TnC Mutant Research Articles

Disease Causing Troponin C Mutations Have Varied Effects on Actin Regulatory States

Cardiomyopathy associated mutations in troponin often affect the actin-activated ATPase activity of myosin and the response of force and ATPase activity to Ca2+. Three hypertrophic cardiomyopathy TnC mutants A8V, C84Y, and D145E were examined for their effects on the distribution of states of regulated actin in vitro. In the absence of activating Ca2+, regulated actin filaments containing the C84Y mutant produced a 2.5x elevation in ATPase activity of skeletal myosin subfragment 1 compared to wild type; the other mutants had normal activity. We examined acrylodan-tropomyosin fluorescence changes that occur following rapid detachment of S1 from regulated actin in the absence of Ca2+. All troponin mutants gave a fluorescence increase indicative of a normal transition into the inactive state. The A8V mutant differed from wild type and other TnC mutants at saturating Ca2+. Actin filaments containing the A8V TnC mutant produced an ATPase rate in Ca2+ that was 1.3x wild type. For comparison, the Δ14TnT mutant that destabilizes the inactive state was 1.7x wild type. Interestingly, the effects of the A8V-TnC and Δ14-TnT mutants were additive leading to a Ca2+-activated rate 2.3x wild type. The A8V TnC mutant appeared to favor the active state relative to wild type but only in the presence of Ca2+. This behavior differed from the Δ14 mutation of TnT where the distribution was also altered at low Ca2+ conditions.

Effects of TNI Inhibitory Peptide Mutation on Troponin Dynamics

Striated muscles are relaxed under low calcium concentration conditions due to actions of the thin filament protein troponin. To investigate this regulatory mechanism, this project studies the dynamic behavior of a mutant troponin in which an 11-residue segment of cardiac troponin I, known as the inhibitory region, has been converted to a Gly-Ala flexible linker. The functional effects of this mutation were previously characterized. (Mouannes Kozaili, et al, JBC 2010). The mutation impairs rather than abolishes troponin's inhibitory function, and thin filaments retain cooperative Ca2+-sensitive properties. Previous work from our lab has mapped the dynamic behavior of the Ca2+-saturated cardiac troponin core domain at 10°C and 25°C using hydrogen/deuterium exchange (HDX)-mass spectrometry. (Kowlessur D, et al, JBC 2010, JBC 2010a). Also, another previous study established the effects of Ca2+ binding to site II of TnC in the NH2 domain on troponin dynamics, using the TnC mutant D65A/E66A (CBMII-TnC) (Manuscript Submitted).To better understand troponin function, the dynamic behavior of site II Ca2+-free troponin (i.e. troponin with CBMII-TnC) containing the TnI inhibitory peptide mutation is investigated in this work. HDX can determine the effect of a specific alteration in a protein on the dynamics of all parts of that protein. The results show that mutation of the inhibitory region alters troponin dynamics both locally and at distance. The inhibitory region replacement slowed the dynamics of some regions, had no effect on others, and increased the dynamic properties of much of the troponin coiled-coil. Some of the effects of Ca2+ removal on troponin dynamics were dependent upon presence of the intact inhibitory region. The current work indicates a clear and complex effect of the inhibitory peptide region on many portions of troponin.

The Influence of Troponin C, Isoform 4 on Drosophila Development, Stretch Activation, and Power Generation

Drosophila indirect flight muscle (IFM) is one of the most rapidly contracting muscle types known. To increase IFM efficiently and power generation many insect species have evolved high degrees of stretch activation and shortening deactivation in their IFMs. In addition to expressing TnC1, a typical calcium binding troponin C, these IFMs express an unusual unique, additional isoform, TnC4. TnC1 has two calcium binding sites, a high-affinity structural site and a lower-affinity regulatory site. TnC4 has only the high-affinity structural binding site. It is hypothesized to respond to stretch rather than calcium concentration to further activate the thin filament. We are investigating the roles of TnC4 and TnC1 in Drosophila using RNAi and by creating a TnC4 null mutant. Using RNAi, we eliminated TnC1 expression, which resulted in upregulation of TnC4. Power generation and stretch activation of IFMs without TnC1 were statistically identical to wild-type. IFMs with undetectable levels of TnC4 expression produced by RNAi do not generate power or display stretch-activation, suggesting that only TnC4 is necessary for normal muscle function. We have also created a TnC4 null, which will allow us to mechanically evaluate transgenically expressed TnC isoforms and mutants in IFM. Preliminary characterization of the TnC4 null indicates that it is homozygous lethal, but viable as a heterozygote. Flight ability of the heterozygotes is reduced, displaying a 10% reduction in wing-beat frequency than wild-type. The homozygous nulls die at the first molt suggesting that TnC4 is also expressed in some larval muscle types and that it plays a vital role in fly development.

