Association of Renal Na,K-ATPase α-Subunit with the β- and γ-Subunits Based on Cryoelectron Microscopy

Purhonen, Pasi; Thomsen, Kristine Rømer; Ab, Maunsbach; Hebert, Hans

doi:10.1007/s00232-006-0056-8

Cited by 10 publications

(8 citation statements)

References 47 publications

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“…As a high-resolution crystal structure of the H,K-ATPase has not been described, the exact location of the β subunit is not known. It is thought that there is close interaction on the luminal surface with the loop between TM7 and TM8 on the outside of the membrane domain [28] and that perhaps the membrane domain of the β subunit is interdigitated between TM9 and TM10 [8,36]. In a crystal of the Na,K-ATPase [30], the β subunit membrane domain is tilted and contacts TM7 and TM10, consistent with the yeast two hybrid and biochemical data obtained for the H,KATPase [28,50].…”

Section: The Structure and Function Of The Gastric Hk-atpasesupporting

confidence: 69%

The gastric HK-ATPase: structure, function, and inhibition

Shin

Munson

Vagin

et al. 2008

Pflugers Arch - Eur J Physiol

220

181

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The gastric H,K-ATPase, a member of the P 2 -type ATPase family, is the integral membrane protein responsible for gastric acid secretion. It is an α,β-heterodimeric enzyme that exchanges cytoplasmic hydronium with extracellular potassium. The catalytic α subunit has ten transmembrane segments with a cluster of intramembranal carboxylic amino acids located in the middle of the transmembrane segments TM4, TM5,TM6, and TM8. Comparison to the known structure of the SERCA pump, mutagenesis, and molecular modeling has identified these as constituents of the ion binding domain. The β subunit has one transmembrane segment with N terminus in cytoplasmic region. The extracellular domain of the β subunit contains six or seven Nlinked glycosylation sites. N-glycosylation is important for the enzyme assembly, maturation, and sorting. The enzyme pumps acid by a series of conformational changes from an E 1 (ion site in) to an E 2 (ion site out) configuration following binding of MgATP and phosphorylation. Several experimental observations support the hypothesis that expulsion of the proton at 160 mM (pH 0.8) results from movement of lysine 791 into the ion binding site in the E 2 P configuration. Potassium access from the lumen depends on activation of a K and Cl conductance via a KCNQ1/KCNE2 complex and Clic6. K movement through the luminal channel in E 2 P is proposed to displace the lysine along with dephosphorylation to return the enzyme to the E 1 configuration. This enzyme is inhibited by the unique proton pump inhibitor class of drug, allowing therapy of acid-related diseases.

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Section: The Structure and Function Of The Gastric Hk-atpasesupporting

confidence: 69%

The gastric HK-ATPase: structure, function, and inhibition

Shin

Munson

Vagin

et al. 2008

Pflugers Arch - Eur J Physiol

220

181

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“…The spectrum was obtained by averaging 16 000 transients per t 1 increment and processed with complex linear prediction in t 1 . Models were generated by using Modeller [20] based on the coordinates for PDB ID: 1VFP [12] and with the sequence alignment proposed by Hebert et al [21] An alternative structure (PDB ID: 1T5S) could also have been used. [13] Model refinement was performed with energy minimisation and simulated annealing, and visualised by using PyMOL (DeLano Scientific LLC).…”

Section: Methodsmentioning

confidence: 99%

Solid‐State NMR Studies of Adenosine 5′‐Triphosphate Freeze‐Trapped in the Nucleotide Site of Na,K‐ATPase

et al. 2009

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The Na,K-ATPase (NKA) maintains the cellular potentials of all animal cells by driving the vectorial transport of Na + and K + across the plasma membrane through two different enzyme conformations (E 1 and E 2 ).[1] NKA exhibits high affinity towards the ATP substrate when sufficient Na + is present to induce the E 1 conformation. [2,3] More than fifty years after the discovery of NKA, the details of how ATP interacts with the enzyme in the E 1 conformation remain unknown, and the only X-ray crystal structure of NKA is for the E 2 conformation in the absence of nucleotide.[4] Solid-state NMR spectroscopy (SSNMR) is a valuable alternative to X-ray diffraction for providing atomistic details of ligands bound to receptors within their native membranes.[ C signals from bound ATP and provide preliminary insights into the nucleotide contacts with the high-affinity site.A critical requirement for the SSNMR experiments is to saturate the high-affinity nucleotide site with ATP whilst avoiding excess free or unspecifically bound nucleotide, because SSNMR spectroscopy cannot readily distinguish these from specifically bound nucleotide. Biochemical measurements of radiolabelled nucleotide binding were therefore carried out to establish such a regime. Equilibrium binding curves show nucleotide binding to a single site with dissociation constants in the range of 0.2-0.5 mm, and a capacity of about 2.9 nmol mg À1 protein for both shark and pig NKA in the buffer used for SSNMR experiments ( Figure S1 in the Supporting Information). Samples for SSNMR analysis contain 130 mm NKA, so we can safely assume that the nucleotide site is saturated by adding 160 mm [U-13 C,15 N]ATP to the membranes; this gives a free nucleotide concentration of about 30 mm. The binding assay also demonstrates the absence of additional unspecific binding to NKA. We thus confirm that at these concentrations more than 80 % of the 13 C NMR signal for the nucleotide will correspond to ATP bound specifically to shark or kidney enzyme. Figure 1 A shows 13 C cross-polarisation magic-angle spinning (CP-MAS) SSNMR spectra at À25 8C for a control shark NKA [a] Dr.

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“…The structure of the Na,K-ATPase complex has been determined at 8 Å resolution by cryoelectron microscopy analysis of two-dimensional crystalline native kidney membranes (Hebert et al 2001;Purhonen et al 2006). In this structure, the electron density map clearly identifies the arrangement of the ten transmembrane helices of the α subunit, as well as distinct regions assigned to the transmembrane domains of the β and FXYD subunits.…”

Section: Structure and Dynamics Of Fxyd1mentioning

confidence: 99%

Structures of the FXYD regulatory proteins in lipid micelles and membranes

Franzin

Gong

Teriete

et al. 2007

J Bioenerg Biomembr

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The FXYD membrane proteins constitute a family of conserved auxiliary subunits of the Na,KATPase, and have been the focus of recent attention due to their ability to finely regulate the activity of the enzyme complex in various physiological settings. In this review we describe the structures of the proteins, as well as their dynamics and their associations with the lipid bilayer membrane, which we have recently determined by NMR spectroscopy. Although the proteins are relatively small, their genes contain as many as six to nine small exons, and the coincidence of structured protein segments with their genetic elements suggests assembly from discrete structural modules through exon shuffling. The three-dimensional structures and backbone dynamics provide the foundation for understanding their intra-membrane association with the Na,K-ATPase α subunit, and the structure of FXYD1 suggests a mechanism whereby the phosphorylation of conserved Ser residues, by protein kinases A and C, could induce a conformational change in the cytoplasmic domain of the protein, to modulate its interaction with the α subunit.

show abstract

Association of Renal Na,K-ATPase α-Subunit with the β- and γ-Subunits Based on Cryoelectron Microscopy

Cited by 10 publications

References 47 publications

The gastric HK-ATPase: structure, function, and inhibition

The gastric HK-ATPase: structure, function, and inhibition

Solid‐State NMR Studies of Adenosine 5′‐Triphosphate Freeze‐Trapped in the Nucleotide Site of Na,K‐ATPase

Structures of the FXYD regulatory proteins in lipid micelles and membranes

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