Na+ pump alpha 2-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).
Golovina
Vera A
VA
Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA. vgolovin@umaryland.edu
Song
Hong
H
James
Paul F
PF
Lingrel
Jerry B
JB
Blaustein
Mordecai P
MP
D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005333:Fetus; D051379:Mice; D018345:Mice, Knockout; D020033:Protein Isoforms / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000235:genetics / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*
D004791:Enzyme Inhibitors; D020033:Protein Isoforms; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; D012964:Sodium; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium