Abstract
Engagement of the IP3 receptor by its ligand releases Ca2+ from intracellular stores of the rat basophilic leukemia (RBL) cell. The IP3 receptor in washed permeabilized cells has high affinity (Kd = 1.2±0.3 nM) for [3H]IP3 and is not sensitive to physiological concentrations of Ca2+. Moreover, washed permeabilized cells only release small amounts of Ca2+ when stimulated with IP3. When [3H]IP3 binding to permeabilized cells is performed in the presence of cytosolic constituents (unwashed cells), the IP3 receptor has a lower affinity for [3H]IP3 (Kd from 20 to 100 nM) and has enhanced Ca2+ release. Cytosolic supernatant, prepared by centrifugation of permeabilized cells and added back to washed permeabilized cells, decreases [3H]IP3 binding in a dose-dependent manner and increases the amount of Ca2+ released by IP3. Depletion of either MgATP or IP3 in the cytosolic supernatant does not affect the supernatant's ability to decrease [3H]IP3 binding. Though MgATP competitively inhibits [3H]IP3 binding, it cannot fully account for the shift in Kd or the modulation of IP3-stimulated Ca2+ release in the presence of cytosol. These findings suggest that components present in the cytosolic supernatant modulate the function of the IP3 receptor by maintaining it in a low affinity state capable of promoting Ca2+ release.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 115-124 |
| Number of pages | 10 |
| Journal | BBA - Biomembranes |
| Volume | 1147 |
| Issue number | 1 |
| DOIs | |
| State | Published - Apr 8 1993 |
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Keywords
- Calcium store
- Inositol 1,4,5-trisphosphate
- Intracellular calcium regulation
- Mast cell
- RBL cell
ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Cell Biology
Cite this
Regulation of inositol 1,4,5-trisphosphate receptors in rat basophilic leukemia cells. II. Modulation of the receptor in permeabilized cells by the cytosolic compartment. / Hershey, Panda E C; Pessah, Isaac N; Mohr, Frederick C.
In: BBA - Biomembranes, Vol. 1147, No. 1, 08.04.1993, p. 115-124.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Regulation of inositol 1,4,5-trisphosphate receptors in rat basophilic leukemia cells. II. Modulation of the receptor in permeabilized cells by the cytosolic compartment
AU - Hershey, Panda E C
AU - Pessah, Isaac N
AU - Mohr, Frederick C
PY - 1993/4/8
Y1 - 1993/4/8
N2 - Engagement of the IP3 receptor by its ligand releases Ca2+ from intracellular stores of the rat basophilic leukemia (RBL) cell. The IP3 receptor in washed permeabilized cells has high affinity (Kd = 1.2±0.3 nM) for [3H]IP3 and is not sensitive to physiological concentrations of Ca2+. Moreover, washed permeabilized cells only release small amounts of Ca2+ when stimulated with IP3. When [3H]IP3 binding to permeabilized cells is performed in the presence of cytosolic constituents (unwashed cells), the IP3 receptor has a lower affinity for [3H]IP3 (Kd from 20 to 100 nM) and has enhanced Ca2+ release. Cytosolic supernatant, prepared by centrifugation of permeabilized cells and added back to washed permeabilized cells, decreases [3H]IP3 binding in a dose-dependent manner and increases the amount of Ca2+ released by IP3. Depletion of either MgATP or IP3 in the cytosolic supernatant does not affect the supernatant's ability to decrease [3H]IP3 binding. Though MgATP competitively inhibits [3H]IP3 binding, it cannot fully account for the shift in Kd or the modulation of IP3-stimulated Ca2+ release in the presence of cytosol. These findings suggest that components present in the cytosolic supernatant modulate the function of the IP3 receptor by maintaining it in a low affinity state capable of promoting Ca2+ release.
AB - Engagement of the IP3 receptor by its ligand releases Ca2+ from intracellular stores of the rat basophilic leukemia (RBL) cell. The IP3 receptor in washed permeabilized cells has high affinity (Kd = 1.2±0.3 nM) for [3H]IP3 and is not sensitive to physiological concentrations of Ca2+. Moreover, washed permeabilized cells only release small amounts of Ca2+ when stimulated with IP3. When [3H]IP3 binding to permeabilized cells is performed in the presence of cytosolic constituents (unwashed cells), the IP3 receptor has a lower affinity for [3H]IP3 (Kd from 20 to 100 nM) and has enhanced Ca2+ release. Cytosolic supernatant, prepared by centrifugation of permeabilized cells and added back to washed permeabilized cells, decreases [3H]IP3 binding in a dose-dependent manner and increases the amount of Ca2+ released by IP3. Depletion of either MgATP or IP3 in the cytosolic supernatant does not affect the supernatant's ability to decrease [3H]IP3 binding. Though MgATP competitively inhibits [3H]IP3 binding, it cannot fully account for the shift in Kd or the modulation of IP3-stimulated Ca2+ release in the presence of cytosol. These findings suggest that components present in the cytosolic supernatant modulate the function of the IP3 receptor by maintaining it in a low affinity state capable of promoting Ca2+ release.
KW - Calcium store
KW - Inositol 1,4,5-trisphosphate
KW - Intracellular calcium regulation
KW - Mast cell
KW - RBL cell
UR - http://www.scopus.com/inward/record.url?scp=0027511716&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0027511716&partnerID=8YFLogxK
U2 - 10.1016/0005-2736(93)90321-P
DO - 10.1016/0005-2736(93)90321-P
M3 - Article
VL - 1147
SP - 115
EP - 124
JO - Biochimica et Biophysica Acta - Biomembranes
JF - Biochimica et Biophysica Acta - Biomembranes
SN - 0005-2736
IS - 1
ER -