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© The Rockefeller University Press, 0022-1295/1998//847/ $5.00
Journal of General Physiology, Volume 111, Number 6, 1998

Article

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

Elena Kaznacheyeva, Vitalie D. Lupu, and Ilya Bezprozvanny

From the Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9040

The inositol (1,4,5)-trisphosphate receptor (InsP3R) mediates Ca2+ release from intracellular stores in response to generation of second messenger InsP3. InsP3R was biochemically purified and cloned, and functional properties of native InsP3-gated Ca2+ channels were extensively studied. However, further studies of InsP3R are obstructed by the lack of a convenient functional assay of expressed InsP3R activity. To establish a functional assay of recombinant InsP3R activity, transient heterologous expression of neuronal rat InsP3R cDNA (InsP3R-I, SI– SII+ splice variant) in HEK-293 cells was combined with the planar lipid bilayer reconstitution experiments. Recombinant InsP3R retained specific InsP3 binding properties (Kd = 60 nM InsP3) and were specifically recognized by anti–InsP3R-I rabbit polyclonal antibody. Density of expressed InsP3R-I was at least 20-fold above endogenous InsP3R background and only 2–3-fold lower than InsP3R density in rat cerebellar microsomes. When incorporated into planar lipid bilayers, the recombinant InsP3R formed a functional InsP3-gated Ca2+ channel with 80 pS conductance using 50 mM Ba2+ as a current carrier. Mean open time of recombinant InsP3-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 InsP3R-I were very similar to properties of native rat cerebellar InsP3R recorded in identical experimental conditions. Recombinant InsP3R also retained bell-shaped dependence on cytosolic Ca2+ concentration and allosteric modulation by ATP, similar to native cerebellar InsP3R. The following conclusions are drawn from these results. (a) Rat neuronal InsP3R-I cDNA encodes a protein that is either sufficient to produce InsP3-gated channel with functional properties identical to the properties of native rat cerebellar InsP3R, or it is able to form a functional InsP3-gated channel by forming a complex with proteins endogenously expressed in HEK-293 cells. (b) Successful functional expression of InsP3R in a heterologous expression system provides an opportunity for future detailed structure–function characterization of this vital protein.

Key Words: calcium channel • ryanodine receptor • planar lipid bilayer • calcium signaling

Address correspondence to Dr. Ilya Bezprozvanny, Department of Physiology, K4.112, UT Southwestern Medical Center at Dallas, Dallas, TX 75235-9040. Fax: 214-648-8685; E-mail: bezprozv{at}utsw.swmed.edu

Abbreviations: InsP3R, inositol (1,4,5)-trisphosphate receptor; RyanR, ryanodine receptor


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