A functional comparison of cardiac troponin C from representatives of three vertebrate taxa: Linking phylogeny and protein function Elizabeth J. Sears, Todd E. Gillis ⁎ Department of Integrative Biology, University of Guelph, Guelph, ON N1G 2W1, Canada Cardiovasclar Research Center, University of Guelph, Canada abstract article info Article history: Received 19 June 2016 Received in revised form 18 July 2016 Accepted 19 July 2016 Available online 22 July 2016 The Ca 2+ affinity of cardiac troponin C (cTnC) from rainbow trout is significantly greater than that of cTnC from mammalian species. This high affinity is thought to enable cardiac function in trout at low physiological temper- atures and is due to residues Asn 2 , Ile 28 , Gln 29 , and Asp 30 (Gillis et al., 2005, Physiol Genomics, 22, 1–7). Interest- ingly, the cTnC of the African clawed frog Xenopus laevis (frog cTnC) contains Gln 29 and Asp 30 but the residues at positions 2 and 28 are those found in all mammalian cTnC isoforms (Asp 2 and Val 28 ). The purpose of this study was to determine the Ca 2+ affinity of frog cTnC, and to determine how these three protein orthologs influence the function of complete troponin complexes. Measurements of Ca 2+ affinity and the rate of Ca 2+ dissociation from the cTnC isoforms and cTn complexes were made by monitoring the fluorescence of anilinonapthalenesulfote iodoacetamide (IAANS) engineered into the cTnC isoforms to report changes in protein conformation. The results demonstrate that the Ca 2+ affinity of frog cTnC is greater than that of trout cTnC and human cTnC. We also found that replacing human cTnC with frog cTnC in a mammalian cTn complex increased the Ca 2+ affinity of the complex by 5-fold, which is also greater than complexes containing trout cTnC. Together these results suggest that frog cTnC has the potential to increase the Ca 2+ sensitivity of force generation by the mammalian heart. © 2016 Elsevier Inc. All rights reserved. Keywords: Protein sequence Protein function Cardiac muscle Ca 2+ activation Thermal stability 1. Introduction Myocyte contraction is initiated when Ca 2+ binds to troponin C (TnC) and triggers a series of conformational changes through the com- ponent proteins of the thin filament that result in the formation of cross-bridges between actin and myosin. Troponin C along with tropo- nin I (TnI) and troponin T (TnT) are the component proteins of the tro- ponin (Tn) complex and it is changes in the interactions between these that lead to the activation of the contractile reaction. Manipulation of the functional characteristics of the troponin complex through either the phosphorylation of specific residues or by manipulation of the amino acid sequence of the component proteins can have a significant influence on the contractile function of striated muscle (Shaffer and Gillis, 2010). For example, the phosphorylation of cardiac TnI (cTnI) by protein kinase A (PKA) following β-adrenergic stimulation decreases the Ca 2+ affinity of the Tn complex and this enables a faster rate of re- laxation between beats (Dong et al., 2007; Fentzke et al., 1999). We have also demonstrated that changes to the amino acid sequence of car- diac TnC (cTnC) that increase its Ca 2+ affinity, increase the Ca 2+ sensi- tivity of force generation by chemically skinned cardiac myocytes (Gillis et al., 2005). More specifically, the Ca 2+ affinity of rainbow trout cardiac TnC (trout cTnC) is approximately two-fold that of bovine cTnC and the residues responsible for this are Asn 2 , Ile 28 , Gln 29 , and Asp 30 (Gillis et al., 2005). When bovine cTnC was mutated to contain these four trout residues, its Ca 2+ affinity was increased two-fold, and when native cTnC in rabbit cardiac myocytes was replaced with this mutant, the Ca 2+ sensitivity of force generation was increased two- fold compared to controls (Gillis et al., 2005). The comparatively high Ca 2+ affinity of trout cTnC is thought to be responsible, in part, for the comparatively high Ca 2+ sensitivity of the trout heart (Gillis et al., 2000). This characteristic is proposed to help enable cardiac function in the trout at low physiological temperatures (Gillis and Tibbits, 2002). Comparative Biochemistry and Physiology, Part B 202 (2016) 8–15 Abbreviations: cTnC, cardiac troponin C; cTnI, cardiac troponin I; cTnT, cardiac troponin T; IAANS, anilinonapthalenesulfote iodoacetamide; k off , rate of Ca 2+ dissociation; k on , rate of Ca 2+ association; cTnC T53C , cTnC mutant where all native cysteines have been replaced with serines and Thr 53 has been mutated to a Cys; cTn, cardiac troponin; nH, Hill coefficient; K F1/2 , Ca 2+ concentration at half-maximum Ca 2+ - dependent fluorescence; C H I R T R , cTn complex composed of human cTnC, rat cTnI and rat cTnT; C F I R T R , cTn complex composed of frog cTnC, rat cTnI and rat cTnT; C T I R T R , cTn com- plex composed of trout cTnC, rat cTnI and rat cTnT; C T I T T R , cTn complex composed of trout cTnC, trout cTnI and rat cTnT. ⁎ Corresponding author. E-mail address: tgillis@uoguelph.ca (T.E. Gillis). URL: http://comparativephys.ca/gillislab/ (T.E. Gillis). http://dx.doi.org/10.1016/j.cbpb.2016.07.004 1096-4959/© 2016 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part B journal homepage: www.elsevier.com/locate/cbpb