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

Abstract

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 (τ = 52 ± 4 s; n= 25). After differentiation for 12-16 days, the [Ca2+]i recovery rate increased by more than threefold (τ = 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.