Interaction between Cardiotonic Steroids and Na,K-ATPase. Effects of pH and Ouabain-Induced Changes in Enzyme Conformation

Cornelius, Flemming; Mahmmoud, Yasser A.

doi:10.1021/bi901212r

Cited by 21 publications

(16 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…First, αM1 and αM2 helices are opened up to accommodate the ouabain molecule, suggesting that initial access of ouabain originate from a state in which the external access channel is opened like in the SERCA1a E 2 :BeF 3− structure [39]. A further movement towards the cytoplasmic end of αM1 and αM2 helices has also been observed in ouabain bound crystal structures [36,37] and functionally suggested by ouabain induced changes of trypsin accessibility in the αM2–αM3 linker [40]. Second, αM4 helix appears to be rather flexible and prompt to adopt changes in conformation upon ouabain binding as observed in our simulations and ouabain bound crystal structures [36,37].…”

Section: Discussionmentioning

confidence: 96%

A Structural Rearrangement of the Na+/K+-ATPase Traps Ouabain within the External Ion Permeation Pathway

Sánchez-Rodríguez

Khalili‐Araghi

Miranda

et al. 2015

Journal of Molecular Biology

View full text Add to dashboard Cite

Using the energy of ATP hydrolysis, the Na+/K+-ATPase is able to transport across the cell membrane Na+ and K+ against their electrochemical gradients. The enzyme is strongly inhibited by ouabain and its derivatives, some that are therapeutically used for patients with heart failure (cardiotonic steroids). Using lanthanide resonance energy transfer (LRET), we trace here the conformational changes occurring on the external side of functional Na+/K+-ATPases induced by the binding of ouabain. Changes in donor/acceptor pair distances are mainly observed within the α subunit of the enzyme. To derive a structural model matching the experimental LRET distances measured with bound ouabain, molecular dynamic simulations were carried out with energy restraints applied simultaneously using a novel methodology with multiple noninteracting fragments. The restrained simulation, initiated from the X-ray structure of the E2(2K+) state, became strikingly similar to the X-ray structure of the sodium-bound state. The final model shows that ouabain is trapped within the external ion permeation pathway of the pump.

show abstract

Section: Discussionmentioning

confidence: 96%

A Structural Rearrangement of the Na+/K+-ATPase Traps Ouabain within the External Ion Permeation Pathway

Sánchez-Rodríguez

Khalili‐Araghi

Miranda

et al. 2015

Journal of Molecular Biology

View full text Add to dashboard Cite

show abstract

“…Treatment with ouabain, a specific inhibitor of Na + /K + -ATPase [39], increases blood-labyrinth barrier permeability by increasing phosphorylation of occludin (Figure 4D–G). A number of regulators of protein phosphorylation, including protein kinase C [26], tyrosine kinase c-Yes [40], and protein phosphatase PP2A [22], have been reported to be involved in this process.…”

Section: Discussionmentioning

confidence: 99%

Na+/K+-ATPase α1 Identified as an Abundant Protein in the Blood-Labyrinth Barrier That Plays an Essential Role in the Barrier Integrity

