Inositol 1,4,5-trisphosphate receptor localization and stability in neonatal cardiomyocytes requires interaction with ankyrin-B

The molecular mechanisms required for inositol 1,4,5-trisphosphate receptor ( InsP(3)R) targeting to specialized endoplasmic reticulum membrane domains are unknown. We report here a direct, high affinity interaction between InsP(3)R and ankyrin- B and demonstrate that this association is critical for InsP(3)R post- translational stability and localization in cultures of neonatal cardiomyocytes. Recombinant ankyrin- B membrane- binding domain directly interacts with purified cerebellar InsP(3)R ( K-d = 2 nM). 220- kDa ankyrin- B co- immunoprecipitates with InsP(3)R in tissue extracts from brain, heart, and lung. Alanine- scanning mutagenesis of the ankyrin- B ANK ( ankyrin repeat) repeat beta- hairpin loop tips revealed that consecutive ANK repeat beta- hairpin loop tips ( repeats 22 - 24) are required for InsP(3)R interaction, thus providing the first detailed evidence of how ankyrin polypeptides associate with membrane proteins. Pulse- chase biosynthesis experiments demonstrate that reduction or loss of ankyrin- B in ankyrin- B (+/-) or ankyrin- B (-/-) neonatal cardiomyocytes leads to similar to 3- fold reduction in half- life of newly synthesized InsP3R. Furthermore, interactions with ankyrin- B are required for InsP(3)R stability as abnormal InsP(3)R phenotypes, including mis- localization, and reduced half-life in ankyrin- B (+/-) cardiomyocytes can be rescued by green fluorescent protein ( GFP)- 220- kDa ankyrin- B but not by GFP- 220- kDa ankyrin- B mutants, which do not associate with InsP(3)R. These new results provide the first physiological evidence of a molecular partner required for early post- translational stability of InsP(3)R.