Haim Garty

Phospholemman (FXYD1) associates with Na,K-ATPase and regulates its transport properties

A family of small, single-span membrane proteins (the FXYD family) has recently been defined based on their sequence and structural homology. Some members of this family have already been identified as tissue-specific regulators of Na,K-ATPase (NKA). In the present study, we demonstrate that phospholemman (PLM) (FXYD1), so far considered to be a heart- and muscle-specific channel or channel-regulating protein, associates specifically and stably with six different α-β isozymes of NKA after coexpression in Xenopus oocytes, and with α1–β, and less efficiently with α2–β isozymes, in native cardiac and skeletal muscles. Stoichiometric association of PLM with NKA occurs posttranslationally either in the Golgi or the plasma membrane. Interaction of PLM with NKA induces a small decrease in the external K+ affinity of α1–β1 and α2–β1 isozymes and a nearly 2-fold decrease in the internal Na+ affinity. In conclusion, this study demonstrates that PLM is a tissue-specific regulator of NKA that may play an essential role in muscle contractility.

CHIF, a member of the FXYD protein family, is a regulator of Na,K-ATPase distinct from the γ-subunit

The biological role of small membrane proteins of the new FXYD family is largely unknown. The best characterized FXYD protein is the γ‐subunit of the Na,K‐ATPase (NKA) that modulates the Na,K‐pump function in the kidney. Here, we report that, similarly to γa and γb splice variants, the FXYD protein CHIF (corticosteroid‐induced factor) is a type I membrane protein which is associated with NKA in renal tissue, and modulates the Na,K‐pump transport when expressed in Xenopus oocytes. In contrast to γa and γb, which both decrease the apparent Na+ affinity of the Na,K‐pump, CHIF significantly increases the Na+ affinity and decreases the apparent K+ affinity due to an increased Na+ competition at external binding sites. The extracytoplasmic FXYD motif is required for stable γ‐subunit and CHIF interaction with NKA, while cytoplasmic, positively charged residues are necessary for the γ‐subunits association efficiency and for CHIFs functional effects. These data document that CHIF is a new tissue‐specific regulator of NKA which probably plays a crucial role in aldosterone‐responsive tissues responsible for the maintenance of body Na+ and K+ homeostasis.

Mechanisms of Aldosterone Action in Tight Epithelia

Since the discovery of aldosterone in the early 1950s (Simpson & Tait, 1952) this adrenocorticosteroid has been recognized as a potent regulator of electrolyte metabolism in all vertebrates (Forman & Mulrow, 1975). In mammals the target tissues for the hormonal action are: the distal segments of the renal tubules, the urinary bladder, the descending colon mucosa, and the salivary and sweat glands. In all of them an aldosterone-induced increase of Na + reabsorption was observed (Crabb6, 1963b; Sharp & Leaf, 1973; Taylor & Palmer, 1982). Most of the present knowledge on the natriferic action of this hormone came, however, from studies on amphibian tight epithelia and in particular the toad urinary bladder. This classical model system for the mammalian distal nephron was chosen by many workers because of its relative histological simplicity and the ease with which its Na + transport can be monitored by short-circuit current recordings (for review see Macknight, DiBona & Leaf, 1980). In biochemical studies, on the other hand, rat and rabbit kidney segments, which provide larger amounts of starting material, were often preferred. An in vitro aldosterone-induced stimulation of Na + transport in toad bladder was first reported by Crabb6 (1961, 1963a). Shortly afterwards it was shown that the hormonal effect involves the induction of protein synthesis and also depends on the availability of metabolic substrates (Edelman, Bogoroch & Porter, 1963; Porter, Bogoroch & Edelman, 1964; Porter & Edelman, 1964; Crabb6 & DeWeer, 1964; Sharp & Leaf, 1964b). Since then research has been carried out in four main areas: 1) Characterization of the cytoplasmic and nuclear aldosterone binding sites.

FXYD7 is a brain-specific regulator of Na,K-ATPase α1–β isozymes

Recently, corticosteroid hormone‐induced factor (CHIF) and the γ‐subunit, two members of the FXYD family of small proteins, have been identified as regulators of renal Na,K‐ATPase. In this study, we have investigated the tissue distribution and the structural and functional properties of FXYD7, another family member which has not yet been characterized. Expressed exclusively in the brain, FXYD7 is a type I membrane protein bearing N‐terminal, post‐translationally added modifications on threonine residues, most probably O‐glycosylations that are important for protein stabilization. Expressed in Xenopus oocytes, FXYD7 can interact with Na,K‐ATPase α1–β1, α2–β1 and α3–β1 but not with α–β2 isozymes, whereas, in brain, it is only associated with α1–β isozymes. FXYD7 decreases the apparent K+ affinity of α1–β1 and α2–β1, but not of α3–β1 isozymes. These data suggest that FXYD7 is a novel, tissue‐ and isoform‐specific Na,K‐ATPase regulator which could play an important role in neuronal excitability.