Effect of Ca2+ binding properties of troponin C on rate of skeletal muscle force redevelopment

To investigate effects of altering troponin (Tn)C Ca(2+) binding properties on rate of skeletal muscle contraction, we generated three mutant TnCs with increased or decreased Ca(2+) sensitivities. Ca(2+) binding properties of the regulatory domain of TnC within the Tn complex were characterized by following the fluorescence of an IAANS probe attached onto the endogenous Cys(99) residue of TnC. Compared with IAANS-labeled wild-type Tn complex, V43QTnC, T70DTnC, and I60QTnC exhibited ∼1.9-fold higher, ∼5.0-fold lower, and ∼52-fold lower Ca(2+) sensitivity, respectively, and ∼3.6-fold slower, ∼5.7-fold faster, and ∼21-fold faster Ca(2+) dissociation rate (k(off)), respectively. On the basis of K(d) and k(off), these results suggest that the Ca(2+) association rate to the Tn complex decreased ∼2-fold for I60QTnC and V43QTnC. Constructs were reconstituted into single-skinned rabbit psoas fibers to assess Ca(2+) dependence of force development and rate of force redevelopment (k(tr)) at 15°C, resulting in sensitization of both force and k(tr) to Ca(2+) for V43QTnC, whereas T70DTnC and I60QTnC desensitized force and k(tr) to Ca(2+), I60QTnC causing a greater desensitization. In addition, T70DTnC and I60QTnC depressed both maximal force (F(max)) and maximal k(tr). Although V43QTnC and I60QTnC had drastically different effects on Ca(2+) binding properties of TnC, they both exhibited decreases in cooperativity of force production and elevated k(tr) at force levels <30%F(max) vs. wild-type TnC. However, at matched force levels >30%F(max) k(tr) was similar for all TnC constructs. These results suggest that the TnC mutants primarily affected k(tr) through modulating the level of thin filament activation and not by altering intrinsic cross-bridge cycling properties. To corroborate this, NEM-S1, a non-force-generating cross-bridge analog that activates the thin filament, fully recovered maximal k(tr) for I60QTnC at low Ca(2+) concentration. Thus TnC mutants with altered Ca(2+) binding properties can control the rate of contraction by modulating thin filament activation without directly affecting intrinsic cross-bridge cycling rates.

Effects of Cardiac Troponin C Mutants on TnC-TnI interaction and its modulation by PKA phosphorylation

We are considering several cardiac TnC mutants as potential therapeutic strategies for cardiomyopathies. To judge their potential to improve in situ function it is important to understand how these mutants affect TnC-TnI interaction and its modulation by PKA-mediated phosphorylation of TnI Ser 23, Ser 24. In this study, we are characterizing two cardiac TnC mutations, Leu48Glu (L48Q) and Ile61Glu (I61Q), with increased and decreased (respectively) Ca2+ binding affinity. In previous studies we showed these mutations resulted in increased (L48Q) and decreased (I61Q) Ca2+ sensitivity of steady state force in skinned rat trabeculae. To determine if these changes in Ca2+ sensitivity were due to altered TnC-TnI interactions we generated a structural marker by attaching IANBD to Cys84 in the N-lobe of cTnC. Half-maximal IANBD fluorescence saturation of Ca2+ binding occurred at pCa7.42 for L48Q cTnC, 7.38 for wild-type (WT) cTnC and 7.30 for I61Q cTnC. In both the absence and presence of saturating Ca2+ (0.6 μM TnC) IANBD fluorescence increased with increasing TnI and saturated at different [TnI] in the order L48Q, WT, I61Q. Fluorescence half-maximal saturation occurred at 0.26μM(saturating Ca2+) and 0.25 μM (no Ca2+)TnI for L48Q cTnC, 0.78 μM and 0.49μM for WT cTnC , and 1.45 μM and 0.69 μM I61Q cTnC according to the exponential function fit of the data. However, preliminary experiments suggest that when PKA phosphorylated [cTnI] was titrated to cTnC, in both the absence and presence of saturating Ca2+, IANBD fluorescence enhancement with L48Q may be impaired. The data thus far suggest that single amino acid mutations that alter Ca2+ binding affinity of TnC can influence interaction with TnI and its modulation by PKA mediated phosphorylation of Ser 23, Ser 24. Supported by HL65497 to MR.

