Regions of the Catalytic α Subunit of Na,K-ATPase Important for Functional Interactions with FXYD 2

Abstract

The ␥ modulator (FXYD 2) is a member of the FXYD family of single transmembrane proteins that modulate the kinetic behavior of Na,K-ATPase. This study concerns the identification of regions in the ␣ subunit that are important for its functional interaction with ␥. An important effect of ␥ is to increase K ؉ antagonism of cytoplasmic Na ؉ activation apparent as an increase in K Na at high. We show that although ␥ associates with ␣1, ␣2, and ␣3 isoforms, it increases the K Na of ␣1 and ␣3 but not ␣2. Accordingly, chimeras of ␣1 and ␣2 were used to identify regions of ␣ critical for the increased K Na . As with ␣1 and ␣2, all chimeras associate with ␥. Kinetic analysis of ␣2 front /␣1 back chimeras indicate that the C-terminal (Lys 907 -Tyr 1018 ) region of ␣1, which includes transmembrane (TM)9 close to ␥, is important for the increase in K Na . However, similar experiments with ␣1 front /␣2 back chimeras indicate a modulatory role of the loop between TMs 7 and 8. Thus, as long as the ␣1 L7/8 loop is present, replacement of TM9 of ␣1 with that of ␣2 does not abrogate the ␥ effect on K Na . In contrast, as long as TM9 is that of ␣1, replacement of L7/8 of ␣1 with that of ␣2 does not abolish the effect. It is suggested that structural association of the TM regions of ␣ and FXYD 2 is not the sole determinant of this effect of FXYD on K Na but is subject to long range modulation by the extramembranous L7/8 loop of ␣.The Na,K-ATPase or sodium pump is an integral membrane protein found in the cells of virtually all higher eukaryotes. It couples the hydrolysis of one molecule of ATP to the electrogenic exchange of three intracellular Na ϩ for two extracellular K ϩ ions against their electrochemical gradients.The sodium pump is an oligomer of two subunits, a large catalytic ␣ subunit and a smaller, highly glycosylated ␤ subunit whose role is to ensure the proper folding, insertion, and maturation of ␣ in the plasma membrane. Four isoforms of ␣ and three of ␤ are expressed in a tissue-and development-specific manner. (for reviews, see Refs. 1 and 2). The sodium pump is subject to diverse modes of regulation by substrates, membrane-associated components, hormones, and neurotransmitters (3). In recent years, considerable attention has been focused on tissue-specific regulation by small transmembrane proteins referred to as FXYD proteins (for reviews, see Refs. 4 -7). These proteins belong to a gene family of small, single transmembrane proteins, with the exception of MAT-8 which has two transmembranes (TM(s)).3 There are at least seven members of which several (FXYDs 1-4 and 7) appear to regulate the kinetic behavior of the sodium pump in distinct and specific ways, in particular the apparent affinity of the enzyme for ligands (Na ϩ , K ϩ , and ATP). Members possess a 35-amino-acid signature sequence that includes 6 invariant amino acids before, in, and after the TM domain. The N-terminal FXYD (Phe-Xaa-Tyr-Asp) motif is invariant, along with two glycines in the TM domain, and a serine in the C-terminal domain. The first FXYD...

Na + /K + -ATPase E960 and phospholemman F28 are critical for their functional interaction

“…However, in contrast to our findings with PLM, E960A-NKA mutation does not alter regulation of the pump's apparent Na + affinity by FXYD2 and FXYD4 (18). One study (21) suggested that the structural association of TM9 with FXYD2 TM domain is not the sole determinant of the FXYD2 effect on the apparent Na + affinity. The authors pointed out that long-range modulation by the extracellular loop between TMs 7 and 8 is also involved (21).…”

“…One study (21) suggested that the structural association of TM9 with FXYD2 TM domain is not the sole determinant of the FXYD2 effect on the apparent Na + affinity. The authors pointed out that long-range modulation by the extracellular loop between TMs 7 and 8 is also involved (21). Such fine differences in the FXYD-NKA-α structural interaction may underlie the intrinsic differences in NKA modulation among various FXYDs.…”

“…The corresponding NKA-α1 residues in rat (full transcript numbering) and shark (posttranslational cleavage numbering) are F956, E960, L964, and F967. Mutational analysis and functional studies suggest that some of these sites may be involved in NKA interaction with FXYD2 (18,21), FXYD4 (18), and FXYD7 (18). However, the relative importance of such sites for NKA-FXYD physical association and functional effects varies among FXYD proteins (18,21) and no prior analysis has addressed PLM (FXYD1), which is also the only mammalian FXYD regulated by phosphorylation.…”

“…Mutational analysis and functional studies suggest that some of these sites may be involved in NKA interaction with FXYD2 (18, 21), FXYD4 (18), and FXYD7 (18). However, the relative importance of such sites for NKA-FXYD physical association and functional effects varies among FXYD proteins (18,21) and no prior analysis has addressed PLM (FXYD1), which is also the only mammalian FXYD regulated by phosphorylation. Here, we combined PLM and NKA mutational analysis, intermolecular FRET, NKA function measurements in live cells, coimmunoprecipitation, and structural modeling to identify key corresponding sites in NKA TM9 and PLM involved in physical association and functional effects.…”

Impaired routing of wild type FXYD2 after oligomerisation with FXYD2-G41R might explain the dominant nature of renal hypomagnesemia

“…Studies indicate that the transmembrane domain of FXYD2 binds to the groove formed by M2, M6, and M9 of the Na,K-ATPase α-subunit [11], whereas the location of the cytoplasmatic part is still questionable [12]. In addition, it appears that the extracellular loop between M7 and M8 is the focal region for γ-α-β interactions [13]. Mahmmoud et al [14] could isolate FXYD2 oligomers when the detergent sodium dodecylsulphate (SDS) was replaced by perfluoro-octanoic acid (PFO, a detergent that tolerates weak interactions) during polyacrylamide gel electrophoresis.…”