Interaction with the Na,K-ATPase and Tissue Distribution of FXYD5 (Related to Ion Channel)

FXYD5 (related to ion channel, dysadherin) is a member of the FXYD family of single span type I membrane proteins. Five members of this group have been shown to interact with the Na,K-ATPase and to modulate its properties. However, FXYD5 is structurally different from other family members and has been suggested to play a role in regulating E-cadherin and promoting metastasis (Ino, Y., Gotoh, M., Sakamoto, M., Tsukagoshi, K., and Hirohashi, S. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 365–370). The goal of this study was to determine whether FXYD5 can modulate the Na,K-ATPase activity, establish its cellular and tissue distribution, and characterize its biochemical properties. Anti-FXYD5 antibodies detected a 24-kDa polypeptide that was preferentially expressed in kidney, intestine, spleen, and lung. In kidney, FXYD5 resides in the basolateral membrane of the connecting tubule, the collecting tubule, and the intercalated cells of the collecting duct. However, there is also labeling of the apical membrane in long thin limb of Henles loop. FXYD5 was effectively immunoprecipitated by antibodies to the α subunit of Na,K-ATPase and the anti-FXYD5 antibody immunoprecipitates α. Co-expressing FXYD5 with the α1 and β1 subunits of the Na,K-ATPase in Xenopus oocytes elicited a more than 2-fold increase in pump activity, measured either as ouabain-blockable outward current or as ouabain-sensitive 86Rb+ uptake. Thus, as found with other FXYD proteins, FXYD5 interacts with the Na,K-ATPase and modulates its properties.

Cloning and Function of the Rat Colonic Epithelial K+ Channel KVLQT1

Abstract. KVLQT1 (KCNQ1) is a voltage-gated K+ channel essential for repolarization of the heart action potential that is defective in cardiac arrhythmia. The channel is inhibited by the chromanol 293B, a compound that blocks cAMP-dependent electrolyte secretion in rat and human colon, therefore suggesting expression of a similar type of K+ channel in the colonic epithelium. We now report cloning and expression of KVLQT1 from rat colon. Overlapping clones identified by cDNA-library screening were combined to a full length cDNA that shares high sequence homology to KVLQT1 cloned from other species. RT-PCR analysis of rat colonic musoca demonstrated expression of KVLQT1 in crypt cells and surface epithelium. Expression of rKVLQT1 in Xenopus oocytes induced a typical delayed activated K+ current, that was further activated by increase of intracellular cAMP but not Ca2+ and that was blocked by the chromanol 293B. The same compound blocked a basolateral cAMP-activated K+ conductance in the colonic mucosal epithelium and inhibited whole cell K+ currents in patch-clamp experiments on isolated colonic crypts. We conclude that KVLQT1 is forming an important component of the basolateral cAMP-activated K+ conductance in the colonic epithelium and plays a crucial role in diseases like secretory diarrhea and cystic fibrosis.

FXYD Proteins: New Tissue‐ and Isoform‐Specific Regulators of Na,K‐ATPase

Abstract: The recently defined FXYD protein family contains seven members that are small, single‐span membrane proteins characterized by a signature sequence containing an FXYD motif and three other conserved amino acid residues. Until recently, the functional role of FXYD proteins was largely unknown, with the exception of the γ subunit of Na,K‐ATPase, which was shown to be a specific regulator of renal α1‐β1 isozymes. We have investigated whether other members of the FXYD family may have a similar role as the γ subunit and have found that CHIF (corticosteroid hormone‐induced factor, FXYD4), FXYD7, as well as phospholemman (FXYD1) specifically associate with Na,K‐ATPase and preferentially with α1‐β isozymes in native tissues, and produce distinct effects on the transport properties of Na,K‐ATPase that are adapted to the physiological demands of the tissues in which they are expressed. These results provide evidence for a unique and novel mode of regulation of Na,K‐ATPase by FXYD proteins that involves a tissue‐specific expression of an auxiliary subunit of distinct Na,K‐ATPase isozymes.

