Calpastatin domain L binds to the IQ domain of cardiac CaV1.2 Ca²⁺ channels

Abstract

Calpastatin (CS), an endogenous inhibitor of the calcium-activated protease calpain, is composed of N-terminal domain L (CS_L) and four repetitive calpain inhibitory domains 1-4, and expressed in virtually all cell types. We have previously found that CS modulates the cardiac CaV1.2 Ca²⁺ channels in inside-out patch mode of patch-clamp technique in guinea-pig cardiomyocytes and that the Ca²⁺ channel regulatory function is located in CS_L. However, how does CS_L modulate the channels activity is not known. In this study, we have determined the interaction site of CS_Lin the channel by the pull-down binding assay using GST-fused fragment peptides of the α₁ subunit. CS_L interacted directly with IQ domain of the proximal C-terminal region of α₁ subunit (a. a. number of guinea-pig α₁: 1648-1672), which is also one of the calmodulin (CaM) binding sites. CS_L bound to IQ domain with a higher affinity in the presence of Ca²⁺ than its absence. CS_L binding to IQ also showed a competitive manner with CaM. These results suggest that CS_L directly interacts with IQ domain of α₁ subunit of the voltage dependant Ca²⁺ channel and inhibits the CaM binding to this domain. A role of CS_L in the regulation of the Ca²⁺ channels will be discussed. [J Physiol Sci. 2007;57 Suppl:S228]