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 (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.