Revealing the Architecture of a K + Channel Pore Through Mutant Cycles with a Peptide Inhibitor

Abstract

Thermodynamic mutant cycles provide a formalism for studying energetic coupling between amino acids on the interaction surface in a protein-protein complex. This approach was applied to the Shaker potassium channel and to a high-affinity peptide inhibitor (scorpion toxin) that binds to its pore entryway. The assignment of pairwise interactions defined the spatial arrangement of channel amino acids with respect to the known inhibitor structure. A strong constraint was placed on the Shaker channel pore-forming region by requiring its amino-terminal border to be 12 to 15 angstroms from the central axis. This method is directly applicable to sodium, calcium, and other ion channels where inhibitor or modulatory proteins bind with high affinity.