Dataset Information

Ca²⁺ and Myosin Cycle States Work as Allosteric Effectors of Troponin Activation.

ABSTRACT: In cardiac muscle, troponin (Tn) and tropomyosin inhibit actin and myosin interactions through the steric blocking of myosin binding to F-actin. Ca²⁺ binding to Tn C modulates this inhibition. Thin filaments become activated upon Ca²⁺ binding, which enables strong binding of myosin with a concomitant release of ATP hydrolysis products and level arm swinging responsible for force generation. Despite this level of description, the current cross-bridge cycle model does not fully define the structural events that take place within Tn during combinatorial myosin and Ca²⁺ interventions. Here, we studied conformational changes within Tn bound to F-actin and tropomyosin by fluorescence lifetime imaging combined with Förster resonance energy transfer. Fluorescent dye molecules covalently bound to the Tn C C-lobe and Tn I C-terminal domain report Ca²⁺- and myosin-induced activation of Tn. Reconstituted thin filaments were deposited on a myosin-coated surface similar to an in vitro motility assay setup without filament sliding involved. Under all the tested conditions, Ca²⁺ was responsible for the most significant changes in Tn activation. Rigor myosin activated Tn at subsaturated Ca²⁺ conditions but not to the degree seen in thin filaments with Ca²⁺. ATP-?-S did not affect Tn activation significantly; however, blebbistatin induced significant activation at subsaturating Ca²⁺ levels. The relation between the extent of Tn activation and its conformational flexibility suggests that active/inactive Tn states coexist in different proportions that depend on the combination of effectors. These results satisfy an allosteric activation model of the thin filament as a function of Ca²⁺ and the myosin catalytic cycle state.

SUBMITTER: Solis C

PROVIDER: S-EPMC6225061 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Ca2+ and Myosin Cycle States Work as Allosteric Effectors of Troponin Activation.

Solís Christopher C Kim Giho H GH Moutsoglou Maria E ME Robinson John M JM

Biophysical journal 20180901 9

In cardiac muscle, troponin (Tn) and tropomyosin inhibit actin and myosin interactions through the steric blocking of myosin binding to F-actin. Ca2+ binding to Tn C modulates this inhibition. Thin filaments become activated upon Ca2+ binding, which enables strong binding of myosin with a concomitant release of ATP hydrolysis products and level arm swinging responsible for force generation. Despite this level of description, the current cross-bridge cycle model does not ful ...[more]

PMID: 30249400

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Project description:The cardiac sodium (Na+)/calcium (Ca2+) exchanger (NCX1) is an electrogenic membrane transporter that regulates Ca2+ homeostasis in cardiomyocytes, serving mainly to extrude Ca2+ during diastole. The direction of Ca2+ transport reverses at membrane potentials near that of the action potential plateau, generating an influx of Ca2+ into the cell. Therefore, there has been great interest in the possible roles of NCX1 in cardiac Ca2+-induced Ca2+ release (CICR). Interest has been reinvigorated by a recent super-resolution optical imaging study suggesting that ~18% of NCX1 co-localize with ryanodine receptor (RyR2) clusters, and ~30% of additional NCX1 are localized to within ~120nm of the nearest RyR2. NCX1 may therefore occupy a privileged position in which to modulate CICR. To examine this question, we have developed a mechanistic biophysically-detailed model of NCX1 that describes both NCX1 transport kinetics and Ca2+-dependent allosteric regulation. This NCX1 model was incorporated into a previously developed super-resolution model of the Ca2+ spark as well as a computational model of the cardiac ventricular myocyte that includes a detailed description of CICR with stochastic gating of L-type Ca2+ channels and RyR2s, and that accounts for local Ca2+ gradients near the dyad via inclusion of a peri-dyadic (PD) compartment. Both models predict that increasing the fraction of NCX1 in the dyad and PD decreases spark frequency, fidelity, and diastolic Ca2+ levels. Spark amplitude and duration are less sensitive to NCX1 spatial redistribution. On the other hand, NCX1 plays an important role in promoting Ca2+ entry into the dyad, and hence contributing to the trigger for RyR2 release at depolarized membrane potentials and in the presence of elevated local Na+ concentration. Whole-cell simulation of NCX1 tail currents are consistent with the finding that a relatively high fraction of NCX1 (~45%) resides in the dyadic and PD spaces, with a dyad-to-PD ratio of roughly 1:2. Allosteric Ca2+ activation of NCX1 helps to "functionally localize" exchanger activity to the dyad and PD by reducing exchanger activity in the cytosol thereby protecting the cell from excessive loss of Ca2+ during diastole.

Dataset Information

Ca²⁺ and Myosin Cycle States Work as Allosteric Effectors of Troponin Activation.

Publications

Ca<sup>2+</sup> and Myosin Cycle States Work as Allosteric Effectors of Troponin Activation.

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