Differentiation induces up-regulation of plasma membrane Ca2+-ATPase and concomitant increase in Ca2+ efflux in human neuroblastoma cell line IMR-32

Precise regulation of intracellular Ca2+ concentration ([Ca2+](i)) is achieved by the coordinated function of Ca2+ channels and Ca2+ buffers. Neuronal differentiation induces up-regulation of Ca2+ channels. However, little is known about the effects of differentiation on the expression of the plasma membrane Ca2+-ATPase (PMCA), the principal Ca2+ extrusion mechanism in neurons. In this study, we examined the regulation of PMCA expression during differentiation of the human neuroblastoma cell line IMR-32. [Ca2+](i) was monitored in single cells using indo-1 microfluorimetry. When the Ca2+-ATPase of the endoplasmic reticulum was blocked by cyclopiazonic acid, [Ca2+](i) recovery after small depolarization-induced Ca2+ loads was governed primarily by PMCAs. [Ca2+](i) returned to baseline by a process described by a monoexponential function in undifferentiated cells (tau = 52 +/- 4 s; n = 25). After differentiation for 12-16 days, the [Ca2+](i) recovery rate increased by more than threefold (tau = 17 +/- 1 s, n = 31). Western blots showed a pronounced increase in expression of three major PMCA isoforms in IMR-32 cells during differentiation, including PMCA2, PMCA3 and PMCA4. These results demonstrate up-regulation of PMCAs on the functional and protein level during neuronal differentiation in vitro. Parallel amplification of Ca2+ influx and efflux pathways may enable differentiated neurons to precisely localize Ca2+ signals in time and space.