Location of the Permeation Pathway in the Recombinant Type 1 Inositol 1,4,5-Trisphosphate Receptor

The inositol 1,4,5-trisphosphate receptor (InsP₃R) forms ligand-regulated intracellular Ca²⁺ release channels in the endoplasmic reticulum of all mammalian cells. The InsP₃R has been suggested to have six transmembrane regions (TMRs) near its carboxyl terminus. A TMR-deletion mutation strategy was applied to define the location of the InsP₃R pore. Mutant InsP₃Rs were expressed in COS-1 cells and single channel function was defined in planar lipid bilayers. Mutants having the fifth and sixth TMR (and the interceding lumenal loop), but missing all other TMRs, formed channels with permeation properties similar to wild-type channels (gCs = 284; gCa = 60 pS; P_Ca/P_Cs = 6.3). These mutant channels bound InsP₃, but ligand occupancy did not regulate the constitutively open pore (P_o > 0.80). We propose that a region of 191 amino acids (including the fifth and sixth TMR, residues 2398–2589) near the COOH terminus of the protein forms the InsP₃R pore. Further, we have produced a constitutively open InsP₃R pore mutant that is ideal for future site-directed mutagenesis studies of the structure–function relationships that define Ca²⁺ permeation through the InsP₃R channel.