Structural Mapping of Divergent Region Domains in the Type 1 Ryanodine Receptor using Two Complementary FRET-based Approaches

Abstract

We used fluorescence resonance energy transfer (FRET) to localize three divergent region domains within the type 1 ryanodine receptor (RyR1), an intracellular Ca2+ channel that mediates skeletal muscle excitation-contraction (EC) coupling. Initial cloning studies of the three RyR isoforms identified three “divergent regions” of primary sequence dissimilarity spanning amino acids 4254-4631 (DR1), 1342-1403 (DR2) and 1872-1923 (DR3) in RyR1. These regions have been implicated in EC coupling as well as in differential sensitivity to pharmacological agonists. Here, we used permeabilized HEK-293T cells expressing recombinant RyR1 to localize these DRs to the cryo electron microscopic (EM) map of RyR1. First, we measured FRET from a green fluorescent protein (GFP) donor fused to either position 1 or 620 of RyR1, to a FRET acceptor, Cy3NTA, targeted to poly-histidine “tags” inserted into DR1 (at position 4429), DR2 (at position1358) or DR3 (at position 1915). While FRET was not detected for His-tagged constructs containing GFP fused at position 1, FRET was observed from GFP fused at position 620 to all 3 His-tagged positions. Second, we targeted a donor to the RyR1 cytoplasmic domain using FKBP12.6 labeled with Alexa Fluor 488, and then measured FRET to Cy3NTA targeted to the His tag sites described above. Donor-FKBPs bound with high-affinity to both recombinant wild type and His-tagged RyRs. FRET was detected from donor conjugated to each of four, well-separated positions on FKBP to Cy3NTA targeted to each divergent region. Since the fused GFPs and FKBP12.6 have already been localized within the cryo EM map of RyR1, we can now triangulate the DR positions to the cryo EM map from these two complementary data sets. Supported by NIH grant R01AR059124 (to JDF, MM, and TG) and R01HL092097 (to RLC).