Modification of intracellular Ca2+ dynamics by laser inactivation of inositol 1,4,5-trisphosphate receptor using membrane-permeant probes.

A membrane-permeant malachite green-conjugated IP3 analog (MGIP3/PM) was synthesized as a probe for small molecule-based CALI (smCALI), and its effect on the Ca2+ signaling in intact DT40 chicken B cells was examined. In DT40 B cells treated with the smCALI probe, laser irradiation inhibited IP3-induced Ca2+ oscillations in response to B cell receptor stimulation, demonstrating that IP3R was acutely inactivated. We then applied smCALI to clarify the mechanism of capacitative Ca2+ entry (CCE), in which involvement of IP3R has been suggested. Despite the inactivation of IP3R by smCALI, thapsigargin-induced CCE remained unaffected, providing evidence that functional IP3R is not required for CCE in DT40 cells. These results demonstrate the potency of the smCALI technique for the study of the roles of IP3R in complex intracellular Ca2+ dynamics.