Downregulation of PMCA2 or PMCA3 reorganizes Ca2+ handling systems in differentiating PC12 cells

Abstract

Changes in PMCA2 and PMCA3 expression during neuronal development are tightly linked to structural and functional modifications in Ca2+ handling machinery. Using antisense strategy we obtained stably transfected PC12 lines with reduced level of PMCA2 or PMCA3, which were then subjected to dibutyryl-cAMP differentiation. Reduced level of neuron-specific PMCAs led to acceleration of differentiation and formation of longer neurites than in control PC12 line. Treatment with dibutyryl-cAMP was associated with retraction of growth cones and intensified formation of varicosities. In PMCA2-reduced cells development of apoptosis and DNA laddering were detected. Higher amounts of constitutive isoforms PMCA1 and PMCA4, their putative extended location to gaps left after partial removal of PMCA2 or PMCA3, together with increased SERCA may indicate the induction of compensatory mechanism in modified cells. Functional studies showed altered expression of certain types of VDCCs in PMCA-reduced cells, which correlated with their higher contribution to Ca2+ influx. The cell response to PMCAs suppression suggests the interplay between transcription level of two opposite calcium-transporting systems i.e. voltage- and store depletion-activated channels facilitating Ca2+ influx and calcium pumps responsible for Ca2+ clearance, as well highlights the role of both neuron-specific PMCA isoforms in the control of PC12 cells differentiation.