Multi-ion distributions in the cytoplasmic domain of inward rectifier potassium channels

Abstract

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 (Mg(2+)) 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 Mg(2+) or spermine (SPM(4+)) show that these ions interact with pore-lining residues, shielding the surface charge and reducing K(+) in both channels. The results also show that SPM(4+) behaves differently inside these two channels. Although SPM(4+) 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 SPM(4+) 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.

Figure 1

Kir channel structure and simulation setup. (A) The full-length Kir2.1/IRK open model. The two opposing positioned subunits of the transmembrane (I and III, residues 57–184) and cytoplasmic (II and IV, residues 185–350) domains are shown to reveal the ion permeation pathway. (Black line) Position of the membrane. The selectivity filter backbone atoms, including the K⁺ ions (magenta spheres), are shown explicitly. Side chains of residues in the cytoplasmic domain that are strong contributors to the electrostatic environment inside the pore of Kir2.1/IRK are also shown (E224, R228, D247, D255, D259, R260, and E299). (B) A snapshot of the all-atom, solvated open model of the Kir2.1/IRK cytoplasmic domain simulation system with ∼1 M KCl. (Magenta) K⁺ ions. (Green) Cl⁻ ions. (Cyan) Water molecules. One of the subunits has been removed to show water and ions inside the pore.

Figure 2

K⁺ and Cl⁻ in Kir1.1/ROMK and Kir2.1/IRK cytoplasmic domains. (A) An example K⁺ ion trajectory from one Kir2.1/IRK simulation. This trajectory and subsequent density plots are shown as functions of radial distance from the pore axis (R = sqrt(X²+Y²)) and height in the simulation box (Z). For a clearer visualization of the cytoplasmic pore structure, all plots are represented for the real values of R > 0 Å, as well as the mirror image defined for R < 0 Å, along the pore-axis symmetry line (red). In this trajectory, the K⁺ ion starts at position (R, Z) = (47.7 Å, −11.3 Å), represented by a symbol (green star) where it travels around the bulk and exits the top of the simulation box. Under periodic boundary conditions, the same ion reenters at the bottom of the cell (i.e., Z = −60 Å and Z = 60 Å are identical) and then finds its way into the cytoplasmic pore (R < 15 Å, −9 Å < Z < 15 Å) where it remains until the end of the simulation (red circle). (B) Two example time series of K⁺ inside the Kir2.1/IRK pore, with radial position R (black) and pore position Z (gray). The top time series is the same trajectory from panel A, showing the ion entering the pore and jumping from stationary sites along the protein surface. The bottom time series is another ion from the same simulation, showing a long-lived interaction, which exits the pore at the end of the simulation. (C) K⁺ and (D) Cl⁻ density distributions in Kir1.1/ROMK and Kir2.1/IRK. The density plots are calculated for each simulation and then averaged between three separate runs. A molecular density of ρ = 6.022 × 10⁻⁴ ions/ų corresponds to 1 M concentration. Contours are shown for densities corresponding to 1 and 2.5 M.

Figure 3

Ion densities in the crystal structure of a closed Kir2.1/IRK cytoplasmic domain, PDB:1U4F. (A) K⁺ and (B) Cl⁻ radial density plots. (C) A snapshot of ions inside the pore, with two of the subunits removed for clarity. Charged residues that line the pore are shown explicitly as in Fig. 1. (D) A closeup view of the hydrated K⁺ ion near the cytoplasmic entrance of the pore that remains throughout the simulation (15 ns).

Figure 4

K⁺ and Cl⁻ densities in the presence of rectification blockers Mg²⁺ and SPM⁴⁺. Density plots of: K⁺ (A) and Cl⁻ (B) with 4 Mg²⁺ ions inside the CD of ROMK (C); K⁺ (D) and Cl⁻ (E) with 4 Mg²⁺ ions inside the CD of IRK (F). Each plot was calculated from an average over three simulations. Density plots of: K⁺ (G), Cl⁻ (H), and SPM⁴⁺ (I) in a single structural model of ROMK; K⁺ (J), Cl⁻ (K), and SPM⁴⁺ (L) in a single structural model of IRK. Averaged density plots for single spermine simulations are shown in Fig. S9. Density plots of K⁺, Cl⁻, and SPM⁴⁺ after an additional spermine molecule is added along the pore from −24 Å < Z < −6 Å and simulated for 10 ns: K⁺ (M), Cl⁻ (N), and SPM⁴⁺ (O) in ROMK, and K⁺ (P), Cl⁻ (Q), and SPM⁴⁺ (R) in IRK. The spermine densities are calculated using the nitrogen atoms (N₁, N₂, N₃, N₄) as the ions. A molecular density of ρ = 6.022 × 10⁻⁴ ions/ų is equivalent to 1 M concentration, and contours are shown for densities corresponding to 1 and 2.5 M. Note that whereas K⁺ and Cl⁻ adequately sample the system and converge to 1 M in the bulk, Mg²⁺ and SPM⁴⁺ remain mainly in the pore throughout the simulations. Therefore, the Mg²⁺ and SPM⁴⁺ distributions can only be interpreted as local densities and do not represent equilibrium.

Figure 5

Spermine structure and position analysis. (A) Kir1.1/ROMK and (B) Kir2.1/IRK. Three probability distributions are shown: (i) the end-to-end distribution calculated between the first and last nitrogen atoms in the polyamine, (ii) the radial position of the center of mass, R, and (iii) the pore position of the spermine center of mass, Z. (iv–vi) The corresponding center of mass trajectories with R (black) and Z (gray). In the Kir2.1/IRK trajectories, two long-lived states of the spermine molecule are observed, denoted as α and β. Snapshots from the simulations corresponding to the α- and β-states are shown on the right. (Silver, blue, red, and yellow) The four subunits. (CPK coloring) The spermine molecule. The residues involved in coordinating the spermine, E224, E299, D255, and D259 are shown explicitly.