Subunit Composition and Role of Na+,K+-ATPases in Ventricular Myocytes

Abstract

Na + ,K + -ATPases are composed of one α and one β subunit; four α and three β isoforms have been found to date. We elucidated which α and β subunits were present in the ventricular myocytes of rat and guinea-pig and what roles the Na + ,K + -ATPase isozymes play in cardiac contraction. The presence of the α1, α2, and α3 subunits and the β1 and β2 subunits in rat and guinea-pig hearts were confirmed at the protein or mRNA level. Immunocytochemistry showed a patchy presence of α1 in the transverse tubules and surface sarcolemma, whereas α2 was distributed continuously in the transverse tubules alone. The α3 isoform was expressed prominently in the guinea-pig intercalated disc and slightly in the rat. On the other hand, the β1 isoform was located in the transverse tubules and surface sarcolemma, whereas the β2 was mainly located in the intercalated disc. The immunocytochemistry and immunoprecipitation findings indicated that the α1 and α2 form heterodimers with β1 and the α3 with β2 in ventricular myocytes. The application of low concentrations of ouabain enhanced the amplitudes of twitch without a change in resting tension in rat and guinea-pig ventricular stripts, whereas that of high concentrations resulted in a decrease in twitch with an increase in the resting tension. We thus conclude that the α2β1 and α3β2 isozymes are selectively located in the transverse tubules and intercalated disc of the ventricular myocytes, respectively, and the α2β1 is involved in the regulation of the Ca 2+ contents in the SR.Key words: transverse tubules, intercalated disc, localization, Na + pump, subunits. Na + , K + -ATPases are heterodimers comprising α and β subunits [1]. There are four α and three β isoforms, and the expression of the α and β subunits have been reported to be developmentally regulated in a cell-specific manner [2]. Although the expression of α and β subunits in oocytes [3] or Sf-9 cells [1], an insect cell line, revealed every combination of α and β subunits was functional, Na + ,K + -ATPases in cells in situ are thought to exist as a specific combination of α and β subunits [4,5]. Furthermore, single cells have been shown to have multiple α subunits, which are distributed differently in the cell membrane [6]. We elucidated that the α1 subunit, which formed a heterodimer with the β3 subunit, was ubiquitously present in the cell membrane of adrenal chromaffin cells, whereas the α2 subunit, forming a heterodimer with the β2, was located in the cell membrane in the vicinity of Ca 2+ store sites [7]. Our functional and morphological analyses revealed that the α2 subunit was involved in the regulation of the Ca 2+ contents in intracellular Ca 2+ store sites and the α3 subunit was not expressed in chromaffin cells [7]. In astrocytes, the α2 subunit has also been shown to take part in regulating the Ca 2+ contents in Ca 2+ store sites, whereas the α1 subunit has only been assumed to play a house-keeping role [8]. On the other hand, a recent knock-in experiment revealed that the α1 subunit could regulate C...

The Apical Localization of Na+, K+-ATPase in Cultured Human Retinal Pigment Epithelial Cells Depends on Expression of the β2 Subunit

“…(a) Na + , K + -ATPase evaluated based on the expression of the β 1 isoform was detected in both the basolateral and apical domains, consistent with the findings of Hu et al ( 1994 ). (b) The predominant combination at the apical membrane domain of polarized ARPE-19 cells is the α 2 β 2 combination, which is consistent with the concept that the α 2 isoform is the preferred binding partner of the β 2 isoform in assembling the Na + , K + -ATPase in different tissues (Lin et al, 2005 ; Harada et al, 2006 ; Tokhtaeva et al, 2012 ) and with the co-localization of α 2 β 2 in human eye sections shown in this work. (c) Polarized ARPE-19 monolayers express Na + , K + -ATPase subunits in a membrane domain-specific pattern: α 1 is detected only at the basolateral domain, α 2 is present only in the apical domain (as seen in Figure 6 ), β 1 subunits are localized in both the basolateral and apical domains, β 2 subunits are preferentially localized in the apical domain, and β 3 subunits are exclusively localized in the basolateral domain.…”

Subunit Isoform Selectivity in Assembly of Na,K-ATPase α-β Heterodimers

“…Therefore, the preferential formation of the ␣ 2 -␤ 2 complexes in the brain is determined not only by cell-specific co-expression of these isoforms, but also by their binding preferences. Preferential formation of the ␣ 2 -␤ 2 was also detected in heart and adrenal medullary cells, where the ␣ 1 subunit is more abundant than the ␣ 2 subunit (26,27), emphasizing preferential ␤ 2 subunit binding to the ␣ 2 subunit.…”

“…These data imply that in cells co-expressing multiple Na,K-ATPase subunits isoforms, various ␣ and ␤ isoforms also assemble in different combinations, dependent on their relative cellular content. However, selective co-immunoprecipitation of the ␣ 2 subunit but not of the ubiquitously expressed ␣ 1 subunit, with the ␤ 2 subunit from mouse and rat brain (19,26), as well as from heart and adrenal medullary cells of guinea pigs and rats (26,27) suggest that the ␣ 2 subunit is the preferred binding partner of the ␤ 2 subunit. In support of this hypothesis, the tissue expres-sion pattern of the ␤ 2 subunit, mainly in muscle and nervous system, is similar to that of the ␣ 2 subunit (3,7).…”

Agrin Regulation of α3 Sodium-Potassium ATPase Activity Modulates Cardiac Myocyte Contraction

“…Whereas staining for agrin was relatively uniform and similar to that seen in embryonic myocytes, ␣3 Na,K-ATPase appeared as intensely labeled foci distributed within a lower, evenly labeled background. Previous studies have also reported low levels of ␣3 subunit expression in adult rodent heart (23,27,28).…”