The subtype and splice variant specific modulation of Inositol 1,4,5-trisphosphate receptors (InsP3R) by interaction with cellular factors plays a fundamental role in defining the characteristics of Ca2+ release in individual cell types. In this study, we investigate the binding properties and functional consequences of the expression of a putative nucleotide binding fold (referred to as the ATPC site) unique to the S2- splice variant of the type-1 InsP3R (InsP3R-1), which is the predominant splice variant in peripheral tissue. A GST fusion protein encompassing amino acids 1574 through 1765 of the S2- InsP3R-1 and including this gycine rich motif, Gly-Tyr-Gly-Glu-Lys-Gly bound ATP specifically as measured by fluorescent TNP-ATP binding. This binding was completely abrogated by a point mutation (Gly-1690-Ala) in the nucleotide binding fold. The functional sensitivity of S2- InsP3R-1 constructs was evaluated in DT40-3KO-M3 cells, a null background for InsP3R, engineered to express muscarinic M3 receptors (M3R). The S2- InsP3R-1 containing the Gly1690Ala mutation was markedly less sensitive to agonist stimulation than wild type (WT) S2- InsP3R-1 or receptors containing similar (GlyAla) mutation in the established nucleotide binding sites in InsP3R-1 (the ATPA and ATPB sites). The ATP sensitivity of InsP3-induced Ca2+ release, however, was not altered by the Gly-1690-Ala mutation when measured in permeabilized DT40-3KO cells suggesting a unique role for the ATPC site. Ca2+ release was dramatically potentiated following activation of PKA in DT40-3KO cells transiently expressing wild-type S2- InsP3R or GlyAla mutations in the ATPA and ATPB sites, but phosphorylation of the receptor and the potentiation of Ca2+ release were absent in cells expressing the Gly-1690-Ala mutation in S2- InsP3R. These data indicate that ATP binding specifically to the ATPC site in S2- InsP3R-1 controls the susceptibility of the receptor to PKA mediated phosphorylation and contributes to the sensitivity of cells to agonist stimulation.