Functional Inositol 1,4,5-Trisphosphate Receptors Assembled from Concatenated Homo- and Heteromeric Subunits

Vertebrate genomes code for three subtypes of inositol 1,4,5-trisphosphate (IP 3 ) receptors (IP 3 R1, -2, and -3). Individual IP 3 R monomers are assembled to form homo- and heterotetrameric channels that mediate Ca 2+ release from intracellular stores. IP 3 R subtypes are regulated differentially by IP 3 , Ca 2+ , ATP, and various other cellular factors and events. IP 3 R subtypes are seldom expressed in isolation in individual cell types, and cells often express different complements of IP 3 R subtypes. When multiple subtypes of IP 3 R are co-expressed, the subunit composition of channels cannot be specifically defined. Thus, how the subunit composition of heterotetrameric IP 3 R channels contributes to shaping the spatio-temporal properties of IP 3 -mediated Ca 2+ signals has been difficult to evaluate. To address this question, we created concatenated IP 3 R linked by short flexible linkers. Dimeric constructs were expressed in DT40–3KO cells, an IP 3 R null cell line. The dimeric proteins were localized to membranes, ran as intact dimeric proteins on SDS-PAGE, and migrated as an ∼1100-kDa band on blue native gels exactly as wild type IP 3 R. Importantly, IP 3 R channels formed from concatenated dimers were fully functional as indicated by agonist-induced Ca 2+ release. Using single channel "on-nucleus" patch clamp, the channels assembled from homodimers were essentially indistinguishable from those formed by the wild type receptor. However, the activity of channels formed from concatenated IP 3 R1 and IP 3 R2 heterodimers was dominated by IP 3 R2 in terms of the characteristics of regulation by ATP. These studies provide the first insight into the regulation of heterotetrameric IP 3 R of defined composition. Importantly, the results indicate that the properties of these channels are not simply a blend of those of the constituent IP 3 R monomers. Background: IP 3 R forms homo- and heterotetrameric channels. However, the impact of the specific composition of the heterotetrameric channel is undefined. Results: Concatenated IP 3 R dimers formed functional tetrameric channels. Heterotetrameric channels containing IP 3 R1 and IP 3 R2 had identical properties to IP 3 R2. Conclusion: Heterotetrameric IP 3 R do not behave as a blend of the constituent monomers. Significance: This study represents the first demonstration of the properties of heterotetrameric IP 3 R of unambiguously defined composition.