An N-terminal Sequence Targets and Tethers Na+ Pump α2 Subunits to Specialized Plasma Membrane Microdomains

Sodium pumps (αβ dimers) with the α1 isoform of the catalytic (α) subunit are expressed in all cells. Additionally, most cells express Na + pumps with a second α isoform. For example, astrocytes and arterial myocytes also express Na + pumps with the α2 isoform. The α2 pumps localize to plasma membrane (PM) microdomains overlying “junctional” sarco-/endoplasmic reticulum (S/ER), but the α1 pumps are more uniformly distributed. To study α2 targeting, we expressed α1/α2 and α2/α1 chimeras and 1-90 and 1-120 amino acid N-terminal peptides in primary cultured mouse astrocytes. Immunocytochemistry revealed that α2/α1 (but not α1/α2) chimeras markedly reduced native α2 ( i.e. were “dominant negatives”). N-terminal (1-120 and 1-90 amino acids) α2 (and α3), but not α1 peptides also targeted to the PM-S/ER junctions and were dominant negative for native α2 in astrocytes and arterial myocytes. Thus α2 and α3 have the same targeting sequence. Ca 2+ (fura-2) signals in astrocytes expressing the 1-90 α2 peptide were comparable to signals in cells from α2 null mutants ( i.e. functionally dominant negative): 1 μ m ATP-evoked Ca 2+ transients were augmented, and 100 n m ouabain-induced amplification was abolished. Amino acid substitutions in the 1-120 α1 and α2 constructs, and in full-length α1, revealed that Leu-27 and Ala-35 are essential for targeting/tethering the constructs to PM-S/ER junctions.