The cardiac Ryanodine receptor (RyR2) is responsible for calcium efflux from the sarcoplasmic reticulum during excitation contraction coupling. Work by ourselves and others has provided evidence for phosphorylation of RyR2 by calcium/calmodulin dependent protein kinase II delta (CaMKIIδ)1. To date however, both the site(s) for phosphorylation and the functional effect of CaMKII-mediated RyR2 phosphorylation remains controversial. To explore this in more detail, we have generated mutations of one putative CaMKIIδ site on RyR2, Ser2815. Both a dominant negative (S2815A) mutation and a constitutively active (S2815D) mutation were generated via site directed mutagenesis and sequencing was used as confirmation of a positive result. Full length clones of the mutant and wild type receptors were then expressed in HEK293 cells following transfection with RyR2 cDNA using the calcium phosphate precipitation method. Since GFP was used as a tag, we were able to monitor RyR2 expression in HEK cells by fluorescence intensity. We have successfully expressed both dominant negative and constitutively active mutants as well as wild-type RyR2 in HEK cells. Membrane fractions from these cells have been used for ryanodine binding assays to assess channel open probability (Po) under various conditions. Initial experiments are being conducted to (i) compare mutant and wild-type ryanodine binding in the presence of different free [Ca2+] and (ii) examine the capacity of CaMKII for modulation of mutant and wild-type RyR2 Po, reflecting the extent to which Ser 2815 is a key modulatory site for CaMKII on RyR2.