Single-channel function of recombinant type 2 inositol 1,4,5-trisphosphatereceptor

A full-length rat type 2 inositol 1,4,5-trisphosphate (InsP(3)) receptor cD NA construct was generated and expressed in COS-1 cells. Targeting of the f ull-length recombinant type 2 receptor protein to the endoplasmic reticulum was confirmed by immunocytochemistry using isoform specific affinity-purif ied antibodies and InsP(3)R-green fluorescent protein chimeras. The recepto r protein was solubilized and incorporated into proteoliposomes for functio nal characterization. Single-channel recordings from proteoliposomes fused into planar lipid bilayers revealed that the recombinant protein formed Ins P(3)- and Ca2+-sensitive ion channels. The unitary conductance (similar to 250 pS; 220/20 mM Cs+ as charge carrier), gating, InsP(3), and Ca2+ sensiti vities were similar to those previously described for the native type 2 Ins P(3)R channel. However, the maximum open probability of the recombinant cha nnel was slightly lower than that of its native counterpart. These data sho w that our lull-length rat type 2 InsP(3)R cDNA construct encodes a protein that forms an ion channel with functional attributes like those of the nat ive type 2 InsP(3)R channel. The possibility of measuring the function of s ingle recombinant type 2 InsP(3)R is a significant step toward the use of m olecular tools to define the determinants of isoform-specific InsP(3)R func tion and regulation.