Ouabain binding kinetics and FXYD7 expression in astrocytes and neurons in primary cultures: implications for cellular contributions to extracellular K+homeostasis?

Abstract

Although Na+,K+-ATPase-mediated K+uptake into astrocytes plays a major role in re-establishing resting extracellular K+following neuronal excitation little information is available about astrocytic Na+,K+-ATPase function, let alone mechanisms returning K+to neurons. The catalytic units of the Na+,K+-ATPase are the astrocyte-specific α2, the neuron-specific α3and the ubiquitously expressed α1. In the present work,BmaxandKDvalues for α1, α2and α3subunits were computed in cultured cerebro-cortical mouse astrocytes and cerebellar granule neurons by non-linear regression as high-affinity (α2, α3) and low-affinity (α1) [3H]ouabain binding sites, which stoichiometrically equal transporter sites. Cellular expression was also determined of the brain- and α1-β1isoform-specific FDYX7, regulating Na+,K+-ATPase efficiency and K+-sensitivity. From ouabain-sensitive K+uptake rates published by ourselves (Walz and Hertz, 1982) or others (Atterwillet al., 1985), Na+,K+-ATPase turnover was determined. Subunits α2and α3showedBmaxof 15–30 pmol/mg protein, with maximum turnover rates of 70–80/s.Bmaxof the α1subunit was low in neurons but very high in astrocytes (645 pmol/mg protein), where turnover rate was slow, reflecting expression of selectively expressed FXYD7, and binding was increased by K+. The role of these characteristics for K+homeostasis are discussed.