Residues within Transmembrane Domains 4 and 6 of the Na,K-ATPase α Subunit Are Important for Na⁺ Selectivity

Abstract

The Na,K- and H,K-ATPases are plasma membrane enzymes responsible for the active exchange of extracellular K(+) for cytoplasmic Na(+) or H(+), respectively. At present, the structural determinants for the specific function of these ATPases remain poorly understood. To investigate the cation selectivity of these ATPases, we constructed a series of Na,K-ATPase mutants in which residues in the membrane spanning segments of the alpha subunit were changed to the corresponding residues common to gastric H,K-ATPases. Thus, mutants were created with substitutions in transmembrane domains TM1, TM4, TM5, TM6, TM7, and TM8 independently or together (designated TMAll). The function of each mutant was assessed after coexpression with the beta subunit in Sf-9 cells using baculoviruses. The enzymatic properties of TM1, TM7, and TM8 mutants were similar to the wild-type Na,K-ATPase, and while TM5 showed modest changes in apparent affinity for Na(+), TM4, TM6, and TMAll displayed an abnormal activity. This resulted in a Na(+)-independent hydrolysis of ATP, a 2-fold higher K(0.5) for Na(+) activation, and the ability to function at low pH. These results suggest a loss of discrimination for Na(+) over H(+) for the enzymes. In addition, TM4, TM6, and TMAll mutants exhibited a 1.5-fold lower affinity for K(+) and a 4-5-fold decreased sensitivity to vanadate. Altogether, these results provide evidence that residues in transmembrane domains 4 and 6 of the alpha subunit of the Na,K-ATPase play an important role in determining the specific cation selectivity of the enzyme and also its E1/E2 conformational equilibrium.

The NA/K-ATPase and its isozymes: what we have learned using the baculovirus expression system

“…This agrees with several other studies that have targeted individual amino acids in those regions of the protein (97)(98)(99)(100)(101)(102)(103)(104)(105), and supports the predictions from homology modeling of the Na/K-ATPase (16,106). Although, the simultaneous replacements in all transmembrane domains exacerbated the inability of the enzyme to select the correct cation, this was not sufficient to convert the Na/K-ATPase into a H,K-ATPase, and the resulting enzyme behaved as a hybrid Na/H,K-ATPase (96). This supports the notion that other regions outside the membrane bilayer participate in the particular properties of each P-type ATPase (98).…”

“…More recently, insect cells have been used to delineate the structural determinants involved in the cation selectivity of the Na/K-ATPase (96). For this, the function of a series of mutants were characterized in which residues in the membrane spanning segments of the α subunit of the Na/K-ATPase, highly conserved across species and isoforms, were changed to the corresponding residues common to the gastric H,K-ATPases of different species.…”

Malaria parasites tolerate a broad range of ionic environments and do not require host cation remodelling

“…Although the P. falciparum genome lacks a clear orthologue of known Na + /K + ATPases, it has many genes for P-type ATPases that could function as Na + and/or K + pumps (Martin et al, 2009;Thever and Saier Jr, 2009). Unfortunately, the precise function of these putative transporters cannot be determined through computational analysis alone because the determinants of solute selectivity within this superfamily are poorly understood (Sanchez and Blanco, 2004). For this and other reasons, it has been tacitly assumed that malaria parasites recognize and use these ions in ways resembling those of their vertebrate hosts (Ginsburg et al, 1986;Lee et al, 1988;Staines et al, 2001;Brand et al, 2003;Singh et al, 2010).…”

Different expression and activity of the α1 and α4 isoforms of the Na,K-ATPase during rat male germ cell ontogeny

“…Cells were fixed in 4% paraformaldehyde (buffered formalin phosphate; Fisher Scientific, Pittsburgh, PA, USA). Samples were then processed for immunocytochemistry, as previously described (Sanchez & Blanco 2004). Briefly, cells were permeabilized with 0.3% Triton X 100 in 25 mM Hepes (pH 7.4), 150 mM NaCl and 1 mM EGTA (HBS).…”