The Structure and Gating of the Epithelial Na+ Channel

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DESCRIPTION (provided by applicant): Epithelial Na+ Channel (ENaC) activity can be modulated by two distinct mechanisms: either by changes in single channel gating properties (i.e., open probability), or by changes in the number of channels expressed in the apical membrane. The mechanism for ENaC gating has yet to be determined. Based on analogy with other cation channels, several mechanisms have been proposed. These mechanisms generally fall into two categories, either channel blocking, or an allosteric transition. In the channel-blocking hypothesis, either a portion of ENaC itself, or another molecule inserts into the channel pore, preventing Na+ transfer. In this hypothesis, changes in residue contacts as the channel transitions from an open to a closed state would be localized to the area, which interacts with the blocker. In the allosteric transition hypothesis, the helices, which form the pore, would undergo a conformational change such that Na+ could no longer traverse the pore. External stimuli could induce such a transition by causing small local changes, which could propogate into global changes, driving the allosteric transition. In this hypothesis, changes in residue contacts as the channel transitions from an open to a closed state would be widespread. We hypothesize that residue contacts made within the pore region of ENaC are altered in the open vs. the closed conformation of the channel. In order to assess these changes, we will exploit the ability of Ni2+ to selectively bind histidine to generate a map of pore region residues. Such a map will help to delineate which amongst the currently proposed models for ENaC gating is most likely