Multi-Ion Distributions in the Cytoplasmic Domain of Inward Rectifier Potassium Channels

Inward rectifier potassium (Kir) channels act as cellular diodes, allowing unrestricted flow of potassium (K+) into the cell while preventing currents of large magnitude in the outward direction. The rectification mechanism by which this occurs involves a coupling between K+ and intracellular blockers—magnesium (Mg2+) or polyamines—that simultaneously occupy the permeation pathway. In addition to the transmembrane pore, Kirs possess a large cytoplasmic domain (CD) that provides a favorable electronegative environment for cations. Electrophysiological experiments have shown that the CD is a key regulator of both conductance and rectification. In this study, we calculate and compare averaged equilibrium probability densities of K+ and Cl− in open-pore models of the CDs of a weak (Kir1.1-ROMK) and a strong (Kir2.1-IRK) rectifier through explicit-solvent molecular-dynamics simulations in ∼1 M KCl. The CD of both channels concentrates K+ ions greater than threefold inside the cytoplasmic pore while IRK shows an additional K+ accumulation region near the cytoplasmic entrance. Simulations carried out with Mg2+ or spermine (SPM4+) show that these ions interact with pore-lining residues, shielding the surface charge and reducing K+ in both channels. The results also show that SPM4+ behaves differently inside these two channels. Although SPM4+ remains inside the CD of ROMK, it diffuses around the entire volume of the pore. In contrast, this polyatomic cation finds long-lived conformational states inside the IRK pore, interacting with residues E224, D259, and E299. The strong rectifier CD is also capable of sequestering an additional SPM4+ at the cytoplasmic entrance near a cluster of negative residues D249, D274, E275, and D276. Although understanding the actual mechanism of rectification blockade will require high-resolution structural information of the blocked state, these simulations provide insight into how sequence variation in the CD can affect the multi-ion distributions that underlie the mechanisms of conduction, rectification affinity, and kinetics.