Carbonic anhydrase-related protein is a novel binding protein for inositol 1,4,5-trisphosphate receptor type 1

The inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) is an intracellular IP₃-gated Ca²⁺ channel that is located on intracellular Ca²⁺ stores and modulates Ca²⁺ signalling. Using the yeast two-hybrid system, we screened a mouse brain cDNA library with bait constructs for mouse IP₃R type 1 (IP₃R1) to identify IP₃R1-associated proteins. In this way, we found that carbonic anhydrase-related protein (CARP) is a novel IP₃R1-binding protein. Western blot analysis revealed that CARP is expressed exclusively in Purkinje cells of the cerebellum, in which IP₃R1 is abundantly expressed. Immunohistochemical analysis showed that the subcellular localization of CARP in Purkinje cells is coincident with that of IP₃R1. Biochemical analysis also showed that CARP is co-precipitated with IP₃R1. Using deletion mutagenesis, we established that amino acids 45-291 of CARP are essential for its association with IP₃R1, and that the CARP-binding site is located within the modulatory domain of IP₃R1 amino acids 1387-1647. CARP inhibits IP₃ binding to IP₃R1 by reducing the affinity of the receptor for IP₃. As reported previously, sensitivity to IP₃ for IP₃-induced Ca²⁺ release in Purkinje cells is low compared with that in other tissues. This could be due to coexpression of CARP with IP₃R in Purkinje cells and its inhibitory effects on IP₃ binding.