Single-Channel Function of Recombinant Type 2 Inositol 1,4,5-Trisphosphate Receptor

AbstractA full-length rat type 2 inositol 1,4,5-trisphosphate (InsP3) receptor cDNA construct was generated and expressed in COS-1 cells. Targeting of the full-length recombinant type 2 receptor protein to the endoplasmic reticulum was confirmed by immunocytochemistry using isoform specific affinity-purified antibodies and InsP3R-green fluorescent protein chimeras. The receptor protein was solubilized and incorporated into proteoliposomes for functional characterization. Single-channel recordings from proteoliposomes fused into planar lipid bilayers revealed that the recombinant protein formed InsP3- and Ca2+-sensitive ion channels. The unitary conductance (∼250 pS; 220/20mM Cs+ as charge carrier), gating, InsP3, and Ca2+ sensitivities were similar to those previously described for the native type 2 InsP3R channel. However, the maximum open probability of the recombinant channel was slightly lower than that of its native counterpart. These data show that our full-length rat type 2 InsP3R cDNA construct encodes a protein that forms an ion channel with functional attributes like those of the native type 2 InsP3R channel. The possibility of measuring the function of single recombinant type 2 InsP3R is a significant step toward the use of molecular tools to define the determinants of isoform-specific InsP3R function and regulation