Single-channel properties of inositol (1,4,5)-trisphosphate receptor heterologously expressed in HEK-293 cells

Elena Kaznacheyeva, Vitalie D. Lupu, Ilya Bezprozvanny

Research output: Contribution to journalArticle

61 Citations (Scopus)

Abstract

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 (K(d) = 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 InsP'3-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.

Original languageEnglish (US)
Pages (from-to)847-856
Number of pages10
JournalJournal of General Physiology
Volume111
Issue number6
DOIs
StatePublished - Jun 1998

Fingerprint

Inositol 1,4,5-Trisphosphate Receptors
HEK293 Cells
Lipid Bilayers
Complementary DNA
Proteins
Second Messenger Systems
Microsomes
Adenosine Triphosphate
Rabbits
Antibodies

Keywords

  • Calcium channel
  • Calcium signaling
  • Planar lipid bilayer
  • Ryanodine receptor

ASJC Scopus subject areas

  • Physiology

Cite this

Single-channel properties of inositol (1,4,5)-trisphosphate receptor heterologously expressed in HEK-293 cells. / Kaznacheyeva, Elena; Lupu, Vitalie D.; Bezprozvanny, Ilya.

In: Journal of General Physiology, Vol. 111, No. 6, 06.1998, p. 847-856.

Research output: Contribution to journalArticle

@article{2dfc37dcc664403ba3d9bb341ec2a5b1,
title = "Single-channel properties of inositol (1,4,5)-trisphosphate receptor heterologously expressed in HEK-293 cells",
abstract = "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 (K(d) = 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 InsP'3-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.",
keywords = "Calcium channel, Calcium signaling, Planar lipid bilayer, Ryanodine receptor",
author = "Elena Kaznacheyeva and Lupu, {Vitalie D.} and Ilya Bezprozvanny",
year = "1998",
month = "6",
doi = "10.1085/jgp.111.6.847",
language = "English (US)",
volume = "111",
pages = "847--856",
journal = "Journal of General Physiology",
issn = "0022-1295",
publisher = "Rockefeller University Press",
number = "6",

}

TY - JOUR

T1 - Single-channel properties of inositol (1,4,5)-trisphosphate receptor heterologously expressed in HEK-293 cells

AU - Kaznacheyeva, Elena

AU - Lupu, Vitalie D.

AU - Bezprozvanny, Ilya

PY - 1998/6

Y1 - 1998/6

N2 - 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 (K(d) = 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 InsP'3-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.

AB - 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 (K(d) = 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 InsP'3-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.

KW - Calcium channel

KW - Calcium signaling

KW - Planar lipid bilayer

KW - Ryanodine receptor

UR - http://www.scopus.com/inward/record.url?scp=0031748535&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0031748535&partnerID=8YFLogxK

U2 - 10.1085/jgp.111.6.847

DO - 10.1085/jgp.111.6.847

M3 - Article

C2 - 9607940

AN - SCOPUS:0031748535

VL - 111

SP - 847

EP - 856

JO - Journal of General Physiology

JF - Journal of General Physiology

SN - 0022-1295

IS - 6

ER -