Visualizing the mapped ion pathway through the Na,K-ATPase pump

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• The Na(+),K(+)-ATPase pump facilitates essential ion transport across cell membranes.
• This pump normally operates with tightly coupled gates, allowing ion access sequentially from one side.
• The marine toxin palytoxin disrupts this gating mechanism, converting the pump into a channel.

Purpose of the Study:

• To investigate the ion translocation pathway of the Na(+),K(+)-ATPase when its gating is disrupted by palytoxin.
• To map the accessible residues within the pump-channel structure using functionalized cysteine mutations and chemical labeling.
• To visualize the open-channel pathway by integrating experimental data with a homology model.

Main Methods:

• Cysteine scanning mutagenesis of Na(+),K(+)-ATPase transmembrane segments (TM1-TM6).
• Functional analysis using Na(+) current recordings through palytoxin-bound pump-channels.
• Accessibility mapping of introduced cysteine residues via reaction with methanethiosulfonate (MTS) reagents.
• Structural visualization using a homology model of Na(+),K(+)-ATPase based on SERCA Ca(2+)-ATPase structure.

Main Results:

• A continuous chain of MTS-reactive positions was identified, spanning from the extracellular surface to the cytoplasm.
• This pathway involves residues from TM1, TM2, TM4, and TM6, and passes through the region of cation binding site II.
• Cavity search analysis of the homology model confirmed an extracellular pathway surrounded by reactive residues, broadening above T806.

Conclusions:

• The findings reveal a structural pathway for cation translocation through the Na(+),K(+)-ATPase when functioning as a channel.
• This provides a molecular basis for understanding ion transport in Na(+),K(+)-ATPase and related P-type pumps.
• The study elucidates the structural consequences of palytoxin binding on pump gating and ion permeation.