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RNA i – induced silencing of the plasma membrane Ca 2+ – ATPase 2 in neuronal cells: effects on Ca 2+ homeostasis and cell viability
Author(s) -
Fernandes Denzyl,
Zaidi Asma,
Bean Jennifer,
Hui Dongwei,
Michaelis Mary L.
Publication year - 2007
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.2007.04592.x
Subject(s) - homeostasis , plasma membrane ca2+ atpase , biology , small interfering rna , small hairpin rna , rna , microbiology and biotechnology , atpase , medicine , endocrinology , biochemistry , enzyme , gene
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. Fluorescence ratio imaging of free [Ca 2+ ] i revealed that primary neurons with reduced PMCA2 expression had higher basal [Ca 2+ ] i , slower recovery from KCl‐induced Ca 2+ transients, and incomplete return to pre‐stimulation Ca 2+ levels. Primary neurons and SH‐SY5Y cells with PMCA2 suppression both exhibited significantly greater vulnerability to the toxicity of various stresses. Our results indicate that a loss of PMCA such as occurs in aging brain likely leads to subtle disruptions in normal Ca 2+ signaling and enhanced susceptibility to stresses that can alter the regulation of Ca 2+ homeostasis.