An N-terminal sequence targets and tethers Na+ pump alpha2 subunits to specialized plasma membrane microdomains.
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.
Song
Hong
H
Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Lee
Moo Yeol
MY
Kinsey
Stephen P
SP
Weber
David J
DJ
Blaustein
Mordecai P
MP
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*
D011993:Recombinant Fusion Proteins; D012964:Sodium