Light-driven sodium transport in sub-bacterial particles of Halobacterium halobium

Light-induced Na+ efflux was observed in sub-bacterial particles of Halobacterium halobium loaded and suspended in 4 M NaCl solution. The Na+ efflux was not ATP driven, since ATPase inhibitors were without effect or even enhanced efflux at low light intensity. Uncouplers, on the other hand, inhibited Na+ efflux, the inhibition being complete at low light intensity. The Na+ efflux was accompanied by proton influx. Both processes were dependent on light intensity, unaffected or enhanced by ATPase inhibitors and similarly affected by uncouplers. Proton influx was not observed in particles loaded with 4 M KCl instead of 4 M NaCl. Na+ transport in the dark could be induced by artificial formation of a pH difference across the membrane; changing the sign of the pH difference reversed the direction of the Na+ transport. Proton influx in the dark followed the artificial formation of a sodium gradient [Na+]in less than [Na+]out). These results may be explained by a Na+/H+ antiport mechanism. The fluxes of Na+ and H+ were of comparable magnitude, but the initial rate of Cl- efflux in the same experiment was one-third of the initial rate of Na+ efflux. Consequently Cl- is not regarded as a participant in the Na+ efflux mechanism.

FXYD Proteins Stabilize Na,K-ATPase AMPLIFICATION OF SPECIFIC PHOSPHATIDYLSERINE-PROTEIN INTERACTIONS

FXYD proteins are a family of seven small regulatory proteins, expressed in a tissue-specific manner, that associate with Na,K-ATPase as subsidiary subunits and modulate kinetic properties. This study describes an additional property of FXYD proteins as stabilizers of Na,K-ATPase. FXYD1 (phospholemman), FXYD2 (γ subunit), and FXYD4 (CHIF) have been expressed in Escherichia coli and purified. These FXYD proteins associate spontaneously in vitro with detergent-soluble purified recombinant human Na,K-ATPase (α1β1) to form α1β1FXYD complexes. Compared with the control (α1β1), all three FXYD proteins strongly protect Na,K-ATPase activity against inactivation by heating or excess detergent (C12E8), with effectiveness FXYD1 > FXYD2 ≥ FXYD4. Heating also inactivates E1 ↔ E2 conformational changes and cation occlusion, and FXYD1 protects strongly. Incubation of α1β1 or α1β1FXYD complexes with guanidinium chloride (up to 6 m) causes protein unfolding, detected by changes in protein fluorescence, but FXYD proteins do not protect. Thus, general protein denaturation is not the cause of thermally mediated or detergent-mediated inactivation. By contrast, the experiments show that displacement of specifically bound phosphatidylserine is the primary cause of thermally mediated or detergent-mediated inactivation, and FXYD proteins stabilize phosphatidylserine-Na,K-ATPase interactions. Phosphatidylserine probably binds near trans-membrane segments M9 of the α subunit and the FXYD protein, which are in proximity. FXYD1, FXYD2, and FXYD4 co-expressed in HeLa cells with rat α1 protect strongly against thermal inactivation. Stabilization of Na,K-ATPase by three FXYD proteins in a mammalian cell membrane, as well the purified recombinant Na,K-ATPase, suggests that stabilization is a general property of FXYD proteins, consistent with a significant biological function.

Direct inhibition of epithelial Na+ channels by a pH-dependent interaction with calcium, and by other divalent ions

SummaryDirect inhibitory effects of Ca2+ and other ions on the epithelial Na+ channels were investigated by measuring the amiloride-blockable22Na+ fluxes in toad bladder vesicles containing defined amounts of mono- and divalent ions. In agreement with a previous report (H.S. Chase, Jr., and Q. Al-Awqati,J. Gen. Physiol.81:643–666, 1983) we found that the presence of micromolar concentrations of Ca2+ in the internal (cytoplasmic) compartment of the vesicles substantially lowered the channel-mediated fluxes. This inhibition, however, was incomplete and at least 30% of the amiloride-sensitive22Na+ uptake could not be blocked by Ca2+ (up to 1mm). Inhibition of channels could also be induced by millimolar concentrations of Ba2+, Sr2+, or VO2+, but not by Mg2+. The Ca2+ inhibition constant was a strong function of pH, and varied from 0.04 μm at pH 7.8 to >10 μm at pH 7.0 Strong pH effects were also demonstrated by measuring the pH dependence of22Na+ uptake in vesicles that contained 0.5 μm Ca2+. This Ca2+ activity produced a maximal inhibition of22Na+ uptake at pH≥7.4 but had no effect at pH≤7.0. The tracer fluxes measured in the absence of Ca2+ were pH independent over this range. The data is compatible with the model that Ca2+ blocks channels by binding to a site composed of several deprotonated groups. The protonation of any one of these groups prevents Ca2+ from binding to this site but does not by itself inhibit transport. The fact that the apical Na+ conductance in vesicles, can effectively be modulated by minor variations of the internal pH near the physiological value, raises the possibility that channels are being regulated by pH changes which alter their apparent affinity to cytoplasmic Ca2+, rather than, or in addition to changes in the cytoplasmic level of free Ca2+.