Effects of cardiac thin filament Ca2+: statistical mechanical analysis of a troponin C site II mutant

Abstract

Cardiac thin filaments contain many troponin C (TnC) molecules, each with one regulatory Ca2+ binding site. A statistical mechanical model for the effects of these sites is presented and investigated. The ternary troponin complex was reconstituted with either TnC or the TnC mutant CBMII, in which the regulatory site in cardiac TnC (site II) is inactivated. Regardless of whether Ca2+ was present, CBMII-troponin was inhibitory in a thin filament-myosin subfragment 1 MgATPase assay. The competitive binding of [3H]troponin and [14C]CBMII-troponin to actin.tropomyosin was measured. In the presence of Mg2+ and low free Ca2+ they had equal affinities for the thin filament. When Ca274+ was added, however, troponin's affinity for the thin filament was 2.2-fold larger for the mutant than for the wild type troponin. This quantitatively describes the effect of regulatory site Ca2+ on troponin's affinity for actin.tropomyosin; the decrease in troponin-thin filament binding energy is small. Application of the theoretical model to the competitive binding data indicated that troponin molecules bind to interdependent rather than independent sites on the thin filament. Ca2+ binding to the regulatory site of TnC has a long-range rather than a merely local effect. However, these indirect TnC-TnC interactions are weak, indicating that the cooperativity of muscle activation by Ca2+ requires other sources of cooperativity.

Cooperative Effect of Calcium Binding to Adjacent Troponin Molecules on the Thin Filament-Myosin Subfragment 1 MgATPase Rate

“…CBMII-Tn binds to actin-tropomyosin with an affinity identical to that of normal troponin in the absence of Ca 2ϩ . This binding, which is very tight for both normal troponin and for CBMII-Tn (4,8,9), permits the present report in which thin filaments are assembled with defined mixtures of normal troponin and CBMII-Tn. In the presence of saturating Ca 2ϩ concentrations, such thin filaments exhibit a fractional saturation of the TnC regulatory sites that is experimentally controllable by varying the relative concentrations of the two forms of troponin.…”

“…If binding were random, then the fraction of adjacent pairs with Ca 2ϩ bound on both elements of the pair would simply equal 2 . However, prior work shows that when the two forms of troponin are present in excess, they compete in a way that implies positive cooperativity and a small tendency for the two forms of troponin to segregate from each other (4). This same tendency must be assumed to exist in the present experiments, which differ in that the troponins are added in a stoichiometric amount relative to the sites on the actin filament.…”

“…1) were repeated using bovine cardiac myosin S1 purified as described previously (10). CBMII-Tn was prepared by reconstitution (13) of the ternary troponin complex from bovine cardiac TnI and TnT (13) and recombinant murine TnC mutant CBMII (4).…”

“…1 are theoretical curves for the fraction of troponin⅐troponin boundaries with Ca 2ϩ bound on both sides, which depends in part upon the degree of cooperativity in the binding of the two forms of troponin to the thin filament. The equilibrium constant Y governs the tendency of troponin and CBMII-Tn to separately cluster on the thin filament rather than bind randomly (3,4). Y also dictates the cooperativity of Ca 2ϩ binding to a thin filament containing only normal troponin, with Y Ͼ 1 indicating positive cooperativity.…”

“…Phalloidin Does Not Alter Cooperative Interactions between Troponin Molecules-The polymerization ability of the troponin⅐tropomyosin complex (18 -20) and atomic models of actin⅐actin contacts in F-actin (4,21) suggest that longitudinal contacts along the thin filament are the most likely source of cooperativity. However, this does not exclude the possibility that cooperativity occurs across rather than along the actin filament.…”