Measurement of calcium dissociation rates from troponin C in rigor skeletal myofibrils

Abstract

Ca2+ dissociation from the regulatory domain of troponin C may influence the rate of striated muscle relaxation. However, Ca(2+) dissociation from troponin C has not been measured within the geometric and stoichiometric constraints of the muscle fiber. Here we report the rates of Ca(2+) dissociation from the N-terminal regulatory and C-terminal structural domains of fluorescent troponin C constructs reconstituted into rabbit rigor psoas myofibrils using stopped-flow technology. Chicken skeletal troponin C fluorescently labeled at Cys 101, troponin C(IAEDANS), reported Ca(2+) dissociation exclusively from the structural domain of troponin C at ∼0.37, 0.06, and 0.07/s in isolation, in the presence of troponin I and in myofibrils at 15°C, respectively. Ca(2+) dissociation from the regulatory domain was observed utilizing fluorescently labeled troponin C containing the T54C and C101S mutations. Troponin [Formula: see text] reported Ca(2+) dissociation exclusively from the regulatory domain of troponin C at >1000, 8.8, and 15/s in isolation, in the presence of troponin I and in myofibrils at 15°C, respectively. Interestingly, troponin [Formula: see text] reported a biphasic fluorescence change upon Ca(2+) dissociation from the N- and C-terminal domains of troponin C with rates that were similar to those reported by troponin [Formula: see text] and troponin C(IAEDANS) at all levels of the troponin C systems. Furthermore, the rate of Ca(2+) dissociation from troponin C in the myofibrils was similar to the rate of Ca(2+) dissociation measured from the troponin C-troponin I complexes. Since the rate of Ca(2+) dissociation from the regulatory domain of TnC in myofibrils is similar to the rate of skeletal muscle relaxation, Ca(2+) dissociation from troponin C may influence relaxation.

Keywords: calcium; dissociation; muscle; myofibril; relaxation; skeletal; troponin.

Figure 1

Characterization of the Ca²⁺ dissociation rates from TnC^IAEDANS in isolation, in the presence of TnI and in myofibrils. (A) Shows the time course of decrease in IAEDANS fluorescence as Ca²⁺ was removed by EGTA from the C-terminal Ca²⁺-binding sites of TnC^IAEDANS ± TnI and in reconstituted myofibrils. TnC^IAEDANS (0.6 μM) ± TnI (1.2 μM) in 10 mM MOPS, 90 mM KCl, 1 mM Mg²⁺, 1 mM DTT at pH 7.0 plus 200 μM Ca²⁺ was rapidly mixed with an equal volume of the same buffer plus 10 mM EGTA at 15°C. Similar experiments were performed with ∼1 mg/ml reconstituted myofibrils except the buffer also contained 0.02% Tween-20. The flat line represents the fluorescence level of Ca²⁺ saturated myofibrils in which EGTA in the second syringe was replaced with 200 μM Ca²⁺. IAEDANS emission fluorescence was monitored through a 420- to 470-nm band-pass filter with excitation at 340 nm. The TnC^IAEDANS and TnC^IAEDANS–TnI complex traces have been normalized to that of the TnC^IAEDANS reconstituted myofibrils trace and staggered for visual clarity. (B) Shows the phase contrast (top panel) and IAEDANS fluorescence (bottom panel) images obtained from a representative rigor psoas myofibril reconstituted with TnC^IAEDANS. The vertical line designates the location of the Z-line, and the horizontal lines designate the location of the A bands.

Figure 2

Calcium binding to ${\text{TnC}}_{\text{IAANS}}^{\text{T54C,C101S}}\pm \text{TnI}\text{.}$ (A) Shows a helical representation of apo TnC and Ca²⁺ saturated TnC ± TnI with T54 depicted in space fill format. The Protein Data Bank files utilized for this figure were 1TOP (apo TnC; Satyshur et al., 1994), 2TN4 (Ca²⁺–TnC; Houdusse et al., 1997), and 1YTZ (Ca²⁺–TnC–TnI, TnT is not shown for clarity; Vinogradova et al., 2005) and were rendered using Rasmol (Sayle and Milner-White, 1995). (B) Shows the Ca²⁺ dependent increase and decrease in IAANS fluorescence for ${\text{TnC}}_{\text{IAANS}}^{\text{T54C,C101S}}$ (●) and the Ca²⁺ dependent decrease in MIANS fluorescence for ${\text{TnC}}_{\text{MIANS}}^{\text{T54C,C101S}}$ (□) as a function of pCa. ${\text{TnC}}_{\text{IAANS}}^{\text{T54C,C101S}}$ fluorescence was fit with the sum of two independent Hill equations. (C) Shows the Ca²⁺ dependent decrease in IAANS and MIANS fluorescence for the ${\text{TnC}}_{\text{IAANS}}^{\text{T54C,C101S}}$ –TnI (●) and ${\text{TnC}}_{\text{MIANS}}^{\text{T54C,C101S}}$ –TnI (□) complexes as a function of pCa. Microliter amounts of Ca²⁺ were added to the different fluorescent TnC constructs (0.6 μM) ± TnI (1.2 μM) in 200 mM MOPS, 90 mM KCl, 2 mM EGTA, 1 mM Mg²⁺, 1 mM DTT, pH 7.0, at 15°C. IAANS fluorescence emission was monitored at 450 nm with excitation at 330 nm, whereas MIANS fluorescence emission was monitored at 435 nm with excitation at 325 nm. Hundred percentage fluorescence corresponds to the highest fluorescent state whereas 0% fluorescence corresponds to the lowest fluorescent state of the two respective conditions (±TnI).

