Structural dynamics of adenine nucleotide potentiation of the human type 2 IP3 receptor

Inositol trisphosphate receptors (IP3R) are intracellular calcium (Ca2+) channels that mediate Ca2+ flux from the endoplasmic reticulum (ER) into the cytosol, playing a critical role in Ca2+ signaling. IP3R activity requires IP3 and Ca2+ and is potentiated by adenine nucleotides through a poorly understood mechanism. Here, we combined single-particle cryo-electron microscopy and all-atom molecular dynamics simulations to investigate the potentiation of IP3Rs by adenine nucleotides. Our structures reveal that ATP and cAMP bind to a conserved site in the juxtamembrane domain, which connects the cytoplasmic IP3- and Ca2+-binding sites with the transmembrane pore. Molecular dynamics simulations predict that the binding of adenine nucleotides rigidifies the juxtamembrane domain, primarily through the coordination of the adenine base. Consistent with the adenine base being critical for potentiation, mutations that disrupt the interactions with the adenine base perturb Ca2+ flux in cells. Taken together, our data suggest that adenine nucleotides potentiate IP3R channel activity by rigidifying the juxtamembrane domain to improve coupling between IP3 and Ca2+ binding and pore opening. Significance StatementInositol-1-4-5-trisphosphate receptors (IP3Rs) are the main intracellular calcium (Ca2+) release channels in non-excitable cells and contribute significantly to intracellular Ca2+ release in excitable cells. Regulation of IP3Rs by inositol-1-4-5-trisphosphate (IP3), adenine nucleotides, and Ca2+ is fundamental to both intracellular Ca2+ homeostasis and signaling. We show that adenine nucleotides modulate IP3R activity by tuning the dynamics of a mechanical fulcrum-like domain, the juxtamembrane domain (JD), which physically couples the IP3- and Ca2+-binding sites in the large regulatory cytoplasmic domain to the channel pore. Using a combination of structural, computational, and functional studies, we show that adenine nucleotides bind to and restrict the movement of the JD of the human type 2 IP3R (hIP3R2). We find that that the coordination of adenine nucleotides is primarily driven by hydrophobic interactions with the adenine moieties of the nucleotides and that these interactions are critical for normal hIP3R2 function. This work establishes the foundation for further research into the physiological role of adenine nucleotide modulation of IP3Rs.