Inwardly rectifying potassium (K IR) channels play important roles in controlling cellular excitability and K + ion homeostasis. Under physiological conditions, K IR channels allow large K + influx at potentials negative to the equilibrium potential of K + but permit little outward current at potentials positive to the equilibrium potential of K + , due to voltage dependent block of outward K + flux by cytoplasmic polyamines. These polycationic molecules enter the K IR channel pore from the intracellular side. They block K + ion movement through the channel at depolarized potentials, thereby ensuring, for instance, the long plateau phase of the cardiac action potential. Key questions concerning how deeply these charged molecules migrate into the pore and how the steep voltage dependence arises remain unclear. Recent MD simulations on GIRK2 (=Kir3.2) crystal structures have provided unprecedented details concerning the conduction mechanism of a K IR channel. Here, we use MD simulations with applied field to provide detailed insights into voltage dependent block of putrescine, using the conductive state of the strong inwardly rectifying K + channel GIRK2 as starting point. Our µs long simulations elucidate details about binding sites of putrescine in the pore and suggest that voltagedependent rectification arises from a dual mechanism.

ATP-sensitive potassium (KATP) channels consist of an inwardly rectifying K+ channel (Kir6.2) pore, to which four ATP-sensitive sulfonylurea receptor (SUR) domains are attached, thereby coupling K+ permeation directly to the metabolic state of the cell. Dysfunction is linked to neonatal diabetes and other diseases. K+ flux through these channels is controlled by conformational changes in the helix bundle region, which acts as a physical barrier for K+ permeation. In addition, the G-loop, located in the cytoplasmic domain, and the selectivity filter might contribute to gating, as suggested by different disease-causing mutations. Gating of Kir channels is regulated by different ligands, like Gβγ, H+, Na+, adenosine nucleotides, and the signaling lipid phosphatidyl-inositol 4,5-bisphosphate (PIP2), which is an essential activator for all eukaryotic Kir family members. Although molecular determinants of PIP2 activation of KATP channels have been investigated in functional studies, structural information of the binding site is still lacking as PIP2 could not be resolved in Kir6.2 cryo-EM structures. In this study, we used Molecular Dynamics (MD) simulations to examine the dynamics of residues associated with gating in Kir6.2. By combining this structural information with functional data, we investigated the mechanism underlying Kir6.2 channel regulation by PIP2.

EAG1 channels belong to the KCNH family of voltage gated potassium channels. They are expressed in several brain regions and increased expression is linked to certain cancer types. Recent cryo-EM structure determination finally revealed the structure of these channels in atomic detail, allowing computational investigations. In this study, we performed molecular dynamics simulations to investigate the ion binding sites and the dynamical behavior of the selectivity filter. Our simulations suggest that sites S2 and S4 form stable ion binding sites, while ions placed at sites S1 and S3 rapidly switched to sites S2 and S4. Further, ions tended to dissociate away from S0 within less than 20 ns, due to increased filter flexibility. This was followed by water influx from the extracellular side, leading to a widening of the filter in this region, and likely non-conductive filter configurations. Simulations with the inactivation-enhancing mutant Y464A or Na ions lead to trapped water molecules behind the SF, suggesting that these simulations captured early conformational changes linked to C-type inactivation.

ATP-sensitive potassium (KATP) channels consist of an inwardly rectifying K+ channel (Kir6.2) pore, to which four ATP-sensitive sulfonylurea receptor (SUR) domains are attached, thereby coupling K+ permeation directly to the metabolic state of the cell. Dysfunction is linked to neonatal diabetes and other diseases. K+ flux through these channels is controlled by conformational changes in the helix bundle region, which acts as a physical barrier for K+ flux. In addition, the G-loop, located in the cytoplasmic domain, and the selectivity filter might contribute to gating, as suggested by different disease-causing mutations. Gating of Kir channels is regulated by different ligands, like Gβγ, H+, Na+, adenosine nucleotides and the signaling lipid phosphatidyl-inositol 4,5-bisphosphate (PIP2), which is an essential activator for all eukaryotic Kir family members. Although molecular determinants of PIP2 activation of KATP channels have been investigated in functional studies, structural information of the binding site is still lacking as PIP2 could not be resolved in Kir6.2 cryo-EM structures. In this study, we used Molecular Dynamics (MD) simulations to examine the dynamics of residues associated with gating in Kir6.2. By combining this structural information with functional data, we investigated the mechanism underlying Kir6.2 channel regulation by PIP2.

MTSET labels residues N190C (Q218C) with a similar time course in both closed and open states. By contrast, extracellular MTSET modifies residues A193 to R201 in KCNQ2 (G219-S228 in KCNQ3) at depolarized potentials, but not at hyperpolarized potentials, suggesting that these residues likely lie buried in the membrane in the closed state. We also use voltage clamp fluorometry (VCF) to determine S4 movement in KCNQ2/3 channels. In homomeric KCNQ channels, the time course and voltage dependence of fluorescence F(V) and ionic current G(V) correlate, as if the S4 and the gate motion were directly coupled. Moreover, in KCNQ2, the F(V) follows the voltage dependence of the modification rate of residue A193C. Our data also indicate that the presence of KCNQ3 subunits affects the S4 and gate of KCNQ2 in heteromeric KCNQ2/3 channels.