Abstract
In striated muscles, the large scaffolding protein obscurin and a small SR-integral membrane protein sAnk1.5 control the retention of longitudinal SR across the sarcomere. How a complex of these proteins facilitates localization of longitudinal SR has yet to be resolved, but we hypothesize that obscurin interacts with a complex of sAnk1.5 proteins. To begin to address this hypothesis, we demonstrate that sAnk1.5 interacts with itself and identify two domains mediating self-association. Specifically, we show by co-precipitation and FLIM-FRET analysis that sAnk1.5 and another small AnkR isoform (sAnk1.6) interact with themselves and each other. We demonstrate that obscurin interacts with a complex of sAnk1.5 proteins and that this complex formation is enhanced by obscurin-binding. Using FLIM-FRET analysis, we show that obscurin interacts with sAnk1.5 alone and with sAnk1.6 in the presence of sAnk1.5. We find that sAnk1.5 self-association is disrupted by mutagenesis of residues Arg64-Arg69, residues previously associated with obscurin-binding. Molecular modeling of two interacting sAnk1.5 monomers facilitated the identification of Gly31-Val36 as an additional site of interaction, which was subsequently corroborated by co-precipitation and FLIM-FRET analysis. In closing, these results support a model in which sAnk1.5 forms large oligomers that interact with obscurin to facilitate the retention of longitudinal SR throughout skeletal and cardiac myocytes.