.0 degrees C and then to 7.0 degrees C, the K(1/2) of BcTnC decreased by 0.13 and 0.32, respectively, whereas the K(1/2) of ScTnC decreased by 0.76 and 0.42, respectively. Increasing pH from 7.0 to 7.3 at 21.0 degrees C increased the K(1/2) of both BcTnC and ScTnC by 0.14, whereas the K(1/2) of both isoforms was increased by 1.35 when pH was raised from 7.0 to 7.6 at 7.0 degrees C.
Gillis
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Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
Marshall
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Xue
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Borgford
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TJ
Tibbits
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GF
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D003080:Cold Temperature; D006863:Hydrogen-Ion Concentration; D009119:Muscle Contraction; D016297:Mutagenesis, Site-Directed; D017686:Oncorhynchus mykiss / Q000378:metabolism*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013045:Species Specificity; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D019209:Troponin C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D019209:Troponin C; D002118:Calcium