Na+ pump α2-subunit expression modulates Ca2+ signaling

The role of the Na+ pump alpha(2)-subunit in Ca2+ 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 [Ca2+] ([Ca2+](cyt)) were measured with sodium-binding benzofuran isophthalate and fura 2 by using digital imaging. Astrocytes express Na+ pumps with both alpha(1)-(approximate to80% of total alpha) and alpha(2)-(approximate to20% of total alpha) subunits. Het astrocytes express approximate to50% 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 [Ca2+](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) Ca2+ pumps and unloads the ER, induces transient (in Ca2+-free media) or sustained (in Ca2+-replete media) elevation of [Ca2+](cyt). These Ca2+ responses to 10 muM CPA were augmented in Het as well as KO cells. When CPA was applied in Ca2+-free media, the reintroduction of Ca2+ induced significantly larger transient rises in [Ca2+](cyt) (due to Ca2+ 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+/Ca2+ 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+/Ca2+ exchange, [Ca2+] in the tiny volume of cytosol between the plasma membrane and ER. This, in turn, augments adjacent ER Ca2+ stores and thereby amplifies Ca2+ signaling without elevating bulk [Na+](cyt).