Thin‐filament regulation of force redevelopment kinetics in rabbit skeletal muscle fibres

Thin-filament regulation of isometric force redevelopment (k(tr)) was examined in rabbit psoas fibres by substituting native TnC with either cardiac TnC (cTnC), a site I-inactive skeletal TnC mutant (xsTnC), or mixtures of native purified skeletal TnC (sTnC) and a site I- and II-inactive skeletal TnC mutant (xxsTnC). Reconstituted maximal Ca(2+)-activated force (rF(max)) decreased as the fraction of sTnC in sTnC: xxsTnC mixtures was reduced, but maximal k(tr) was unaffected until rF(max) was <0.2 of pre-extracted F(max). In contrast, reconstitution with cTnC or xsTnC reduced maximal k(tr) to 0.48 and 0.44 of control (P < 0.01), respectively, with corresponding rF(max) of 0.68 +/- 0.03 and 0.25 +/- 0.02 F(max). The k(tr)-pCa relation of fibres containing sTnC: xxsTnC mixtures (rF(max) > 0.2 F(max)) was little effected, though k(tr) was slightly elevated at low Ca(2+) activation. The magnitude of the Ca(2+)-dependent increase in k(tr) was greatly reduced following cTnC or xsTnC reconstitution because k(tr) at low levels of Ca(2+) was elevated and maximal k(tr) was reduced. Solution Ca(2+) dissociation rates (k(off)) from whole Tn complexes containing sTnC (26 +/- 0.1 s(-1)), cTnC (38 +/- 0.9 s(-1)) and xsTnC (50 +/- 1.2 s(-1)) correlated with k(tr) at low Ca(2+) levels and were inversely related to rF(max). At low Ca(2+) activation, k(tr) was similarly elevated in cTnC-reconstituted fibres with ATP or when cross-bridge cycling rate was increased with 2-deoxy-ATP. Our results and model simulations indicate little or no requirement for cooperative interactions between thin-filament regulatory units in modulating k(tr) at any [Ca(2+)] and suggest Ca(2+) activation properties of individual troponin complexes may influence the apparent rate constant of cross-bridge detachment.

Kinetics of Cardiac Thin-Filament Activation Probed by Fluorescence Polarization of Rhodamine-Labeled Troponin C in Skinned Guinea Pig Trabeculae

A genetically engineered cardiac TnC mutant labeled at Cys-84 with tetramethylrhodamine-5-iodoacetamide dihydroiodide was passively exchanged for the endogenous form in skinned guinea pig trabeculae. The extent of exchange averaged nearly 70%, quantified by protein microarray of individual trabeculae. The uniformity of its distribution was verified by confocal microscopy. Fluorescence polarization, giving probe angle and its dispersion relative to the fiber long axis, was monitored simultaneously with isometric tension. Probe angle reflects underlying cTnC orientation. In steady-state experiments, rigor cross-bridges and Ca 2+ with vanadate to inhibit cross-bridge formation produce a similar change in probe orientation as that observed with cycling cross-bridges (no Vi). Changes in probe angle were found at [Ca 2+] well below those required to generate tension. Cross-bridges increased the Ca 2+ dependence of angle change (cooperativity). Strong cross-bridge formation enhanced Ca 2+ sensitivity and was required for full change in probe position. At submaximal [Ca 2+], the thin filament regulatory system may act in a coordinated fashion, with the probe orientation of Ca 2+-bound cTnC significantly affected by Ca 2+ binding at neighboring regulatory units. The time course of the probe angle change and tension after photolytic release [Ca 2+] by laser photolysis of NP-EGTA was Ca 2+ sensitive and biphasic: a rapid component ∼10 times faster than that of tension and a slower rate similar to that of tension. The fast component likely represents steps closely associated with Ca 2+ binding to site II of cTnC, whereas the slow component may arise from cross-bridge feedback. These results suggest that the thin filament activation rate does not limit the tension time course in cardiac muscle.

