Spatial and temporal alterations in intracellular calcium [Ca 2+ ] i play a pivotal role in a wide array of neuronal functions. Disruption in Ca 2+ homeostasis has been implicated in the decline in neuronal function in brain aging and in neurodegenerative disorders. The plasma membrane Ca 2+ -ATPase (PMCA) is a high affinity Ca 2+ transporter that plays a crucial role in the termination of [Ca 2+ ] i signals and in the maintenance of low [Ca 2+ ] i essential for signaling. Recent evidence indicates that PMCA is uniquely sensitive to its lipid environment and is stimulated by lipids with ordered acyl chains. Here we show that both PMCA and its activator calmodulin (CaM) are partitioned into liquid-ordered, cholesterol-rich plasma membrane microdomains or 'lipid rafts' in primary cultured neurons. Association of PMCA with rafts was demonstrated in preparations isolated by sucrose density gradient centrifugation and in intact neurons by confocal microscopy. Total raftassociated PMCA activity was much higher than the PMCA activity excluded from these microdomains. Depletion of cellular cholesterol dramatically inhibited the activity of the raft-associated PMCA with no effect on the activity of the non-raft pool. We propose that association of PMCA with rafts represents a novel mechanism for its regulation and, consequently, of Ca 2+ signaling in the central nervous system.

Oxidative stress leads to the disruption of calcium homeostasis in brain neurons; however, the direct effects of oxidants on proteins that regulate intracellular calcium [Ca 2+ ] i are not known. The calmodulin (CaM) -stimulated plasma membrane Ca 2+ -ATPase (PMCA) plays a critical role in regulating [Ca 2+ ] i . Our previous in vitro studies showed that PMCA present in brain synaptic membranes is readily inactivated by a variety of reactive oxygen species (ROS). The present studies were conducted to determine the vulnerability of PMCA to ROS generated in neurons as would likely occur in vivo. Primary cortical neurons were exposed to paraquat (PQ), a redox cycling agent that generates intracellular ROS. Low concentrations of PQ (5-10 μM) increased PMCA basal activity by 2-fold but abolished its sensitivity to CaM. Higher concentrations (25-100 μM) inhibited both components of PMCA activity. Immunoblots showed the formation of high molecular weight PMCA aggregates. Additionally, PMCA showed evidence of proteolytic degradation. PMCA proteolysis was prevented by a calpain inhibitor, suggesting a role for calpain. Our findings suggest that PMCA is a sensitive target of oxidative stress in primary neurons. Inactivation of this Ca 2+ transporter under prolonged oxidative stress could alter neuronal Ca 2+ signaling.

Intraneuronal calcium ([Ca 2+ ] i ) regulation is altered in aging brain, possibly because of the changes in critical Ca 2+ transporters. We previously reported that the levels of the plasma membrane Ca 2+-ATPase (PMCA) and the V max for enzyme activity are significantly reduced in synaptic membranes in aging rat brain. The goal of these studies was to use RNA i techniques to suppress expression of a major neuronal isoform, PMCA2, in neurons in culture to determine the potential functional consequences of a decrease in PMCA activity. Embryonic rat brain neurons and SH-SY5Y neuroblastoma cells were transfected with in vitro -transcribed short interfering RNA or a short hairpin RNA expressing vector, respectively, leading to 80% suppression of PMCA2 expression within 48 h.