Probing the Extracellular Access Channel of the Na,K-ATPase

When the Na,K-ATPase pumps at each turnover two K⁺ ions into the cytoplasm, this translocation consists of several reaction steps. First, the ions diffuse consecutively from the extracellular phase through an access pathway to the binding sites where they are coordinated. In the next step, the enzyme is dephosphorylated and the ions are occluded inside the membrane domain. The subsequent transition to the E₁ conformation produces a deocclusion of the binding sites to the cytoplasmic side of the membrane and allows in the last steps ion dissociation and diffusion to the aqueous phase. The interaction and competition of K⁺ with various quaternary organic ammonium ions have been used to gain insight into the molecular mechanism of the ion binding process from the extracellular side in the P-E₂ conformation of the enzyme. Using the electrochromic styryl dye RH421, evidence has been obtained that the access pathway consists of a wide and water-filled funnel-like part that is accessible also for bulky cations such as the benzyltriethylammonium ion, and a narrow part that permits passage only of small cations such as K⁺ and NH₄⁺ in a distinct electrogenic way. Benzyltriethylammonium ions inhibit K⁺ binding in a competitive manner that can be explained by a stopper-like function at the interface between the wide and narrow parts of the access pathway. In contrast to other quaternary organic ammonium ions, benzyltriethylammonium ions show a specific binding to the ion pump in a position inside the access pathway where it blocks effectively the access to the binding sites

Modulation of the Na,K-ATPase by Magnesium Ions

Since the beginning of investigations of the Na,K-ATPase, it has been well-known that Mg²⁺ is an essential cofactor for activation of enzymatic ATP hydrolysis without being transported through the cell membrane. Moreover, experimental evidence has been collected through the years that shows that Mg²⁺ ions have a regulatory effect on ion transport by interacting with the cytoplasmic side of the ion pump. Our experiments allowed us to reveal the underlying mechanism. Mg²⁺ is able to bind to a site outside the membrane domain of the protein’s α subunit, close to the entrance of the access channel to the ion-binding sites, thus modifying the local concentration of the ions in the electrolyte, of which Na⁺, K⁺, and H⁺ are of physiological interest. The decrease in the concentration of these cations can be explained by electrostatic interaction and estimated by the Debye–Hückel theory. This effect provokes the observed apparent reduction of the binding affinity of the binding sites of the Na,K-ATPase in the presence of various Mg²⁺ concentrations. The presence of the bound Mg²⁺, however, does not affect the reaction kinetics of the transport function of the ion pump. Therefore, stopped-flow experiments could be performed to gain the first insight into the Na⁺ binding kinetics on the cytoplasmic side by Mg²⁺ concentration jump experiments

TtOmp85, a β‑Barrel Assembly Protein, Functions by Barrel Augmentation

Outer membrane proteins are vital for Gram-negative bacteria and organisms that inherited organelles from them. Proteins from the Omp85/BamA family conduct the insertion of membrane proteins into the outer membrane. We show that an eight-stranded outer membrane β-barrel protein, TtoA, is inserted and folded into liposomes by an Omp85 homologue. Furthermore, we recorded the channel conductance of this Omp85 protein in black lipid membranes, alone and in the presence of peptides comprising the sequence of the two N-terminal and the two C-terminal β-strands of TtoA. Only with the latter could a long-living compound channel that exhibits conductance levels higher than those of the Omp85 protein alone be observed. These data support a model in which unfolded outer membrane protein after docking with its C-terminus penetrates into the transmembrane β-barrel of the Omp85 protein and augments its β-sheet at the first strand. Augmentation with successive β-strands leads to a compound, dilated barrel of both proteins