Direct association of ligand-binding and pore domains in homo- and heterotetrameric inositol 1,4,5-trisphosphate receptors

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Abstract
Inositol 1,4,5‐trisphosphate receptors (IP3Rs) are a family of intracellular Ca2+ channels that exist as homo‐ or heterotetramers. In order to determine whether the N‐terminal ligand‐binding domain is in close physical proximity to the C‐terminal pore domain, we prepared microsomal membranes from COS‐7 cells expressing recombinant type I and type III IP3R isoforms. Trypsin digestion followed by cross‐linking and co‐immunoprecipitation of peptide fragments suggested an inter‐subunit N‐ and C‐terminal interaction in both homo‐ and heterotetramers. This observation was further supported by the ability of in vitro translated C‐terminal peptides to interact specifically with an N‐terminal fusion protein. Using a 45Ca2+ flux assay, we provide functional evidence that the ligand‐binding domain of one subunit can gate the pore domain of an adjacent subunit. We conclude that common structural motifs are shared between the type I and type III IP3Rs and propose that the gating mechanism of IP3R Ca2+ channels involves the association of the N‐terminus of one subunit with the C‐terminus of an adjacent subunit in both homo‐ and heterotetrameric complexes.