IP(3)-mediated Ca(2+) signals in human neuroblastoma SH-SY5Y cells with exogenous overexpression of type 3 IP(3) receptor.
Human neuroblastoma SH-SY5Y cells, predominantly expressing type 1 inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), were stably transfected with IP(3)R type 3 (IP(3)R3) cDNA. Immunocytochemistry experiments showed a homogeneous cytoplasmic distribution of type 3 IP(3)Rs in transfected and selected high expression cloned cells. Using confocal Ca(2+) imaging, carbachol (CCh)-induced Ca(2+) release signals were studied. Low CCh concentrations (< or = 750 nM) evoked baseline Ca(2+) oscillations. Transfected cells displayed a higher CCh responsiveness than control or cloned cells. Ca(2+) responses varied between fast, large Ca(2+) spikes and slow, small Ca(2+) humps, while in the clone only Ca(2+) humps were observed. Ca(2+) humps in the transfected cells were associated with a high expression level of IP(3)R3. At high CCh concentrations (10 microM) Ca(2+) transients in transfected and cloned cells were similar to those in control cells. In the clone exogenous IP(3)R3 lacked the C-terminal channel domain but IP(3)-binding capacity was preserved. Transfected cells mainly expressed intact type 3 IP(3)Rs but some protein degradation was also observed. We conclude that in transfected cells expression of functional type 3 IP(3)Rs causes an apparent higher affinity for IP(3). In the clone, the presence of degraded receptors leads to an efficient cellular IP(3) buffer and attenuated IP(3)-evoked Ca(2+) release.
Van Acker
K
K
Laboratory of Physiology, Katholieke Universiteit Leuven, B-3000, Leuven, Belgium
Nadif Kasri
N
N
De Smet
P
P
Parys
J B
JB
De Smedt
H
H
Missiaen
L
L
Callewaert
G
G
D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics* / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000235:genetics*; D002217:Carbachol / Q000494:pharmacology; D002455:Cell Division / Q000235:genetics; D018679:Cholinergic Agonists / Q000494:pharmacology; D002999:Clone Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005057:Eukaryotic Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007150:Immunohistochemistry; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D009447:Neuroblastoma; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D015220:Calcium Channels; D018679:Cholinergic Agonists; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear; D015544:Inositol 1,4,5-Trisphosphate; D002217:Carbachol; D002118:Calcium