Unique Regulatory Properties of Heterotetrameric Inositol 1,4,5-Trisphosphate Receptors Revealed by Studying Concatenated Receptor Constructs

The ability of inositol 1,4,5-trisphosphate receptors (IP₃R) to precisely initiate and generate a diverse variety of intracellular Ca²⁺ signals is in part mediated by the differential regulation of the three subtypes (R1, R2, and R3) by key functional modulators (IP₃, Ca²⁺, and ATP). However, the contribution of IP₃R heterotetramerization to Ca²⁺ signal diversity has largely been unexplored. In this report, we provide the first definitive biochemical evidence of endogenous heterotetramer formation. Additionally, we examine the contribution of individual subtypes within defined concatenated heterotetramers to the shaping of Ca²⁺ signals. Under conditions where key regulators of IP₃R function are optimal for Ca²⁺ release, we demonstrate that individual monomers within heteromeric IP₃Rs contributed equally toward generating a distinct ''blended'' sensitivity to IP₃ that is likely dictated by the unique IP₃ binding affinity of the heteromers. However, under suboptimal conditions where ATP] were varied, we found that one subtype dictated the ATP regulatory properties of heteromers. We show that R2 monomers within a heterotetramer were both necessary and sufficient to dictate the ATP regulatory properties. Finally, the ATP-binding site B in R2 critical for ATP regulation was mutated and rendered non-functional to address questions relating to the stoichiometry of IP₃R regulation. Two intact R2 monomers were sufficient to maintain ATP regulation in R2 homotetramers. In summary, we demonstrate that heterotetrameric IP₃R do not necessarily behave as the sum of the constituent subunits, and these properties likely extend the versatility of IP₃-induced Ca²⁺ signaling in cells expressing multiple IP₃R isoforms.