IP 3-mediated Ca 2+ signals in human neuroblastoma SH-SY5Y cells with exogenous overexpression of type 3 IP 3 receptor

Abstract

Human neuroblastoma SH-SY5Y cells, predominantly expressing type 1 inositol 1,4,5-trisphosphate (IP 3) receptor (IP 3R), were stably transfected with IP 3R type 3 (IP 3R3) cDNA. Immunocytochemistry experiments showed a homogeneous cytoplasmic distribution of type 3 IP 3Rs 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 (≤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 3R3. At high CCh concentrations (10 μM) Ca 2+ transients in transfected and cloned cells were similar to those in control cells. In the clone exogenous IP 3R3 lacked the C-terminal channel domain but IP 3-binding capacity was preserved. Transfected cells mainly expressed intact type 3 IP 3Rs but some protein degradation was also observed. We conclude that in transfected cells expression of functional type 3 IP 3Rs 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.