Figure 3

Comparison of the calcium dependence of skeletal muscle force generation by native and TnC reconstituted fibers. The Ca²⁺ dependence of isometric force generation in native (■ TnC^Endog) single rabbit skinned psoas fibers is compared to that of fibers reconstituted with TnC^T54C,C101S (●) or ${\text{TnC}}_{\text{IAANS}}^{\text{T54C,C101S}}$ (▲) as a function of pCa. The experimental conditions are described in “Experimental Procedures.” Each data point represents the mean ± SE from at least four separate fibers individually normalized and fit with a logistic sigmoid function mathematically equivalent to the Hill equation. The inset shows the phase contrast (top of paired panels) and IAANS or MIANS fluorescence (bottom of paired panels) images obtained from representative rigor psoas myofibrils reconstituted with ${\text{TnC}}_{\text{IAANS}}^{\text{T54C,C101S}}$ or ${\text{TnC}}_{\text{MIANS}}^{\text{T54C,C101S}}$. The vertical lines designate the location of the Z-lines, and the horizontal lines designate the location of the A bands.

Figure 4

Comparison of calcium dissociation kinetics from isolated ${\text{TnC}}_{\text{IAANS}}^{\text{T54C,C101S}}$, TnC^T54C,C101S, and${\text{TnC}}_{\text{MIANS}}^{\text{T54C,C101S}}$. (A) Shows the time course of decrease in IAANS fluorescence as Ca²⁺ was removed by EGTA from the N-terminal regulatory and C-terminal structural [(A) inset] Ca²⁺-binding sites of ${\text{TnC}}_{\text{IAANS}}^{\text{T54C,C101S}}$. ${\text{TnC}}_{\text{IAANS}}^{\text{T54C,C101S}}$ (0.6 μM) in 10 mM MOPS, 90 mM KCl, 1 mM Mg²⁺, 1 mM DTT at pH 7.0 plus 200 μM Ca²⁺ was rapidly mixed with an equal volume of the same buffer plus 10 mM EGTA at 4 and 15°C. IAANS emission fluorescence was monitored through a 420- to 470-nm band-pass filter with excitation at 330 nm. (B) Shows the time course of increase in Quin-2 fluorescence as Ca²⁺ was removed from the N-terminal regulatory and C-terminal structural [(B) inset] Ca²⁺-binding sites of TnC^T54C,C101S by Quin-2. TnC^T54C,C101S (6 μM) in 10 mM MOPS, 90 mM KCl, 1 mM Mg²⁺, 1 mM DTT at pH 7.0 plus 60 μM Ca²⁺ was rapidly mixed with an equal volume of the same buffer plus 150 μM Quin-2 at 4 and 15°C. Control experiments where Ca²⁺ (60 μM) was rapidly mixed with an equal volume of Quin-2 (150 μM) were flat lines. Quin-2 emission fluorescence was monitored through a 510-nm broad band-pass interference filter with excitation at 330 nm. (C) Shows the time course of increase in MIANS fluorescence as Ca²⁺ was removed by EGTA from the N-terminal regulatory Ca²⁺-binding sites of ${\text{TnC}}_{\text{MIANS}}^{\text{T54C,C101S}}$. ${\text{TnC}}_{\text{MIANS}}^{\text{T54C,C101S}}$ (0.6 μM) in 10 mM MOPS, 90 mM KCl, 1 mM Mg²⁺, 1 mM DTT at pH 7.0 plus 200 μM Ca²⁺ was rapidly mixed with an equal volume of the same buffer plus 10 mM EGTA at 4 and 15°C. MIANS emission fluorescence was monitored through a 420- to 470-nm band-pass filter with excitation at 325 nm. The traces in (A) through (C) have been staggered for visual clarity.

