The sodium-potassium (Na/K) pump is a P-type ATPase that generates Na+ and K+ concentration gradients across the cell membrane. For each ATP molecule, the pump extrudes three Na+ and imports two K+ by alternating between outward- and inward-facing conformations that preferentially bind K+ or Na+, respectively. Remarkably, the selective K+ and Na+ binding sites share several residues, and how the pump is able to achieve the selectivity required for the functional cycle is unclear. Here, free energy perturbation molecular dynamics (FEP/MD) simulations based on the crystal structures of the Na/K pump in a K+-loaded state (E2·Pi) reveal that protonation of the high-field acidic side-chains involved in the binding sites is critical to achieve the proper K+ selectivity. This prediction is tested with electrophysiological experiments showing that the selectivity of the E2P state for K+ over Na+ is affected by extracellular pH.

The Na/K pump is a P-type ATPase that exchanges three intracellular Na + ions for two extracellular K + ions through the plasmalemma of nearly all animal cells. The mechanisms involved in cation selection by the pump's ion-binding sites (site I and site II bind either Na + or K + ; site III binds only Na + ) are poorly understood. We studied cation selectivity by outward-facing sites (high K + affinity) of Na/K pumps expressed in like organic cation transport challenges the concept of rigid structural models in which ion specificity at site I and site II arises from a precise and unique arrangement of coordinating ligands. Furthermore, actions by guanidinium + derivatives suggest that Na + binds to site III in a hydrated form and that the inward current observed without external Na + and K + represents cation transport when normal occlusion at sites I and II is impaired. These results provide insights on external ion selectivity at the three binding sites.he Na/K pump uses the free energy from ATP hydrolysis to export three Na + ions against a steep electrochemical gradient in exchange for the import of two K + ions. The pump alternates between two major conformational states, E1 and E2 (1), and its function is explained via a ping-pong (2) alternate-access mechanism (Fig. S1). Under physiological conditions, the E2P (phosphorylated) state, with extracellular-facing ion-binding sites, must select K + in the presence of more than 10-fold greater external Na + concentration. On the other hand, the E1 state, with cytoplasmic-facing ion-binding sites, selects Na + over K + , which is present at a 10-fold higher concentration.Of the three ion-binding sites, sites I and II, or shared sites, can bind either Na + or K + , but site III exclusively binds Na + . External release of Na + ions from these three sites occurs sequentially (3, 4). In the outward-facing conformation, Na + release is followed by binding of K + in the normal forward operation of the Na/K pump, inducing an outwardly directed pump current that can be studied under voltage clamp. Rebinding of Na Most of the cycle's voltage dependence is believed to arise from the release (rebinding in the backward reaction) of the first Na + ion through a high-field access channel when leaving its binding site (3, 4, 7). There are indications that the Na + -exclusive site III releases Na + before the shared sites (6,8,9) and that the voltagedependent rebinding of this first externally released Na + blocks release of the other two Na + ions from the shared sites. Concordantly, at saturating [K + o ], where Na + competition for the shared sites should be negligible, the Na/K pump current still presents significant VDI in the presence of external Na + (10) (Fig. S2). In addition, several studies have proposed a noncanonical mode of transport in which protons move in the inward direction down their electrochemical gradient at very negative voltages (11) through a pathway that passes through site III (9, 10, 12, 13).These observations raise a number of critical questions ...

Objectives: To compare the effectiveness of both vancomycin powder and antibiotic bead placement to irrigation and debridement alone in prevention of infection in a contaminated open fracture model in rats. Methods: In a previously described model of contaminated open fractures, 45 rats had simulated open fractures created, stabilized, and contaminated with Staphylococcus aureus. They were then treated 6 hours later with 3 interventions: irrigation and debridement alone (control group) or in combination with placement of polymethyl methacrylate beads containing vancomycin and tobramycin powders (antibiotic bead group) or placement of 10 mg of intrawound vancomycin powder (powder group). Rats were allowed to recover and then killed 14 days later for harvest of femurs and plates. Femurs and plates were both incubated overnight, and bacterial colonies were counted in each group for comparison. Results: Quantitative counts of bacteria in bone showed significantly reduced growth in both bead and powder groups when compared with control group (P < 0.0001). Quantitative counts of bacteria in plates showed significantly reduced growth in both bead and powder groups when compared with control group (P < 0.0003; 0.029). No significant differences were seen in bacterial growth between bead and powder groups for either bones (P = 0.13) or plates (P = 0.065). Conclusions: When compared with irrigation and debridement alone, placement of intrawound vancomycin powder significantly decreased bacterial load in a contaminated open fracture model in rats similar to placing antibiotic beads. This may provide an additional adjuvant treatment that does not require a secondary surgery for bead removal.

The sodium–potassium (Na/K) pump plays an essential role in maintaining cell volume and secondary active transport of other solutes by establishing the Na+ and K+ concentration gradients across the plasma membrane of animal cells. The recently determined crystal structures of the Na/K pump to atomic resolution provide a new impetus to investigate molecular determinants governing the binding of Na+ and K+ ions and conformational transitions during the functional cycle. The pump cycle is generally described by the alternating access mechanism, in which the pump toggles between different conformational states, where ions can bind from either the intracellular or the extracellular side. However, important issues concerning the selectivity of the Na/K pump remain to be addressed. In particular, two out of the three binding sites are shared between Na+ and K+ and it is not clear how the protein is able to select K+ over Na+ when it is in the outwardly facing phosphorylated conformation (E2P), and Na+ over K+ when it is in the inwardly facing conformation (E1). In this review article, we will first briefly review the recent advancement in understanding the microscopic mechanism of K+ selectivity in the Na/K pump at the E2·Pi state and then outline the remaining challenges to be addressed about ion selectivity.