Interaction of FXYD10 (PLMS) with Na,K-ATPase from Shark Rectal Glands

Abstract

FXYD domain-containing proteins are tissue-specific regulators of the Na,K-ATPase that have been shown to have significant physiological implications. Information about the sites of interaction between some FXYD proteins and subunits of the Na,K-ATPase is beginning to emerge. We previously identified an FXYD protein in plasma membranes from shark rectal gland cells and demonstrated that this protein (FXYD10) modulates shark Na,K-ATPase activity. The present study was undertaken to identify the location of the C-terminal domain of FXYD10 on the ␣-subunit of Na,K-ATPase, using covalent cross-linking combined with proteolytic cleavage. Treatment of Na,K-ATPase-enriched membranes with the homobifunctional thiol cross-linker 1,4-bismaleimidyl-2,3-dihydroxybutane resulted in cross-linking of FXYD10 to the ␣-subunit. Cross-linking was not affected by preincubation with sodium or potassium but was significantly reduced after pre-incubation with the non-hydrolyzable ATP analog ␤,␥-methyleneadenosine 5-triphosphate (AMP-PCP). A peptic assay was developed, in which pepsin treatment of Na,K-ATPase at low pH resulted in extensive cleavage of the ␣-subunit while FXYD10 was left intact. Proteolytic fragments of control and cross-linked preparations were isolated by immunoprecipitation and analyzed by gel electrophoresis. A proteolytic fragment containing FXYD10 cross-linked to a fragment from the ␣-subunit could be localized on SDS gels. Sequencing of this fragment showed the presence of FXYD10 as well as a fragment within the A domain of the ␣-subunit comprising 33 amino acids, including a single Cys residue, Cys 254 . Thus, regulation of Na,KATPase by FXYD10 occurs in part via cytoplasmic interaction of FXYD10 with the A domain of the shark ␣-subunit.

A third Na + -binding site in the sodium pump

“…We have recently provided evidence for an interaction between the transmembrane segment of FYXD proteins and the ninth transmembrane segment of the ␣-subunit (22). A similar position of FXYD protein is supported by other approaches (31) while other locations have also been proposed recently (32). The role of the ninth transmembrane helix in Na ϩ transport and the close interaction between the transmembrane domain of FXYD proteins and the ninth transmembrane helix suggest that interaction between these transmembrane segments could be responsible for the functional effects of the association of these proteins with the ␣͞␤ complex, even if interactions between cytoplasmic domains may also affect Na,K-ATPase function.…”

Expression Profiles of Branchial FXYD Proteins in the Brackish Medaka Oryzias dancena: A Potential Saltwater Fish Model for Studies of Osmoregulation

“…The first reported FXYD protein in fish was a phospholemman-like protein (i.e., FXYD10) from the piked dogfish ( Squalus acanthias ) [29]. Subsequent studies revealed the function of FXYD10 and its interaction with NKA [30], [31].…”

FXYD Proteins Reverse Inhibition of the Na+-K+ Pump Mediated by Glutathionylation of Its β1 Subunit

“…Fig. 6A shows a model of the predicted spatial relationship between C1 and C2 and the ␣ 1 subunit derived from the crystal structure of shark rectal gland Na ϩ -K ϩ ATPase (7) and from cross-linking experiments (27). The model suggests that C2 comes into close proximity to basic amino acids of helix 4 of the ␣ subunit.…”