Figure 5

Comparison of calcium dissociation kinetics from ${\text{TnC}}_{\text{IAANS}}^{\text{T54C,C101S}}$, ${\text{TnC}}_{\text{MIANS}}^{\text{T54C,C101S}}$, and TnC^T54C,C101S in the presence of TnI. (A) Shows the time course of increase in IAANS fluorescence as Ca²⁺ was removed by EGTA from the N-terminal regulatory and C-terminal structural Ca²⁺-binding sites of the ${\text{TnC}}_{\text{IAANS}}^{\text{T54C,C101S}}$ –TnI complex. ${\text{TnC}}_{\text{IAANS}}^{\text{T54C,C101S}}$ (0.6 μM) plus TnI (1.2 μM) in 10 mM MOPS, 90 mM KCl, 1 mM Mg²⁺, 1 mM DTT at pH 7.0 plus 200 μM Ca²⁺ was rapidly mixed with an equal volume of the same buffer plus 10 mM EGTA at 4 and 15°C. IAANS emission fluorescence was monitored as described in the legend of Figure 4A. (B) Shows the time course of increase in MIANS fluorescence as Ca²⁺ was removed by EGTA from the N-terminal regulatory Ca²⁺-binding sites of the ${\text{TnC}}_{\text{MIANS}}^{\text{T54C,C101S}}$ –TnI complex. ${\text{TnC}}_{\text{MIANS}}^{\text{T54C,C101S}}$ (0.6 μM) plus TnI (1.2 μM) in 10 mM MOPS, 90 mM KCl, 1 mM Mg²⁺, 1 mM DTT at pH 7.0 plus 200 μM Ca²⁺ was rapidly mixed with an equal volume of the same buffer plus 10 mM EGTA at 4 and 15°C. MIANS emission fluorescence was monitored as described in the legend of Figure 4C. (C) Shows the time course of increase in Quin-2 fluorescence as Ca²⁺ was removed from the N-terminal regulatory [(C) inset shows expanded 15°C trace] and C-terminal structural Ca²⁺-binding sites of the TnC^T54C,C101S–TnI complex by Quin-2. TnC^T54C,C101S (6 μM) plus TnI (9 μM) in 10 mM MOPS, 90 mM KCl, 1 mM Mg²⁺, 1 mM DTT at pH 7.0 plus ∼30 μM Ca²⁺ was rapidly mixed with an equal volume of the same buffer plus 150 μM Quin-2 at 4 and 15°C. Control experiments where Ca²⁺ (30 μM) was rapidly mixed with an equal volume of Quin-2 (150 μM) were flat lines. Quin-2 emission fluorescence was monitored as described in the legend of Figure 4B. The traces in (A) through (C) have been staggered for visual clarity.

Figure 6

Comparison of calcium dissociation kinetics from ${\text{TnC}}_{\text{IAANS}}^{\text{T54C,C101S}}$and${\text{TnC}}_{\text{MIANS}}^{\text{T54C,C101S}}$ reconstituted myofibrils. (A) Shows the time course of decrease in IAANS fluorescence as Ca²⁺ was removed by EGTA from the N-terminal regulatory and C-terminal structural Ca²⁺-binding sites of ${\text{TnC}}_{\text{IAANS}}^{\text{T54C,C101S}}$ reconstituted rigor rabbit psoas myofibrils. ∼1 mg/ml ${\text{TnC}}_{\text{IAANS}}^{\text{T54C,C101S}}$ reconstituted myofibrils in 10 mM MOPS, 90 mM KCl, 1 mM Mg²⁺, 1 mM DTT, 0.02% Tween-20 at pH 7.0 plus 200 μM Ca²⁺ was rapidly mixed with an equal volume of the same buffer plus 10 mM EGTA at 4 and 15°C. The flat line represents the 15°C fluorescence level of Ca²⁺ saturated myofibrils in which EGTA in the second syringe was replaced with 200 μM Ca^2+. IAANS emission fluorescence was monitored as described in the legend of Figure 4A. (B) Shows the time course of decrease in MIANS fluorescence as Ca²⁺ was removed by EGTA from the N-terminal regulatory Ca²⁺-binding sites of ${\text{TnC}}_{\text{MIANS}}^{\text{T54C,C101S}}$ reconstituted rigor rabbit psoas myofibrils. ∼1 mg/ml ${\text{TnC}}_{\text{MIANS}}^{\text{T54C,C101S}}$ reconstituted myofibrils in 10 mM MOPS, 90 mM KCl, 1 mM Mg²⁺, 1 mM DTT, 0.02% Tween-20 at pH 7.0 plus 200 μM Ca²⁺ was rapidly mixed with an equal volume of the same buffer plus 10 mM EGTA at 4 [(B) inset] and 15°C. MIANS emission fluorescence was monitored as described in the legend of Figure 4C. The 4°C trace in (A) has been staggered for visual clarity.