Grant: $6,737 - National Institutes of Health - Jun. 4, 2009
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Award Description: This application is for a supplement to support an outstanding student, Mauro Caffarelli, selected for the RU SURF program. Mauro will join our ongoing effort to understand in molecular detail how Na/K-ATPase pumps work. The Na/K pump generates transmembrane gradients of Na and K ions vital to animal cells. It is the target of clinically important drugs, and inherited Na/K pump mutations cause genetic diseases. Using powerful electrophysiological and molecular structural methods previously successfully applied in ion channels, we have established the view of the Na/K pump as a specialized ion pathway with tightly coupled cytoplasmic and extracellular gates that open and close alternately to allow access to the ion binding pocket from one side of the membrane at a time. Coupling of this alternating gating cycle to autophosphorylation and dephosphorylation reactions enables a single Na/K pump to export 3 Na ions and import 2 K ions for each ATP hydrolyzed. By exploiting the marine toxin, palytoxin, which disrupts the coupling between the Na/K pump?s two gates, so transforming it into a channel, we used cysteine scanning mutagenesis and hydrophilic thiol-specific methanethiosulfonate reagents to identify the ion translocation pathway through the pump?s transmembrane domain. Mapping reactive positions onto atomic models of the Na/K pump structure showed that they line a single unbranched pathway bounded by transmembrane helices TM1, TM2, TM4, and TM6 that spans the full distance across the membrane. Those results substantially advance the first of the parent grant?s three specific aims: (1) to determine the location, structure, and physicochemical characteristics of the ion-translocation pathway (or pathways) traversed by the transported Na and K ions, (2) to determine the location and structure of the Na/K pump's two principal gates, and (3) to examine the mechanisms controlling opening and closing of the gates. Mauro will address an outstanding question to help complete aim (1). An uncoupled current, believed carried by protons, flows through the Na/K pump in the absence of external Na and K ions, but the route of proton travel is unknown. Mauro will use two-microelectrode voltage-clamp recording in mutated ouabain-resistant Na/K pumps expressed in Xenopus oocytes, together with target cysteine modification and other biochemical manipulation, to test whether this uncoupled proton current flows along the principal action pathway that we identified in palytoxin-bound Na/K pump-channels.
Project Description: As defined in the Award Description field.
Jobs Summary: Student intern (Total jobs reported: 1)
Project Status: More than 50% Completed
This award's data was last updated on Jun. 4, 2009. Help expand these official descriptions using the wiki below.