Sorting of α2 and α3 Na+ Pumps in Glia and Neurons: Linkage with Na/Ca Exchanger‐1

Abstract

The α2 Na+ pump amino acid (AA) sorting sequence in astrocytes and arterial myocytes is: L27DELKKEVA35 (Song et al., J Biol Chem 281:12929, 2006). Plasmids encoding this sequence, with an N‐terminal Green Fluorescent Protein and C‐terminal Flag (f)‐tag (GFP‐LDELKKEVA‐f), when transfected (Lipofectamine) into mouse primary cultured astrocytes, were dominant negative (DN) for normal α2 expression (by immunocytochemistry). Mutations of AAs 28–34 to Ala did not prevent the DN effect, but L27A, A35S (as in α1 pumps) or K31P were not DN. Thus, the α2 sorting sequence corresponds to two turns of an α‐helix with attachment sites at Leu‐27 and Ala‐35. Loss of the DN effect by the K31P mutant is consistent because Pro disrupts α‐helices. Adenoviral transfection of α2(1‐130)/α1‐f chimera in astrocytes knocks down α2 Na+ pumps and, simultaneously, Na/Ca exchanger 1 (NCX1) expression, with a ~12 hr half‐time. NCX1 silencer RNA and α2 Na+ pump knock‐out mouse data also reveal linkage of α2 and NCX1 expression in astrocytes.The homologous neuronal α3 Na+ pump N‐terminal sequence is: LDDLKKEVA. We generated plasmids coding for the N‐terminal 120 AAs of α1 and α3 Na+ pumps with mCherry and flag tags [mCherry‐α1(1‐120)‐f and mCherry‐α3(1‐120)‐f]. Transfection and expression of the latter, but not the former, was DN for α3 Na+ pumps in rat primary cultured hippocampal neurons. This suggests that LDDLKKEVA is the α3 sorting sequence.