Käthi Geering

The Functional Role of β Subunits in Oligomeric P-Type ATPases

Na,K-ATPase and gastric and nongastric H,K-ATPases are the only P-type ATPases of higher organisms that are oligomeric and are associated with a β subunit, which is obligatory for expression and function of enzymes. Topogenesis studies suggest that β subunits have a fundamental and unique role in K+-transporting P-type ATPases in that they facilitate the correct membrane integration and packing of the catalytic α subunit of these P-type ATPases, which is necessary for their resistance to cellular degradation, their acquisition of functional properties, and their routing to the cell surface. In addition to this chaperone function, β subunits also participate in the determination of intrinsic transport properties of Na,K- and H,K-ATPases. Increasing experimental evidence suggests that β assembly is a highly ordered, β isoform-specific process, which is mediated by multiple interaction sites that contribute in a coordinate, multistep process to the structural and functional maturation of Na,K- and H,K-ATPases.

Regulation of Ca2+ channel expression at the cell surface by the small G-protein kir/Gem.

Voltage-dependent calcium (Ca2+) channels are involved in many specialized cellular functions, and are controlled by intracellular signals such as heterotrimeric G-proteins, protein kinases and calmodulin (CaM). However, the direct role of small G-proteins in the regulation of Ca2+ channels is unclear. We report here that the GTP-bound form of kir/Gem, identified originally as a Ras-related small G-protein that binds CaM, inhibits high-voltage-activated Ca2+ channel activities by interacting directly with the β-subunit. The reduced channel activities are due to a decrease in α1-subunit expression at the plasma membrane. The binding of Ca2+/CaM to kir/Gem is required for this inhibitory effect by promoting the cytoplasmic localization of kir/Gem. Inhibition of L-type Ca2+ channels by kir/Gem prevents Ca2+-triggered exocytosis in hormone-secreting cells. We propose that the small G-protein kir/Gem, interacting with β-subunits, regulates Ca2+ channel expression at the cell surface.

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.

The gamma subunit is a specific component of the Na,K-ATPase and modulates its transport function.

The role of small, hydrophobic peptides that are associated with ion pumps or channels is still poorly understood. By using the Xenopus oocyte as an expression system, we have characterized the structural and functional properties of the γ peptide which co‐purifies with Na,K‐ATPase. Immuno‐radiolabeling of epitope‐tagged γ subunits in intact oocytes and protease protection assays show that the γ peptide is a type I membrane protein lacking a signal sequence and exposing the N‐terminus to the extracytoplasmic side. Co‐expression of the rat or Xenopus γ subunit with various proteins in the oocyte reveals that it specifically associates only with isozymes of Na,K‐ATPase. The γ peptide does not influence the formation and cell surface expression of functional Na,K‐ATPase α–β complexes. On the other hand, the γ peptide itself needs association with Na,K‐ATPase in order to be stably expressed in the oocyte and to be transported efficiently to the plasma membrane. γ subunits do not associate with individual α or β subunits but only interact with assembled, transport‐competent α–β complexes. Finally, electrophysiological measurements indicate that the γ peptide modulates the K+ activation of Na,K pumps. These data document for the first time the membrane topology, the specificity of association and a potential functional role for the γ subunit of Na,K‐ATPase.

Functional roles of Na,K-ATPase subunits.

Purpose of reviewNa,K-ATPase is an oligomeric protein composed of α subunits, β subunits and FXYD proteins. The catalytic α subunit hydrolyzes ATP and transports the cations. Increasing experimental evidence suggest that β subunits and FXYD proteins essentially contribute to the variable physiological needs of Na,K-ATPase function in different tissues. Recent findingsBeta subunits have a crucial role in the structural and functional maturation of Na,K-ATPase and modulate its transport properties. The chaperone function of the β subunit is essential, for example, in the formation of tight junctions and cell polarity. Recent studies suggest that β subunits also have inherent functions, which are independent of Na,K-ATPase activity and which may be involved in cell–cell adhesiveness and in suppression of cell motility. As for FXYD proteins, they modulate Na,K-ATPase activity in a tissue-specific way, in some cases in close cooperation with posttranslational modifications such as phosphorylation. SummaryA better understanding of the multiple functional roles of the accessory subunits of Na,K-ATPase is crucial to appraise their influence on physiological processes and their implication in pathophysiological states.

The functional role of the β-subunit in the maturation and intracellular transport of Na,K-ATPase

The minimal functional enzyme unit of Na,K‐ATPase consists of an α—β complex. The α‐subunit bears all functional domains of the enzyme and so far a regulatory role for the β‐subunit in the catalytic cycle has not been established. On the other hand, increasing experimental evidence suggests that the β‐subunit is an indispensable element for the structural and functional maturation of the enzyme as well as its intracellular transport to the plasma membrane. This brief review summarizes the experimental data supporting the hypothesis that assembly of the β‐subunit is needed for the α‐subunit to acquire the correct, stable configuration necessary for the acquisition of functional properties and its exit from the ER.

Regulation of the sodium pump: how and why?

Abstract The sodium pump, Na + , K + -ATPase, controls (directly or indirectly) many essential cellular functions, such as cell volume, heat production, intracellular pH, free calcium concentration and membrane potential. In epithelia, due to its asymmetric cell surface distribution, the sodium pump represents the major driving force for the transepithelial transport of ions, solutes and water, thereby controlling the extracellular volume and its ionic composition. By modulating the concentration of cytosolic calcium in excitable cells, Na + , K + -ATPase regulates the efficiency of muscle contraction in cardiac cells, and the release or uptake of neurotransmitters in neurons. How and why Na + ,K + -ATPase is regulated in response to acute or chronic environmental changes are the questions we address in this review. Alterations in regulation may play key roles in pathological processes such as heart failure, hypertension, or neurological disorders.

