Na+ pump α2-subunit expression modulates Ca2+ signaling

The role of the Na(+) pump alpha(2)-subunit in Ca(2+) signaling was examined in primary cultured astrocytes from wild-type (alpha(2)+/+ = WT) mouse fetuses and those with a null mutation in one alpha(2)+/- = heterozygote (Het) or both alpha(2)-/- = knockout (KO) alpha(2) genes. Na(+) pump catalytic (alpha) subunit expression was measured by immunoblot; cytosol Na(+) (Na(+)(cyt)) and Ca(2+) (Ca(2+)(cyt)) were measured with sodium-binding benzofuran isophthalate and fura 2 by using digital imaging. Astrocytes express Na(+) pumps with both alpha(1)- ( approximately 80% of total alpha) and alpha(2)- ( approximately 20% of total alpha) subunits. Het astrocytes express approximately 50% of normal alpha(2); those from KO express none. Expression of alpha(1) is normal in both Het and KO cells. Resting Na(+)(cyt) = 6.5 mM in WT, 6.8 mM in Het (P > 0.05 vs. WT), and 8.0 mM in KO cells (P < 0.001); 500 nM ouabain (inhibits only alpha(2)) equalized Na(+)(cyt) at 8 mM in all three cell types. Resting Ca(2+)(cyt) = 132 nM in WT, 162 nM in Het, and 196 nM in KO cells (both P < 0.001 vs. WT). Cyclopiazonic acid (CPA), which inhibits endoplasmic reticulum (ER) Ca(2+) pumps and unloads the ER, induces transient (in Ca(2+)-free media) or sustained (in Ca(2+)-replete media) elevation of Ca(2+)(cyt). These Ca(2+) responses to 10 microM CPA were augmented in Het as well as KO cells. When CPA was applied in Ca(2+)-free media, the reintroduction of Ca(2+) induced significantly larger transient rises in Ca(2+)(cyt) (due to Ca(2+) entry through store-operated channels) in Het and KO cells than in WT cells. These results correlate with published evidence that alpha(2) Na(+) pumps and Na(+)/Ca(2+) exchangers are confined to plasma membrane microdomains that overlie the ER. The data suggest that selective reduction of alpha(2) Na(+) pump activity can elevate local Na(+) and, via Na(+)/Ca(2+) exchange, Ca(2+) in the tiny volume of cytosol between the plasma membrane and ER. This, in turn, augments adjacent ER Ca(2+) stores and thereby amplifies Ca(2+) signaling without elevating bulk Na(+)(cyt).