TY - JOUR
T1 - The α2β2 isoform combination dominates the astrocytic Na+/K+-ATPase activity and is rendered nonfunctional by the α2.G301R familial hemiplegic migraine type 2-associated mutation
AU - Stoica, Anca
AU - Larsen, Brian Roland
AU - Assentoft, Mette
AU - Holm, Rikke
AU - Holt, Leanne Melissa
AU - Vilhardt, Frederik
AU - Vilsen, Bente
AU - Lykke-Hartmann, Karin
AU - Olsen, Michelle Lynne
AU - Macaulay, Nanna
PY - 2017/11
Y1 - 2017/11
N2 - Synaptic activity results in transient elevations in extracellular K+, clearance of which is critical for sustained function of the nervous system. The K+ clearance is, in part, accomplished by the neighboring astrocytes by mechanisms involving the Na+/K+-ATPase. The Na+/K+-ATPase consists of an α and a β subunit, each with several isoforms present in the central nervous system, of which the α2β2 and α2β1 isoform combinations are kinetically geared for astrocytic K+ clearance. While transcript analysis data designate α2β2 as predominantly astrocytic, the relative quantitative protein distribution and isoform pairing remain unknown. As cultured astrocytes altered their isoform expression in vitro, we isolated a pure astrocytic fraction from rat brain by a novel immunomagnetic separation approach in order to determine the expression levels of α and β isoforms by immunoblotting. In order to compare the abundance of isoforms in astrocytic samples, semi-quantification was carried out with polyhistidine-tagged Na+/K+-ATPase subunit isoforms expressed in Xenopus laevis oocytes as standards to obtain an efficiency factor for each antibody. Proximity ligation assay illustrated that α2 paired efficiently with both β1 and β2 and the semi-quantification of the astrocytic fraction indicated that the astrocytic Na+/K+-ATPase is dominated by α2, paired with β1 or β2 (in a 1:9 ratio). We demonstrate that while the familial hemiplegic migraine-associated α2.G301R mutant was not functionally expressed at the plasma membrane in a heterologous expression system, α2+/G301R mice displayed normal protein levels of α2 and glutamate transporters and that the one functional allele suffices to manage the general K+ dynamics.
AB - Synaptic activity results in transient elevations in extracellular K+, clearance of which is critical for sustained function of the nervous system. The K+ clearance is, in part, accomplished by the neighboring astrocytes by mechanisms involving the Na+/K+-ATPase. The Na+/K+-ATPase consists of an α and a β subunit, each with several isoforms present in the central nervous system, of which the α2β2 and α2β1 isoform combinations are kinetically geared for astrocytic K+ clearance. While transcript analysis data designate α2β2 as predominantly astrocytic, the relative quantitative protein distribution and isoform pairing remain unknown. As cultured astrocytes altered their isoform expression in vitro, we isolated a pure astrocytic fraction from rat brain by a novel immunomagnetic separation approach in order to determine the expression levels of α and β isoforms by immunoblotting. In order to compare the abundance of isoforms in astrocytic samples, semi-quantification was carried out with polyhistidine-tagged Na+/K+-ATPase subunit isoforms expressed in Xenopus laevis oocytes as standards to obtain an efficiency factor for each antibody. Proximity ligation assay illustrated that α2 paired efficiently with both β1 and β2 and the semi-quantification of the astrocytic fraction indicated that the astrocytic Na+/K+-ATPase is dominated by α2, paired with β1 or β2 (in a 1:9 ratio). We demonstrate that while the familial hemiplegic migraine-associated α2.G301R mutant was not functionally expressed at the plasma membrane in a heterologous expression system, α2+/G301R mice displayed normal protein levels of α2 and glutamate transporters and that the one functional allele suffices to manage the general K+ dynamics.
U2 - 10.1002/glia.23194
DO - 10.1002/glia.23194
M3 - Journal article
C2 - 28787093
SN - 0894-1491
VL - 65
SP - 1777
EP - 1793
JO - GLIA
JF - GLIA
IS - 11
ER -