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.

Role of beta-subunit domains in the assembly, stable expression, intracellular routing, and functional properties of Na,K-ATPase.

The β-subunit of Na,K-ATPase (βNK) interacts with the catalytic α-subunit (αNK) in the ectodomain, the transmembrane, and the cytoplasmic domain. The functional significance of these different interactions was studied by expressing αNK inXenopus oocytes along with N-terminally modified βNK or with chimeric βNK/βH,K-ATPase (βHK). Complete truncation of the βNK N terminus allows for cell surface-expressed, functional Na,K-pumps that exhibit, however, reduced apparent K+ and Na+ affinities as assessed by electrophysiological measurements. A mutational analysis suggests that these functional effects are not related to a direct interaction of the β N terminus with the αNK but rather that N-terminal truncation induces a conformational change in another functionally relevant β domain. Comparison of the functional properties of αNK·βNK, αNK·βHK, or αNK·βNK/βHK complexes shows that the effect of the βNK on K+ binding is mainly mediated by its ectodomain. Finally, βHK/NK containing the transmembrane domain of βHK produces stable but endoplasmic reticulum-retained αNK·β complexes, while αNK/βHK complexes can leave the ER but exhibit reduced ouabain binding capacity and transport function. Thus, interactions of both the transmembrane and the ectodomain of βNK with αNK are necessary to form correctly folded Na,K-ATPase complexes that can be targeted to the plasma membrane and/or become functionally competent. Furthermore, the β N terminus plays a role in the β-subunit’s folding necessary for correct interactions with the α-subunit.

FXYD Proteins: New Tissue-Specific Regulators of the Ubiquitous Na,K-ATPase

Maintenance of the Na+ and K+ gradients between the intracellular and extracellular milieus of animal cells is a prerequisite for basic cellular homeostasis and for functions of specialized tissues. The Na,K-ATPase, an oligomeric P-type adenosine triphosphatase (ATPase), is composed of a catalytic α subunit and a regulatory β subunit and is the main player that fulfils these tasks. A variety of regulatory mechanisms are necessary to guarantee appropriate Na,K-ATPase expression and activity adapted to changing physiological demands. Recently, a regulatory mechanism was defined that is mediated by interaction of Na,K-ATPase with small proteins of the FXYD family, which possess a single transmembrane domain and so far have been considered as channels or regulators of ion channels. The mammalian FXYD proteins FXYD1 through FXYD7 exhibit tissue-specific distribution. Phospholemman (FXYD1) in heart and skeletal muscle, the γ subunit of Na,K-ATPase (FXYD2) and corticosteroid hormone-induced factor (FXYD4, also known as CHIF) in the kidney, and FXYD7 in the brain associate preferentially with the widely expressed Na,K-ATPase α1-β1 isozyme and modulate its transport activity in a way that conforms to tissue-specific requirements. Thus, tissue- and isozyme-specific interaction of Na,K-ATPase with FXYD proteins contributes to proper handling of Na+ and K+ by the Na,K-ATPase, and ensures correct function in such processes as renal Na+-reabsorption, muscle contraction, and neuronal excitability. The Na,K-ATPase is a plasma membrane enzyme that is responsible for maintaining the Na+ and K+ gradients between the intra- and extracellular milieu of animal cells. It consists of a catalytic α subunit and a regulatory β subunit. Na,K-ATPase transports Na+ out of the cell and K+ into the cell against their respective chemical gradients by using energy derived from the hydrolysis of ATP. Maintenance of the Na+ and K+ gradients by the Na,K-ATPase is essential for basic cellular homeostasis as well as for specialized functions of various tissues. Rigorous control of the Na+ and K+ gradients by Na,K-ATPase is necessary for preservation of cell volume, maintenance of membrane potential, and activity of secondary transporters that provide the cell with nutrients or regulate cellular solute concentrations. Moreover, in renal epithelial cells, the Na,K-ATPase, exclusively located in the basolateral membrane, is the driving force for the Na+ reabsorption that maintains extracellular volume and, hence, blood pressure. In addition, in heart and skeletal muscle, the activity of Na,K-ATPase is tightly coupled to the activity of a Na+/Ca2+-exchanger that controls muscle contraction. Finally, in the nervous system, Na,K-ATPase contributes to the re-establishment of the basal Na+ and K+ gradients both during action potentials and consequent to neuronal excitation. The Na,K-ATPase must be finely regulated to fulfil its important tasks under changing physiological conditions. Tissue-specific differences in Na,K-ATPase activity are achieved by the expression of four α and three β isoforms, which potentially can form 12 Na,K-ATPase isozymes with different functional properties. Moreover, various hormones and neurotransmitters are involved in the short- and long-term control of Na,K-ATPase; they regulate its activity and/or expression through protein kinase phosphorylation or transcriptional control. In this review, we discuss a novel regulatory mechanism of Na,K-ATPase that is mediated by the interaction of Na,K-ATPase with small membrane proteins of the FXYD family that so far have been considered as channels or regulators of ion channels. The FXYD proteins FXYD1 through FXYD7 exhibit tissue-specific distribution. FXYD1 (phospholemman) in heart and skeletal muscle, FXYD2 (the γ subunit of Na,K-ATPase) and FXYD4 [corticosteroid hormone-induced factor (CHIF)] in the kidney, and FXYD7 in the brain associate preferentially with the Na,K-ATPase α1-β1 isozyme and modulate its transport properties in a way that conforms to tissue-specific requirements. Thus, FXYD proteins contribute to proper handling of Na+ and K+ by the most widely expressed Na,K-ATPase α1-β1 isozymes, and ensure correct function in such mechanisms as renal Na+ reabsorption, muscle contraction, and neuronal excitability