ELSEVIER PII S0361-9230(96)00042-2 BrainResearchBulletin, Vol. 40, No. 3, pp. 167-174, 1996 Copyright© 1996Elsevier ScienceInc. Printedin the USA.All rightsreserved 0361-9230/96$15.00 + .00 Expression of the fll and fl2(AMOG) Subunits of the Na, K-ATPase in Neural Tissues: Cellular and Developmental Distribution Patterns EMILIA LECUONA,* SONIA LUQUJN, t JULIO AVILA,* LUIS M. GARCiA-SEGURA t AND PABLO MARTJN-VASALLO .1 *Laboratorio de Biologfa del Desarrollo, Departamento de Bioqufmica y Biologfa Molecular, Universidad de La Laguna, 38206 La Laguna, Tenerife, Spain and tlnstituto Cajal, C.S.I.C. Dr. Arce 37, 28002 Madrid, Spain [Received 23 May 1995; Accepted 23 January 1996] ABSTRACT: We have used isoform-specific antisera against the Na,K-ATPase/]1 (SpETbl) and/~2(AMOG) (SpETb2) subunit iso- forms in order to establish their specific cellular and subcellular localization in several developmental stages of the rat central nervous system. Immunocytochemical preparations revealed/~1 isoform protein in most neural cells, being predominantly lo- cated in the soma of neurons and astrocytes, with no apprecia- ble developmental variations. In the newborn rat,/~2(AMOG) im- munoreactivity was present in cellular processes of astroglia and in the somas of neurons and decreasing in intensity with maturation until adulthood, where no/~2 isoform was detected in neurons. The differential location of these isoforms, both de- velopmentally and at the cellular level suggest a complex reg- ulation of their genes expression and mechanisms of subcellular distribution, as well as functional differences. KEY WORDS: Na,K-ATPase/~ subunit isoforms, AMOG, Adhe- sion molecule on glia. INTRODUCTION The Na,K-ATPase is the plasma membrane-bound enzyme that, utilizing ATP as an energy source, is responsible for maintaining the K ÷ and Na ÷ concentrations, osmotic balance, and membrane electrical potentials characteristic of most animal cells. Na,K- ATPase is present in high concentrations in the central nervous system (CNS), maintaining the cation gradients necessary for neural impulse conduction in neurons and potassium buffering and neurotransmitter uptake in glial cells. The enzyme is com- posed of two subunits, (called a and/5). The a, ( 112 kDa), or catalytic subunit, contains the binding sites for ATP and the ions implicated in the pumping process and serves as the receptor for cardiac glycosides. The/5 (45 kDa) subunit is a highly glyco- sylated protein and has no known role in ion transport, although the expression of both c~ and/5 subunits is required for Na,K- ATPase activity [ 20,15 ]. Two different isoforms of the/5 subunit (/~1 and f12) have been described in mammals [14] and a/53 isoform in Xenopus laevis [10] and Bufo marinus [11]. The/52 isoform is an adhesion molecule on glial cells (AMOG) and is specifically involved in neuron-astrocyte adhesion [7], with no To whom requests for reprints should be addressed. obvious adhesion function in other cell types. No adhesion func- tion has been reported for the/~ 1 isoform. A role of the/5 subunit as the receptor for the newly synthesized a-subunit has been proposed [12], and interdependency of a and/5 subunit seems to be necessary for their transport out of the endoplasmic retic- ulum [6]. While a and /5-subunit mRNA expression [14,23], immu- noblotting [4], 3H-ouabain binding [3], and a subunit immu- nolocalization in neural tissue [15,4] and cultured neural cells [ 3 ] have been broadly described, very little is known about the specific cellular and subcellular localization of each/5 isoform in neural cells and their developmental patterns "in vivo". In order to further understand the expression, localization, and functional implications of these proteins, isoform-specific antibodies against the human Na,K-ATPase /51 (SpETbl) and /52(AMOG) (SpETb2) isoforms [8,9] have been used to probe inununoblots of brain microsomes and to perform immunohistochemistry on preparations of central nervous system from newborn, 6-day-, 10- day-, 15-day-, and 90-day-old (adult) rats. EXPERIMENTAL PROCEDURES Antibodies and Reagents Antibodies specific for truncated protein Na,K-ATPase /51 (SpETbl) and/52(AMOG) (SpETb2) subunits [8,9] have been described. Both sera (S) were raised against the ectodomain of the human Na,K-ATPase /51 and/52(AMOG) subunits which had been generated as truncated proteins by pET expression vec- tors in E. coli [ 8,9,19 ]. Western Blots Microsomal fractions were prepared from whole brains of newborn and adult rats. Protein concentration was determined according to Bradford [ 1 ]. Na,K-ATPase activity was determined as described by B. Forbush, III [5]. One Na,K-ATPase unit = /~mol Pi h ~ per mg of protein. Protein samples, loaded so that each lane contained between 0.15 and 0.30 Na, K-ATPase units, were subjected to electrophoresis in 10% polyacrylamide-SDS 167