Measurement of Calcium Fluxes in Permeabilized Cells Using a 45Ca2+ Uptake and Release Assay

Many cell surface receptors activate phosphoinositidase(s) C, via G proteins that catalyze the hydrolysis of phosphatidylinositol 4,5-biphosphate to produce the second messengers, inositol(1,4,5)trisphosphate [Ins(1,4,5)P3 ] and diacylglycerol (1 ). Ins(1,4,5)P3 interacts with specific receptor populations of ligand-gated channels to mobilize nonmitochondrial intracellular calcium (Ca2+ ) stores (1 ). Because Ins(1,4,5)P3 is very hydrophilic, it cannot readily cross the intact plasma membrane. Consequently, Ins(1,4,5)P3 -induced Ca2+ release was initially demonstrated in permeabilized pancreatic acinar cells (2 ), and all subsequent studies in cells have involved the introduction of Ins(1,4,5)P3 by rendering a cell population permeable (3 ), using microinjection techniques (4 ) or by the presentation of chemically modified membrane-permeable Ins(1,4,5)P3 analogs, such as photolabile “caged Ins(1,4,5)P3 ” (5 ). An alternative approach involves disruption of the plasma membrane and preparation of microsomes from the intracellular vesicular Ca2+ stores (6 ,7 ), however, these preparations exhibit a loss of Ins(1,4,5)P3 responsiveness compared to cells. The author will describe a 45 Ca2+ -release assay used to monitor Ins(1,4,5)P3 -induced Ca2+ mobilization from nonmitochondrial intracellular Ca2+ stores using “cytosol-like” buffer (CLB) and permeabilized SH-SY5Y neuroblastoma cell populations.