Using concatenated subunits to investigate the functional consequences of heterotetrameric inositol 1,4,5-trisphosphate receptors

Abstract

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are a family of ubiquitous, ER localized, tetrameric Ca2+ release channels. There are three subtypes of the IP3Rs (R1, R2, R3), encoded by three distinct genes, that share ∼60-70% sequence identity. The diversity of Ca2+ signals generated by IP3Rs is thought to be largely the result of differential tissue expression, intracellular localization and subtype-specific regulation of the three subtypes by various cellular factors, most significantly InsP3, Ca2+ and ATP. However, largely unexplored is the notion of additional signal diversity arising from the assembly of both homo and heterotetrameric InsP3Rs. In the present article, we review the biochemical and functional evidence supporting the existence of homo and heterotetrameric populations of InsP3Rs. In addition, we consider a strategy that utilizes genetically concatenated InsP3Rs to study the functional characteristics of heterotetramers with unequivocally defined composition. This approach reveals that the overall properties of IP3R are not necessarily simply a blend of the constituent monomers but that specific subtypes appear to dominate the overall characteristics of the tetramer. It is envisioned that the ability to generate tetramers with defined wild type and mutant subunits will be useful in probing fundamental questions relating to IP3R structure and function.

FIG 1. Immunodepletion experiments supporting the existence of heteromeric IP₃Rs in vivo

(A) Schematic representation of strategy for immunodepletion experiment. (B) Mouse pancreas lysates were subject to sequential immunoprecipitation, first with α-InsP₃R2 (left blot, middle lane) followed by α-InsP₃R1 (left blot, right lane) to maximally deplete R2 and R1, respectively (right blot). Equivalent supernatants, before (Input) and after immunodepletion (Post-ID), were separated using SDS-PAGE and probed with the indicated antibodies. (C) A histogram comparing R3 immunoreactivity post R2 and post R2 + R1 immunodepletion. Quantitative densitometry revealed ~11% of R3 exists as homotetramers. Adapted from [28] with permission.

Fig 2. Generation of concatenated IP₃R constructs and their stable expression DT40-3KO IP₃R null cells

(A) InsP₃R ‘head’ and ‘tail’ subunits were subcloned into pJAZZ_mamm, a linear vector plasmid developed from N15 coliphage (Lucigen). (B) Representative western blots showing that concatenated dimer constructs were stably transfected and expressed into DT40-3KO cells. (C) Representative blue native gels demonstrating that DT40-3KO cells express monomeric or dimeric constructs are assembled to form tetramers. Adapted from [28] with permission.

Fig 3. Activity of concatenated heterodimers is dominated by R2

(A-F) Activation of B cell Receptor in DT40-3KO cells stably expressing R1 and R2 containing monomeric and dimeric InsP₃Rs results in characteristic Ca²⁺ signals. R2 properties appear to dominate. (G-I) Representative single channel K⁺ current recordings of R1R1, R2R2 and R1R2 dimers, measured using on-nucleus patch clamp, at 1 or 10 μM InsP₃ in the presence of 200 nM free Ca²⁺ and either 100 μM or 5 mM ATP. R2 properties appear to dominate. Adapted from [28] with permission.