Single-channel properties of inositol (1,4,5)-trisphosphate receptor heterologously expressed in HEK-293 cells.
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.
Kaznacheyeva
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Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9040, USA.
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Bezprozvanny
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D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D015220:Calcium Channels / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002531:Cerebellum / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007293:Inosine Triphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D015640:Ion Channel Gating / Q000502:physiology; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D019837:Ryanodine Receptor Calcium Release Channel / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D015220:Calcium Channels; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D007293:Inosine Triphosphate