Association of the type 1 inositol (1,4,5)-trisphosphate receptor with 4.1N protein in neurons

The type 1 inositol (1,4,5)-trisphosphate receptor (InsP₃R1) is an intracellular calcium (Ca²⁺) release channel that plays an important role in neuronal function. In yeast two-hybrid screen of rat brain cDNA library with the InsP₃R1 carboxy-terminal bait we isolated multiple clones of neuronal cytoskeletal protein 4.1N. We mapped the 4.1N-interaction site to a short fragment (50 amino acids) within the carboxy-terminal tail of the InsP₃R1 and the InsP₃R1-interaction site to the carboxy-terminal domain (CTD) of 4.1N. We established that InsP₃R1 carboxy-terminal binds selectively to the CTDΔ alternatively spliced form of the 4.1N protein. In biochemical experiments we demonstrated that 4.1N and InsP₃R1 specifically associate in vitro. We showed that both 4.1N and InsP₃R1 were enriched in synaptic locations and immunoprecipitated the 4.1N-InsP₃R1 complex from rat brain synaptosomes. In biochemical experiments we demonstrated a possibility of InsP₃R1-4.1N-CASK-syndecan-2 quaternary complex formation. From our findings we hypothesize that InsP₃R1-4.1N association may play a role in InsP₃R1 localization or Ca²⁺ signaling in neurons.