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.
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1 June 1998
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June 01 1998
Single-Channel Properties of Inositol (1,4,5)-Trisphosphate Receptor Heterologously Expressed in HEK-293 Cells
Elena Kaznacheyeva,
Elena Kaznacheyeva
From the Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9040
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Vitalie D. Lupu,
Vitalie D. Lupu
From the Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9040
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Ilya Bezprozvanny
Ilya Bezprozvanny
From the Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9040
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Elena Kaznacheyeva
From the Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9040
Vitalie D. Lupu
From the Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9040
Ilya Bezprozvanny
From the Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9040
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: [email protected]
Drs. Kaznacheyeva and Lupu contributed equally to this work and should be considered co-first authors.
Received:
December 30 1997
Accepted:
March 26 1998
Online ISSN: 1540-7748
Print ISSN: 0022-1295
1998
J Gen Physiol (1998) 111 (6): 847–856.
Article history
Received:
December 30 1997
Accepted:
March 26 1998
Citation
Elena Kaznacheyeva, Vitalie D. Lupu, Ilya Bezprozvanny; Single-Channel Properties of Inositol (1,4,5)-Trisphosphate Receptor Heterologously Expressed in HEK-293 Cells . J Gen Physiol 1 June 1998; 111 (6): 847–856. doi: https://doi.org/10.1085/jgp.111.6.847
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