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

Abstract

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 mice displayed normal protein levels of α2 and glutamate transporters and that the one functional allele suffices to manage the general K dynamics.

Serotonin, norepinephrine, and acetylcholine differentially affect astrocytic potassium clearance to modulate somatosensory signaling in male mice

“…Therefore, it is crucial to clear potassium rapidly and efficiently. It is now widely accepted that activity‐induced changes in [K + ] e are rapidly managed by the astrocytic potassium inward rectifying channel subtype 4.1 (Kir4.1) (Bellot‐Saez et al, ; Butt & Kalsi, ; Chever, Djukic, McCarthy, & Amzica, ; D'Ambrosio, Gordon, & Winn, ; Larsen et al, ; Larsen & MacAulay, ; Sibille et al, ) and the astrocyte‐specific α2β2 isoform of the Na + /K + ‐ATPase (NKA) (D'Ambrosio et al, ; Larsen et al, ; Pellerin & Magistretti, ; Stoica et al, ). Due to the highly negative resting membrane potential of astrocytes, Kir4.1 is highly responsive to increases in [K + ] e with local inward K + uptake into astrocytes (Sibille et al, , ) whereas the astrocyte‐specific NKA is additionally responsive to changes in intracellular Na + driven by the synapse‐dependent glutamate transporter (Larsen, Holm, Vilsen, & MacAulay, ; Larsen, Stoica, et al, ; Pellerin & Magistretti, ) and/or the Na + /Ca 2+ exchanger in response to astrocyte Ca 2+ transients (Wang, Smith, et al, ).…”

Developmental maturation of activity‐induced K+ and pH transients and the associated extracellular space dynamics in the rat hippocampus

“…; Stoica et al . ). Following stimulus end, K + returns to the neurons, presumably via the neuronal α3β1/α1β1 isoforms of the Na + /K + ‐ATPase (Ransom et al .…”

“…The specificity of antibodies targeting the α subunit isoforms of the Na + /K + ‐ATPase was demonstrated previously upon expression of the three isoforms in Xenopus laevis oocytes followed by tests of cross‐reactivity (Stoica et al . ). For the semi‐quantitative analysis of Na + /K + ‐ATPase isoforms, polyhistidine‐tagged versions of Na + /K + ‐ATPase isoforms α1, α2 and α3 expressed in X. laevis oocytes were used as standards for normalization as described previously (Stoica et al .…”

“…For the semi‐quantitative analysis of Na + /K + ‐ATPase isoforms, polyhistidine‐tagged versions of Na + /K + ‐ATPase isoforms α1, α2 and α3 expressed in X. laevis oocytes were used as standards for normalization as described previously (Stoica et al . ).…”

“…; Stoica et al . ). Following termination of neuronal activity, K + exits the astrocytic compartment and re‐enters the neurons, presumably via the action of the neuronal α3 Na + /K + ‐ATPase isoform (Coles & Schneider‐Picard, ; Ransom et al .…”