Cloning and Expression of Salmon Cardiac Troponin C:  Titration of the Low-Affinity Ca²⁺-Binding Site Using a Tryptophan Mutant

Abstract

Activation of cardiac actomyosin ATPase requires the occupation of the single low-affinity Ca(2+)-binding site of troponin C (cTnC). Previously, we demonstrated pronounced differences between mammals and cold-water salmonid fish in the Ca2+ sensitivity of cardiac preparations, particularly in relation to temperature [Churcotte, C., Moyes, C. D., Baldwin, K., Bressler, B., & Tibbits, G. F. (1994) Am. J. Physiol. 267, R62-R70]. In this study, we examine the extent to which cTnC structure could account for the observed differences in myofibrillar Ca2+ sensitivity. Salmonid (Oncorhynchus mykiss) cTnC was cloned, sequenced, and expressed in Escherichia coli as a maltose-binding protein fusion. The coding region has 87% homology with human cTnC cDNA and differs in 13 of 161 amino acid residues from the human/bovine/porcine isoform. The sequence corresponding to the single regulatory Ca(2+)-binding site II is completely homologous to that of mammals. The protein expressed exhibits optical properties similar (circular dichroism, intrinsic fluorescence) to those of cTnC purified from salmonid (Salmo salar) and bovine ventricle. A single tryptophan residue was introduced into the inactive Ca(2+)-binding site I (ScTnC-FW27) to facilitate Ca2+ titration. The Ca(2+)-binding constant (K1/2 = 5.33 pCa units) was within the range reported for the low-affinity sites of mammalian cTnC. Although differences in TnC primary structure are striking, Ca2+ affinity of intact cardiac myofibrils is likely influenced by interactions with other troponin proteins.

Increasing mammalian cardiomyocyte contractility with residues identified in trout troponin C

“…2B). These data demonstrate that Asn 2 , Ile 28 , Gln 29 , and Asp 30 are responsible for the high Ca 2ϩ affinity of F27W ScTnC as determined in vitro. The Ca 2ϩ affinity of the full-length F27W McTnC mutants was found to increase, relative to F27W ScTnC, in a manner quantitatively similar to that of the NH 2 -terminal mutants (Fig.…”

Sequence mutations in teleost cardiac troponin C that are permissive of high Ca2+ affinity of site II

“…Fluorescent probes engineered into TnC through Phe to Trp mutation in site I have been used previously to study the Ca 2ϩ binding dynamics of this molecule (8,19,21,27,29,32,38). We have demonstrated the effectiveness of Trp at residue 27 in reporting Ca 2ϩ binding to site II in McTnC and ScTnC without significantly affecting the ␣-helical content of the protein, which reflects the general structure of the molecule, using far UV circular dichroism spectra (27).…”

“…We have demonstrated the effectiveness of Trp at residue 27 in reporting Ca 2ϩ binding to site II in McTnC and ScTnC without significantly affecting the ␣-helical content of the protein, which reflects the general structure of the molecule, using far UV circular dichroism spectra (27). Additionally, the K1⁄2 values of Ca 2ϩ binding to the F27W cNTnC mutants and full-length IFcTnC in the present study are within the range previously reported for that of Ca 2ϩ -triggered tension generation in cardiac myocytes under similar conditions (16,18,23,24,28,39).…”

“…We have demonstrated that the high Ca 2ϩ sensitivity of trout myofibrils is due, in part, to the differences in the affinity of mammalian and trout cTnC for Ca 2ϩ (10). Although the amino acid sequence of trout cTnC (ScTnC) is 91% identical to mammalian cTnC (McTnC) (27), and site II is completely conserved, ScTnC has 2.3-fold higher Ca 2ϩ affinity than McTnC at 21°C (10). This difference in affinity was maintained when McTnC and ScTnC Ca 2ϩ binding were compared at 7 and 37°C and over a range of pH values.…”

Temperature and the expression of seven muscle-specific protein genes during embryogenesis in the Atlantic codGadus morhuaL.

“…In birds, mammals and amphibians, a fast-fibre type and a type specific to both slow and cardiac fibres have been identified (Reinach and Karlsson, 1988;Gahlmann and Kedes, 1990;Parmacek et al, 1990;Jin et al, 1995;Tiso et al, 1997;Warkman and Atkinson, 2002). Numerous studies have investigated aspects of TnC function in teleosts at the protein level (Demaille et al, 1974;McCubbin et al, 1982;Gerday et al, 1984;Feller and Gerday, 1989;Crockford and Johnston, 1993;Francois et al, 1997), but until now the only published nucleotide sequences were those from zebrafish (showing highest identity to Xenopus fast-type; Xu et al, 2000) and trout (showing highest identity to Xenopus slow/cardiactype; Moyes et al, 1996). Because protein sequences with homology to both fast-skeletal and slow/cardiac forms have been isolated from fish, it has been supposed that their nature is equivalent to those in birds and mammals (Yuasa et al, 1998).…”