Polarized distribution of Na+, K+-ATPase α-subunit isoforms in electrocyte membranes

We have previously demonstrated that Na + ,K + -ATPase activity is present in both differentiated plasma membranes from Electrophorus electricus (L.) electrocyte. Considering that the a subunit is responsible for the catalytic properties of the enzyme, the aim of this work was to study the presence and localization of a isoforms (a1 and a2) in the electrocyte. Dose–response curves showed that non-innervated membranes present a Na + ,K + -ATPase activity 2.6-fold more sensitive to ouabain (I50=1.0F0.1 AM) than the activity of innervated membranes (I50=2.6F0.2 AM). As depicted in [ 3 H]ouabain binding experiments, when the [ 3 H]ouabain–enzyme complex was incubated in a medium containing unlabeled ouabain, reversal of binding occurred differently: the bound inhibitor dissociated 32% from Na + ,K + -ATPase in non-innervated membrane fractions within 1 h, while about 50% of the ouabain bound to the enzyme in innervated membrane fractions was released in the same time. These data are consistent with the distribution of a1 and a2 isoforms, restricted to the innervated and noninnervated membrane faces, respectively, as demonstrated by Western blotting from membrane fractions and immunohistochemical analysis of the main electric organ. The results provide direct evidence for a distinct distribution of Na + ,K + -ATPase a-subunit isoforms in the differentiated membrane faces of the electrocyte, a characteristic not yet described for any polarized cell. D 2003 Elsevier B.V. All rights reserved.