Single-Channel Properties of Inositol (1,4,5)-Trisphosphate Receptor Heterologously Expressed in HEK-293 Cells

The inositol (1,4,5)-trisphosphate receptor (InsP(3)R) mediates Ca(2+) release from intracellular stores in response to generation of second messenger InsP(3). InsP(3)R was biochemically purified and cloned, and functional properties of native InsP(3)-gated Ca(2+) channels were extensively studied. However, further studies of InsP(3)R are obstructed by the lack of a convenient functional assay of expressed InsP(3)R activity. To establish a functional assay of recombinant InsP(3)R activity, transient heterologous expression of neuronal rat InsP(3)R cDNA (InsP(3)R-I, SI− SII+ splice variant) in HEK-293 cells was combined with the planar lipid bilayer reconstitution experiments. Recombinant InsP(3)R retained specific InsP(3) binding properties (K (d) = 60 nM InsP(3)) and were specifically recognized by anti–InsP(3)R-I rabbit polyclonal antibody. Density of expressed InsP(3)R-I was at least 20-fold above endogenous InsP(3)R background and only 2–3-fold lower than InsP(3)R density in rat cerebellar microsomes. When incorporated into planar lipid bilayers, the recombinant InsP(3)R formed a functional InsP(3)-gated Ca(2+) channel with 80 pS conductance using 50 mM Ba(2+) as a current carrier. Mean open time of recombinant InsP(3)-gated channels was 3.0 ms; closed dwell time distribution was double exponential and characterized by short (18 ms) and long (130 ms) time constants. Overall, gating and conductance properties of recombinant neuronal rat InsP(3)R-I were very similar to properties of native rat cerebellar InsP(3)R recorded in identical experimental conditions. Recombinant InsP(3)R also retained bell-shaped dependence on cytosolic Ca(2+) concentration and allosteric modulation by ATP, similar to native cerebellar InsP(3)R. The following conclusions are drawn from these results. (a) Rat neuronal InsP(3)R-I cDNA encodes a protein that is either sufficient to produce InsP(3)-gated channel with functional properties identical to the properties of native rat cerebellar InsP(3)R, or it is able to form a functional InsP(3)-gated channel by forming a complex with proteins endogenously expressed in HEK-293 cells. (b) Successful functional expression of InsP(3)R in a heterologous expression system provides an opportunity for future detailed structure–function characterization of this vital protein.