A Structural Model of a Kir Channel in the Open State Derived from Mutagenic Scanning of the Pore Gating Energetics

Specific stimuli (e.g. intracellular pH, PIP2) cause Kir channels to undergo a reversible transition between the closed and open state. The precise rearrangement of the pore structure (e.g. the TM regions) during these gating transitions is currently unknown in Kir channels owning to the lack of an open state crystal structure and validating functional data. We employed systematic alanine scanning mutagenesis on the entire Kir1.1 pore structure (residues 51 - 192) and determined the IC50 values for pH inhibition for each mutant. We reasoned that a shift in the IC50 value should represent a change in the free energy of the open state relative to the closed state assuming that the mutations did not affect the cytoplasmatic pH sensor directly. We identified 26 mutations that produced a marked shift in the pH sensitivity, intriguingly, 24 of these mutations increased the pH sensitivity suggesting that the open state is structurally more optimised than the closed state. Specifically, we expected that in the open state the IC50 shifting residues would interact with each other explaining why the alanine substitution would preferentially destabilise this state. We used this expectation as conceptual basis to develop scoring methods to evaluate structural models of Kir1.1 in the open state that we generated from existing crystallographic open state structures of other K+ channels (KvAP, NaK, KirBac3.1) using homology modelling and MD simulations. This analysis revealed an excellent agreement of our functional data with one particular open state model of Kir1.1. In this model more than 80% of the IC50 shifting residues are part of a tightly packed network of interacting residues that largely disintegrates upon channel closure.