Abstract
Inward-rectifier potassium channels (Kirs) are lipid-gated ion channels that differ from other K+ channels in that they allow K+ ions to flow more easily into, rather than out of, the cell. Inward rectification is known to result from endogenous magnesium ions or polyamines (e.g., spermine) binding to Kirs, resulting in a block of outward potassium currents, but questions remain regarding the structural and dynamic basis of the rectification process and lipid-dependent channel activation. Here, we present the results of long-timescale molecular dynamics simulations starting from a crystal structure of phosphatidylinositol 4,5-bisphosphate (PIP2)-bound chicken Kir2.2 with a non-conducting pore. After introducing a mutation (G178R) that is known to increase the open probability of a homologous channel, we were able to observe transitions to a stably open, ion-conducting pore, during which key conformational changes occurred in the main activation gate and the cytoplasmic domain. PIP2 binding appeared to increase stability of the pore in its open and conducting state, as PIP2 removal resulted in pore closure, with a median closure time about half of that with PIP2 present. To investigate structural details of inward rectification, we simulated spermine binding to and unbinding from the open pore conformation at positive and negative voltages, respectively, and identified a spermine-binding site located near a previously hypothesized site between the pore cavity and the selectivity filter. We also studied the effects of long-range electrostatics on conduction and spermine binding by mutating charged residues in the cytoplasmic domain and found that a finely tuned charge density, arising from basic and acidic residues within the cytoplasmic domain, modulated conduction and rectification.