Human ether-à-go-go related gene (hERG) 1 channels conduct the rapid delayed rectifier K⁺current (I_Kr) and are essential for the repolarization of the cardiac action potential. hERG1 inhibition by structurally diverse drugs may lead to life threatening arrhythmia. Putative binding determinants of hERG1 channel blockers include T623, S624 and V625 on the pore helix and residues G648, Y652 and F656, located on segment S6. We and others have previously hypothesized that additional binding determinants may be located on helix S5, which is in close contact with the S6 segments. In order to test this hypothesis, we performed a detailed investigation combining ionic current measurements with two-microelectrode voltage clamp and molecular modeling techniques. We identified a novel aromatic high affinity binding determinant for blockers located in helix S5, F557, which is equally potent as Y652. Modeling supports a direct interaction with the outer pore helix.

Inward rectifier potassium (Kir) channel activity is controlled by plasma membrane lipids. Phosphatidylinositol-4,5-bisphosphate (PIP₂) binding to a primary site is required for opening of classic inward rectifier Kir2.1 and Kir2.2 channels, but interaction of bulk anionic phospholipid (PL⁻) with a distinct second site is required for high PIP₂ sensitivity. Here we show that introduction of a lipid-partitioning tryptophan at the second site (K62W) generates high PIP₂ sensitivity, even in the absence of PL⁻. Furthermore, high-resolution x-ray crystal structures of Kir2.2[K62W], with or without added PIP₂ (2.8- and 2.0-Å resolution, respectively), reveal tight tethering of the C-terminal domain (CTD) to the transmembrane domain (TMD) in each condition. Our results suggest a refined model for phospholipid gating in which PL⁻ binding at the second site pulls the CTD toward the membrane, inducing the formation of the high-affinity primary PIP₂ site and explaining the positive allostery between PL⁻ binding and PIP₂ sensitivity.

Tuned calcium entry through voltage-gated calcium channels is a key requirement for many cellular functions. This is ensured by channel gates which open during membrane depolarizations and seal the pore at rest. The gating process is determined by distinct sub-processes: movement of voltage-sensing domains (charged S4 segments) as well as opening and closure of S6 gates. Neutralization of S4 charges revealed that pore opening of CaV1.2 is triggered by a “gate releasing” movement of all four S4 segments with activation of IS4 (and IIIS4) being a rate-limiting stage. Segment IS4 additionally plays a crucial role in channel inactivation. Remarkably, S4 segments carrying only a single charged residue efficiently participate in gating. However, the complete set of S4 charges is required for stabilization of the open state. Voltage clamp fluorometry, the cryo-EM structure of a mammalian calcium channel, biophysical and pharmacological studies, and mathematical simulations have all contributed to a novel interpretation of the role of voltage sensors in channel opening, closure, and inactivation. We illustrate the role of the different methodologies in gating studies and discuss the key molecular events leading CaV channels to open and to close.

Inwardly rectifying potassium (Kir) channels play a key role in controlling membrane potentials in excitable and unexcitable cells, thereby regulating a plethora of physiological processes. G-protein–gated Kir channels control heart rate and neuronal excitability via small hyperpolarizing outward K⁺ currents near the resting membrane potential. Despite recent breakthroughs in x-ray crystallography and cryo-EM, the gating and conduction mechanisms of these channels are poorly understood. MD simulations have provided unprecedented details concerning the gating and conduction mechanisms of voltage-gated K⁺ and Na⁺ channels. Here, we use multi-microsecond–timescale MD simulations based on the crystal structures of GIRK2 (Kir3.2) bound to phosphatidylinositol-4,5-bisphosphate to provide detailed insights into the channel’s gating dynamics, including insights into the behavior of the G-loop gate. The simulations also elucidate the elementary steps that underlie the movement of K⁺ ions through an inward-rectifier K⁺ channel under an applied electric field. Our simulations suggest that K⁺ permeation might occur via direct knock-on, similar to the mechanism recently shown for K_v channels.

Potassium ion conduction through open potassium channels is essential to control of membrane potentials in all cells. To elucidate the open conformation and hence the mechanism of K⁺ion conduction in the classic inward rectifier Kir2.2, we introduced a negative charge (G178D) at the crossing point of the inner helix bundle, the location of ligand-dependent gating. This “forced open” mutation generated channels that were active even in the complete absence of phosphatidylinositol-4,5-bisphosphate (PIP₂), an otherwise essential ligand for Kir channel opening. Crystal structures were obtained at a resolution of 3.6 Å without PIP₂bound, or 2.8 Å in complex with PIP₂. The latter revealed a slight widening at the helix bundle crossing (HBC) through backbone movement. MD simulations showed that subsequent spontaneous wetting of the pore through the HBC gate region allowed K⁺ion movement across the HBC and conduction through the channel. Further simulations reveal atomistic details of the opening process and highlight the role of pore-lining acidic residues in K⁺conduction through Kir2 channels.