Simulations of the Helix Bundle Crossing Gate Opening in Kir Channels

Abstract

Inwardly rectifying K+ (Kir) channels are gated by the phospholipid PIP2. Along the ion permeation pathway, three relatively narrow regions (the selectivity filter -SF, the inner helix bundle crossing (HBC), and the intracellular G-loop) may serve as gates to control ion permeation. A crystal structure of a Kir3.1 chimera [Nishida et al., 2007] captured the cytosolic G-loop gate in “closed/constricted” or “open/dilated” conformations. 100 ns Molecular Dynamics (MD) simulations studying the PIP2-driven Kir channel activation of the Kir3.1 chimera led us to propose a molecular mechanism of the G-loop gate opening [Meng et al., 2012]. However, opening of the HBC gate was not observed throughout this simulation. Mutagenesis and single-channel recording studies in our lab showed that a proline mutation on the inner helix of the Kir3.4 channel dramatically increased the open probability of the channel [Jin et al., 2002]. We introduced the corresponding M170P mutation on the Kir3.1 chimera structure and ran 100 ns long simulations of four mutant channel systems: dilated and constricted M170P Kir3.1 chimera in the presence (holo) and absence (apo) of PIP2, using the GROMACS program [Hess et al., 2008]. Three potassium ions present in the SF passed through the HBC gate in the system of the holo dilated M170P Kir3.1 chimera within the 100 ns simulation time. Minimal distance measurements indicated that the HBC gate was able to open only when PIP2 was present and the G-loop gate was stabilized in the open state. Principal component analysis revealed coupled conformational changes in the Slide helix, DE- and LM-loops, possibly related to the opening of the HBC gate. Moreover, unique residue interactions within the transmembrane domains were observed in the dilated holo system. Predictions of these models are being tested experimentally.