Myofibrillar determinants of rate of relaxation in skinned skeletal muscle fibers.

The influence of Ca2+ dissociation rate from TnC and decreased cross-bridge detachment rate on the time course of relaxation induced by flash photolysis of diazo-2 in rabbit skinned psoas fibers was investigated at 15 degrees C. A TnC mutant (M82Q TnC) that exhibited increased Ca2+ sensitivity caused by a decreased Ca2+ dissociation rate in solution also increased the Ca2+ sensitivity of force and decreased the rate of relaxation in fibers approximately 2-fold. In contrast, a TnC mutant (NHdel TnC) with decreased Ca2+ sensitivity caused by an increased Ca2+ dissociation rate in solution decreased Ca2+ sensitivity of force but did not accelerate relaxation. Decreasing the rate of cross-bridge kinetics by reducing [Pi] slowed relaxation -2-fold and led to two phases of relaxation, a linear phase followed by an exponential phase. In fibers, M82Q TnC further slowed relaxation in low [Pi] approximately 2-fold whereas NHdel TnC had no significant effect on relaxation. These results are consistent with the interpretation that the Ca2+ dissociation rate and cross-bridge detachment rate are similar in fast twitch skeletal muscle such that decreasing either rate slows relaxation but accelerating Ca2+ dissociation has little effect on relaxation.

Characterization of the biologically important interaction between troponin C and the N-terminal region of troponin I.

The N-terminal regulatory region of Troponin I, residues 1-40 (TnI 1-40, regulatory peptide) has been shown to have a biologically important function in the interactions of troponin I and troponin C. Truncated analogs corresponding to shorter versions of the N-terminal region (1-30, 1-28, 1-26) were synthesized by solid-phase methodology. Our results indicate that residues 1-30 of TnI comprises the minimum sequence to retain full biological activity as measured in the acto-S1-TM ATPase assay. Binding of the TnI N-terminal regulatory peptides (TnI 1-30 and the N-terminal regulatory peptide (residues 1-40) labeled with the photoprobe benzoylbenzoyl group, BBRp) were studied by gel electrophoresis and photochemical cross-linking experiments under various conditions. Fluorescence titrations of TnI 1-30 were carried out with TnC mutants that carry a single tryptophan fluorescence probe in either the N- or C-domain (F105W, F105W/C domain (88-162), F29W and F29W/N domain (1-90)) (Fig. 1). Low Kd values (Kd < 10(-7) M) were obtained for the interaction of F105W and F105W/C domain (88-162) with TnI 1-30. However, there was no observable change in fluorescence when the fluorescence probe was located at the N-domain of the TnC mutant (F29W and F29W/N domain (1-90)). These results show that the regulatory peptide binds strongly to the C-terminal domain of TnC.

Regulation of force and unloaded sliding speed in single thin filaments: effects of regulatory proteins and calcium.

1. Measurements of the unloaded sliding speed of and isometric force exerted on single thin filaments in in vitro motility assays were made to evaluate the role of regulatory proteins in the control of unloaded thin filament sliding speed and isometric force production. 2. Regulated actin filaments were reconstituted from rabbit F-actin, native bovine cardiac tropomyosin (nTm), and either native bovine cardiac troponin (nTn), troponin containing a TnC mutant, CBMII, in which the sole regulatory site in cardiac TnC (site II) is inactivated (CBMII-Tn), or troponin containing a point mutation in TnT (I79N, where isoleucine at position 79 is replaced with asparagine) associated with familial hypertrophic cardiomyopathy (FHC). 3. Addition of regulatory proteins to the thin filament increases both the unloaded sliding speed and the isometric force exerted by myosin heads on the thin filaments. 4. Variation of thin filament activation by varying [Ca2+] or the fraction of CBMII/TnC bound to the thin filament at pCa 5, had little effect on the unloaded filament sliding speed until the fraction of the thin filament containing calcium bound to TnC was less than 0.15. These results suggest that [Ca2+] primarily affects the number of attached and cycling crossbridges. 5. The presence of the FHC TnT mutant increased the thin filament sliding speed but reduced the isometric force that heavy meromyosin exerted on regulated thin filaments. These latter results, together with the increased sliding speed and isometric force seen in the presence of regulatory proteins, suggest that thin filament regulatory proteins exert significant allosteric effects on the interaction of crossbridges with the thin filament.

