An N-terminal sequence targets and tethers Na⁺ pump α2 subunits to specialized plasma membrane microdomains

Sodium pumps (αβdimers) 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. Ca²⁺ (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 μM ATP-evoked Ca²⁺ transients were augmented, and 100 nM 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.