Mutations of Hydrophobic Residues in the N-terminal Domain of Troponin C Affect Calcium Binding and Exchange with the Troponin C-Troponin I96–148 Complex and Muscle Force Production

Abstract

Interactions between troponin C and troponin I play a critical role in the regulation of skeletal muscle contraction and relaxation. We individually substituted 27 hydrophobic Phe, Ile, Leu, Val, and Met residues in the regulatory domain of the fluorescent troponin C F29W with polar Gln to examine the effects of these mutations on: (a) the calcium binding and dynamics of troponin C F29W complexed with the regulatory fragment of troponin I (troponin I 96 -148 ) and (b) the calcium sensitivity of force production. Troponin I 96 -148 was an accurate mimic of intact troponin I for measuring the calcium dynamics of the troponin C F29W -troponin I complexes. The calcium affinities of the troponin C F29W -troponin I 96 -148 complexes varied ϳ243-fold, whereas the calcium association and dissociation rates varied ϳ38-and ϳ33-fold, respectively. Interestingly, the effect of the mutations on the calcium sensitivity of force development could be better predicted from the calcium affinities of the troponin C F29W -troponin I 96 -148 complexes than from that of the isolated troponin C F29W mutants. Most of the mutations did not dramatically affect the affinity of calcium-saturated troponin C F29W for troponin I 96 -148 . However, the Phe 26 to Gln and Ile 62 to Gln mutations led to >10-fold lower affinity of calcium-saturated troponin C F29W for troponin I 96 -148 , causing a drastic reduction in force recovery, even though these troponin C F29W mutants still bound to the thin filaments. In conclusion, elucidating the determinants of calcium binding and exchange with troponin C in the presence of troponin I provides a deeper understanding of how troponin C controls signal transduction.Troponin C (TnC) 1 regulates striated muscle contraction and relaxation through the binding and release of Ca 2ϩ (for review see Refs. 1-3). Skeletal muscle TnC (ϳ18 kDa) consists of globular N-and C-terminal domains connected by a 31-residue ␣-helix (for review see Refs. 4 and 5). Both domains bind two Ca 2ϩ ions through a pair of EF hand Ca 2ϩ -binding motifs. Each pair of EF hands interacts with one another through a short antiparallel ␤-sheet connecting the two Ca 2ϩ -binding loops (Ref. 6 and references within). The EF hands are numbered I-IV, and the helices flanking the loops are designated A-H, with an additional N-terminal 14-residue ␣-helix (Fig. 1, N helix), which is absent in the closely related EF hand Ca 2ϩ -binding protein calmodulin.Much is known about the cation binding properties of TnC in solution. Each EF hand system binds Ca 2ϩ and Mg 2ϩ competitively, with the two C-terminal EF hands possessing higher Ca 2ϩ and Mg 2ϩ affinities (6 -8). In fact, the Ca 2ϩ -binding sites of the C-domain of TnC possess ϳ10-fold higher Ca 2ϩ affinity with a greater than 100-fold slower Ca 2ϩ dissociation rate compared with those in the N-domain (6, 9). In part because of its high Ca 2ϩ and Mg 2ϩ affinities and slow Ca 2ϩ exchange rates (as compared with the kinetics of muscle contraction and relaxation), the C-domain is thought to play a struc...

Engineered Troponin C Constructs Correct Disease-related Cardiac Myofilament Calcium Sensitivity

“…TnC, TnI, and TnT were purified as described previously (15,16). Rabbit skeletal actin and bovine ventricular cardiac tropomyosin (Tm) were purified from acetone powders as described previously (34,35).…”

“…Different isoforms of the troponin complex (Tn) within an organism and between different species help to tune the response of TnC to Ca 2ϩ to meet developmental and environmental demands of the heart (11)(12)(13)(14)(15). In this regard, TnC does not behave like a simple switch because the Ca 2ϩ binding properties of the regulatory EF-hand of TnC are modulated by interactions with its protein binding partner, troponin I (TnI) (16,17). The response of TnC to Ca 2ϩ can be further adjusted by additional myofilament proteins (troponin T, actin, tropomyosin, and myosin) and by an assortment of posttranslational modifications to many of these proteins (5,(17)(18)(19).…”

“…Unfortunately, there are no pharmacological compounds that just target TnC (31). However, by directly engineering TnC, our laboratory has developed several TnC constructs, which behave as Ca 2ϩ sensitizers or desensitizers in biochemical model systems and in muscle (16,32,33). By utilizing different design principles (32,33), the intrinsic Ca 2ϩ binding properties of TnC can be finely or grossly tuned.…”

Designing Calcium-sensitizing Mutations in the Regulatory Domain of Cardiac Troponin C

“…To follow Ca 2 ϩ binding and exchange with the regulatory domain of cTnC, we utilized the intrinsic fluorescence of cTnC F27W , which has been previously used to study Ca 2ϩ binding to the N-domain site of cTnC and its mutants or isoforms (26 -29). This mutation is analogous to sTnC F29W , which enabled numerous studies of Ca 2ϩ and target protein binding to the N-domain of sTnC and its mutants (6,8,22,25,42).…”

Cardiac Troponin T Isoforms Affect the Ca2+ Sensitivity of Force Development in the Presence of Slow Skeletal Troponin I

“…The Tn samples were excited using a 150-watt xenon arc source. k off was determined using the fluorescent Ca 2ϩ chelator Quin-2 (19,20). Quin-2 was excited at 330 nm with its emission monitored through a 510-nm broad bandpass interference filter (Oriel (Stratford, CT)).…”