The Role of the NH2- and COOH-terminal Domains of the Inhibitory Region of Troponin I in the Regulation of Skeletal Muscle Contraction

The role of the inhibitory region of troponin (Tn) I in the regulation of skeletal muscle contraction was studied with three deletion mutants of its inhibitory region: 1) complete (TnI-(Delta96-116)), 2) the COOH-terminal domain (TnI-(Delta105-115)), and 3) the NH(2)-terminal domain (TnI-(Delta95-106)). Measurements of Ca(2+)-regulated force and relaxation were performed in skinned skeletal muscle fibers whose endogenous TnI (along with TnT and TnC) was displaced with high concentrations of added troponin T. Reconstitution of the Tn-displaced fibers with a TnI.TnC complex restored the Ca(2+) sensitivity of force; however, the levels of relaxation and force development varied. Relaxation of the fibers (pCa 8) was drastically impaired with two of the inhibitory region deletion mutants, TnI-(Delta96-116).TnC and TnI-(Delta105-115).TnC. The TnI-(Delta95-106).TnC mutant retained approximately 55% relaxation when reconstituted in the Tn-displaced fibers. Activation in skinned skeletal muscle fibers was enhanced with all TnI mutants compared with wild-type TnI. Interestingly, all three mutants of TnI increased the Ca(2+) sensitivity of contraction. None of the TnI deletion mutants, when reconstituted into Tn, could inhibit actin-tropomyosin-activated myosin ATPase in the absence of Ca(2+), and two of them (TnI-(Delta96-116) and TnI-(Delta105-115)) gave significant activation in the absence of Ca(2+). These results suggest that the COOH terminus of the inhibitory region of TnI (residues 105-115) is much more critical for the biological activity of TnI than the NH(2)-terminal region, consisting of residues 95-106. Presumably, the COOH-terminal domain of the inhibitory region of TnI is a part of the Ca(2+)-sensitive molecular switch during muscle contraction.

Tryptophan mutants of troponin C from skeletal muscle--an optical probe of the regulatory domain.

We have generated a series of chicken skeletal muscle troponin C mutants to study the conformation of the regulatory domain in the N-terminal half of the molecule. These mutants each contained a single Trp at position 22 (helix A), 52 (linker of helices B and C), or 90 (central helix). Some of these mutants also contained additional mutations to introduce a single Cys at a desired position. The mutants were characterized by molecular graphics and CD and found to have a minimum of structural perturbations when compared with the native structure. They also retained the ability to regulate myofibrillar ATPase activity. The fluorescence of Trp22 was sensitive to Ca2+ binding only to the regulatory sites, whereas Trp52 and Trp90 responded to Ca2+ binding to both the regulatory and the Ca2+/Mg2+ sites. The tryptophan quantum yield (Q) of all Trp22-containing mutants was very high (0.33) in the absence of bound Ca2+, compared to that of L-tryptophan in aqueous solution (0.14). Q decreased 25% upon binding of Ca2+ to the regulatory sites. The quantum yields of Trp52 and Trp90 in apo mutants were close to 0.14. In the presence of bound Ca2+ at the regulatory sites, the quantum yield of Trp52 decreased 16%, whereas that of Trp90 increased 25%. Results from acrylamide quenching of the fluorescence of the three Trp residues indicated that Trp22 was the least exposed and Trp52 was the most exposed, consistent with other spectral data that Trp22 was in a relatively nonpolar environment and Trp52 was in a highly polar environment. The ability of Trp52 and Trp90 to sense Ca2+ binding to sites located at both domains suggests inter-domain communication in the protein. These single Trp TnC mutants provide specific signals for probing Ca2+-induced conformational changes in the regulatory domain.