et al. 2011

View full text Add to dashboard Cite

BackgroundThe endothelial-blood/tissue barrier is critical for maintaining tissue homeostasis. The ear harbors a unique endothelial-blood/tissue barrier which we term “blood-labyrinth-barrier”. This barrier is critical for maintaining inner ear homeostasis. Disruption of the blood-labyrinth-barrier is closely associated with a number of hearing disorders. Many proteins of the blood-brain-barrier and blood-retinal-barrier have been identified, leading to significant advances in understanding their tissue specific functions. In contrast, capillaries in the ear are small in volume and anatomically complex. This presents a challenge for protein analysis studies, which has resulted in limited knowledge of the molecular and functional components of the blood-labyrinth-barrier. In this study, we developed a novel method for isolation of the stria vascularis capillary from CBA/CaJ mouse cochlea and provided the first database of protein components in the blood-labyrinth barrier as well as evidence that the interaction of Na+/K+-ATPase α1 (ATP1A1) with protein kinase C eta (PKCη) and occludin is one of the mechanisms of loud sound-induced vascular permeability increase.Methodology/Principal FindingsUsing a mass-spectrometry, shotgun-proteomics approach combined with a novel “sandwich-dissociation” method, more than 600 proteins from isolated stria vascularis capillaries were identified from adult CBA/CaJ mouse cochlea. The ion transporter ATP1A1 was the most abundant protein in the blood-labyrinth barrier. Pharmacological inhibition of ATP1A1 activity resulted in hyperphosphorylation of tight junction proteins such as occludin which increased the blood-labyrinth-barrier permeability. PKCη directly interacted with ATP1A1 and was an essential mediator of ATP1A1-initiated occludin phosphorylation. Moreover, this identified signaling pathway was involved in the breakdown of the blood-labyrinth-barrier resulting from loud sound trauma.Conclusions/SignificanceThe results presented here provide a novel method for capillary isolation from the inner ear and the first database on protein components in the blood-labyrinth-barrier. Additionally, we found that ATP1A1 interaction with PKCη and occludin was involved in the integrity of the blood-labyrinth-barrier.

show abstract

“…With another approach, we aimed to modify the conformational poise by exposing myocytes to the Na ϩ ionophore monensin to enhance Na ϩ influx and increase its intracellular concentration (18). We also exposed myocytes to ouabain that binds to the Na ϩ -K ϩ pump in E2 and E2P conformations (19). Glutathionylation of ␤1 Na ϩ -K ϩ pump subunits was induced from base line by exposing myocytes to the chemical oxidant ONOO Ϫ or to angiotensin II (Ang II).…”

Section: Glutathionylation Of Namentioning

confidence: 99%

Susceptibility of β1 Na+-K+ Pump Subunit to Glutathionylation and Oxidative Inhibition Depends on Conformational State of Pump

Liu¹,

García²,

Mahmmoud

et al. 2012

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Glutathionylation of cysteine 46 of the β1 subunit of the Na+-K+ pump causes pump inhibition. However, the crystal structure, known in a state analogous to an E2·2K+·Pi configuration, indicates that the side chain of cysteine 46 is exposed to the lipid bulk phase of the membrane and not expected to be accessible to the cytosolic glutathione. We have examined whether glutathionylation depends on the conformational changes in the Na+-K+ pump cycle as described by the Albers-Post scheme. We measured β1 subunit glutathionylation and function of Na+-K+-ATPase in membrane fragments and in ventricular myocytes. Signals for glutathionylation in Na+-K+-ATPase-enriched membrane fragments suspended in solutions that preferentially induce E1ATP and E1Na3 conformations were much larger than signals in solutions that induce the E2 conformation. Ouabain further reduced glutathionylation in E2 and eliminated an increase seen with exposure to the oxidant peroxynitrite (ONOO−). Inhibition of Na+-K+-ATPase activity after exposure to ONOO− was greater when the enzyme had been in the E1Na3 than the E2 conformation. We exposed myocytes to different extracellular K+ concentrations to vary the membrane potential and hence voltage-dependent conformational poise. K+ concentrations expected to shift the poise toward E2 species reduced glutathionylation, and ouabain eliminated a ONOO−-induced increase. Angiotensin II-induced NADPH oxidase-dependent Na+-K+ pump inhibition was eliminated by conditions expected to shift the poise toward the E2 species. We conclude that susceptibility of the β1 subunit to glutathionylation depends on the conformational poise of the Na+-K+ pump.

show abstract

Interaction between Cardiotonic Steroids and Na,K-ATPase. Effects of pH and Ouabain-Induced Changes in Enzyme Conformation

Cited by 21 publications

References 37 publications

A Structural Rearrangement of the Na+/K+-ATPase Traps Ouabain within the External Ion Permeation Pathway

A Structural Rearrangement of the Na+/K+-ATPase Traps Ouabain within the External Ion Permeation Pathway

Na+/K+-ATPase α1 Identified as an Abundant Protein in the Blood-Labyrinth Barrier That Plays an Essential Role in the Barrier Integrity

Susceptibility of β1 Na+-K+ Pump Subunit to Glutathionylation and Oxidative Inhibition Depends on Conformational State of Pump

Contact Info

Product

Resources

About