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)P
3
] and diacylglycerol (
1
). Ins(1,4,5)P
3
interacts with specific receptor populations of ligand-gated channels to mobilize nonmitochondrial intracellular calcium (Ca
2+
) stores (
1
). Because Ins(1,4,5)P
3
is very hydrophilic, it cannot readily cross the intact plasma membrane. Consequently, Ins(1,4,5)P
3
-induced Ca
2+
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)P
3
by rendering a cell population permeable (
3
), using microinjection techniques (
4
) or by the presentation of chemically modified membrane-permeable Ins(1,4,5)P
3
analogs, such as photolabile “caged Ins(1,4,5)P
3
” (
5
). An alternative approach involves disruption of the plasma membrane and preparation of microsomes from the intracellular vesicular Ca
2+
stores (
6
,
7
), however, these preparations exhibit a loss of Ins(1,4,5)P
3
responsiveness compared to cells. The author will describe a
45
Ca
2+
-release assay used to monitor Ins(1,4,5)P
3
-induced Ca
2+
mobilization from nonmitochondrial intracellular Ca
2+
stores using “cytosol-like” buffer (CLB) and permeabilized SH-SY5Y neuroblastoma cell populations.