Effect of cTnC Mutations on the Response of Reconstituted Thin Filaments to Calcium Upon cTnI Phosphorylation

Abstract

The objective of this work is to examine the effect of calcium sensitizing and desensitizing cTnC mutations on the response of reconstituted thin filaments to calcium upon cTnI phosphorylation. M45Q cTnC mutation increases calcium sensitivity of the cTn complex reconstituted into thin filaments by decreasing the rate of calcium dissociation. On the other hand, the D73N mutation (removal of an X-acid pair from the second EF-hand of cTnC) reduces the calcium sensitivity of the cTn complex reconstituted into thin filaments by accelerating the rate of calcium dissociation. Pseudo-phosphorylation of cTnI by PKA led to ∼2.8-fold decrease in the calcium sensitivity of the cTn complex reconstituted into thin filaments. In addition, pseudo-phosphorylation of cTnI led to ∼3.3-fold faster rate of calcium dissociation from the cTn complex reconstituted into thin filaments. However, the D73N mutation substantially diminished the extent of the decrease in calcium sensitivity and of the acceleration in calcium dissociation from the cTn complex reconstituted into thin filaments associated with pseudo-phosphorylation of cTnI by PKA. In contrast, M45Q mutation did not abolish the decrease in the calcium sensitivity and the acceleration in calcium dissociation from the cTn complex reconstituted into thin filaments induced by the pseudo-phosphorylation of cTnI by PKA. In fact, M45Q mutation enhanced the effect of cTnI pseudo-phosphorylation on the rate of Ca2+ dissociation from the cTn complex reconstituted into thin filaments. These results suggest that calcium desensitizing cTnC mutations diminish the effect of phosphorylation of cTnI on calcium binding and exchange with reconstituted thin filaments. A deeper understanding of how altered calcium sensitivity of cTnC affects the transmission of PKA phosphorylation signal from cTnI to cTnC could lead to the rational design of novel treatments for heart disease.