title: An N-terminal sequence targets and tethers Na+ pump alpha2 subunits to specialized plasma membrane microdomains.
abstract: Sodium pumps (alphabeta dimers) with the alpha1 isoform of the catalytic (alpha) subunit are expressed in all cells. Additionally, most cells express Na+ pumps with a second alpha isoform. For example, astrocytes and arterial myocytes also express Na+ pumps with the alpha2 isoform. The alpha2 pumps localize to plasma membrane (PM) microdomains overlying "junctional" sarco-/endoplasmic reticulum (S/ER), but the alpha1 pumps are more uniformly distributed. To study alpha2 targeting, we expressed alpha1/alpha2 and alpha2/alpha1 chimeras and 1-90 and 1-120 amino acid N-terminal peptides in primary cultured mouse astrocytes. Immunocytochemistry revealed that alpha2/alpha1 (but not alpha1/alpha2) chimeras markedly reduced native alpha2 (i.e. were "dominant negatives"). N-terminal (1-120 and 1-90 amino acids) alpha2 (and alpha3), but not alpha1 peptides also targeted to the PM-S/ER junctions and were dominant negative for native alpha2 in astrocytes and arterial myocytes. Thus alpha2 and alpha3 have the same targeting sequence. Ca2+ (fura-2) signals in astrocytes expressing the 1-90 alpha2 peptide were comparable to signals in cells from alpha2 null mutants (i.e. functionally dominant negative): 1 microM ATP-evoked Ca2+ transients were augmented, and 100 nM ouabain-induced amplification was abolished. Amino acid substitutions in the 1-120 alpha1 and alpha2 constructs, and in full-length alpha1, revealed that Leu-27 and Ala-35 are essential for targeting/tethering the constructs to PM-S/ER junctions.
authors.affiliation: Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Song
authors.forename: Moo Yeol
authors.initials: MY
authors.lastname: Lee
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Kinsey
authors.forename: David J
authors.initials: DJ
authors.lastname: Weber
authors.forename: Mordecai P
authors.initials: MP
authors.lastname: Blaustein
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D008297:Male; D021962:Membrane Microdomains / Q000737:chemistry*; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000737:chemistry*
chemical_list: D011993:Recombinant Fusion Proteins; D012964:Sodium