Regulation of Ca2+ signaling by Na+ pump alpha-2 subunit expression

The role of the Na(+) pump alpha2 subunit in Ca(2+) signaling was examined in primary cultured astrocytes from wild type (WT) mouse fetuses and those with a null mutation in one (Het) or both (KO) alpha2 genes. Cytosol Na(+) and Ca(2+) concentrations ([Na(+)](CYT) and [Ca(2+)](CYT)) were measured with benzofuran isophthalate (SBFI) and Fura-2. Het astrocytes express approximately 50% of normal alpha2; KO cells express none. Resting [Na(+)](CYT) = 6.5 mM in WT, 6.8 mM in Het, and 8.0 mM in KO cells; 500 nM ouabain (which inhibits only alpha2) equalized [Na(+)](CYT) at 8 mM in all genotypes. Resting [Ca(2+)](CYT) = 132 nM in WT, 162 nM in Het and 196 nM in KO cells. Cyclopiazonic acid (CPA), which inhibits endoplasmic reticulum (ER) Ca(2+) pumps, induces elevation of [Ca(2+)]CYT. These Ca(2+) responses are augmented in Het and KO cells. This correlates with alpha2 Na(+) pump and Na(+)/Ca(2+) exchanger (NCX) localization in plasma membrane (PM) microdomains that overlie the ER. Selective reduction of alpha2 Na(+) pump activity apparently elevates local [Na(+)] and, via NCX, [Ca(2+)] in the tiny cytosolic space between the PM and ER. This augments adjacent ER Ca(2+) stores and amplifies Ca(2+) signaling without elevating bulk [Na(+)](CYT).