Conotoxin κM-RIIIJ, a tool targeting asymmetric heteromeric Kv1 channels.

The vast complexity of native heteromeric K+ channels is largely unexplored. Defining the composition and subunit arrangement of individual subunits in native heteromeric K+ channels and establishing their physiological roles is experimentally challenging. Here we systematically explored this "zone of ignorance" in molecular neuroscience. Venom components, such as peptide toxins, appear to have evolved to modulate physiologically relevant targets by discriminating among closely related native ion channel complexes. We provide proof-of-principle for this assertion by demonstrating that κM-conotoxin RIIIJ (κM-RIIIJ) from Conus radiatus precisely targets "asymmetric" Kv channels composed of three Kv1.2 subunits and one Kv1.1 or Kv1.6 subunit with 100-fold higher apparent affinity compared with homomeric Kv1.2 channels. Our study shows that dorsal root ganglion (DRG) neurons contain at least two major functional Kv1.2 channel complexes: a heteromer, for which κM-RIIIJ has high affinity, and a putative Kv1.2 homomer, toward which κM-RIIIJ is less potent. This conclusion was reached by (i) covalent linkage of members of the mammalian Shaker-related Kv1 family to Kv1.2 and systematic assessment of the potency of κM-RIIIJ block of heteromeric K+ channel-mediated currents in heterologous expression systems; (ii) molecular dynamics simulations of asymmetric Kv1 channels providing insights into the molecular basis of κM-RIIIJ selectivity and potency toward its targets; and (iii) evaluation of calcium responses of a defined population of DRG neurons to κM-RIIIJ. Our study demonstrates that bioactive molecules present in venoms provide essential pharmacological tools that systematically target specific heteromeric Kv channel complexes that operate in native tissues.

Insights into the function of ion channels by computational electrophysiology simulations.

Ion channels are of universal importance for all cell types and play key roles in cellular physiology and pathology. Increased insight into their functional mechanisms is crucial to enable drug design on this important class of membrane proteins, and to enhance our understanding of some of the fundamental features of cells. This review presents the concepts behind the recently developed simulation protocol Computational Electrophysiology (CompEL), which facilitates the atomistic simulation of ion channels in action. In addition, the review provides guidelines for its application in conjunction with the molecular dynamics software package GROMACS. We first lay out the rationale for designing CompEL as a method that models the driving force for ion permeation through channels the way it is established in cells, i.e., by electrochemical ion gradients across the membrane. This is followed by an outline of its implementation and a description of key settings and parameters helpful to users wishing to set up and conduct such simulations. In recent years, key mechanistic and biophysical insights have been obtained by employing the CompEL protocol to address a wide range of questions on ion channels and permeation. We summarize these recent findings on membrane proteins, which span a spectrum from highly ion-selective, narrow channels to wide diffusion pores. Finally we discuss the future potential of CompEL in light of its limitations and strengths. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.

Direct knock-on of desolvated ions governs strict ion selectivity in K+ channels.

The seeming contradiction that K+ channels conduct K+ ions at maximal throughput rates while not permeating slightly smaller Na+ ions has perplexed scientists for decades. Although numerous models have addressed selective permeation in K+ channels, the combination of conduction efficiency and ion selectivity has not yet been linked through a unified functional model. Here, we investigate the mechanism of ion selectivity through atomistic simulations totalling more than 400 µs in length, which include over 7,000 permeation events. Together with free-energy calculations, our simulations show that both rapid permeation of K+ and ion selectivity are ultimately based on a single principle: the direct knock-on of completely desolvated ions in the channels' selectivity filter. Herein, the strong interactions between multiple 'naked' ions in the four filter binding sites give rise to a natural exclusion of any competing ions. Our results are in excellent agreement with experimental selectivity data, measured ion interaction energies and recent two-dimensional infrared spectra of filter ion configurations.

Ion permeation in K⁺ channels occurs by direct Coulomb knock-on.

Potassium channels selectively conduct K(+) ions across cellular membranes with extraordinary efficiency. Their selectivity filter exhibits four binding sites with approximately equal electron density in crystal structures with high K(+) concentrations, previously thought to reflect a superposition of alternating ion- and water-occupied states. Consequently, cotranslocation of ions with water has become a widely accepted ion conduction mechanism for potassium channels. By analyzing more than 1300 permeation events from molecular dynamics simulations at physiological voltages, we observed instead that permeation occurs via ion-ion contacts between neighboring K(+) ions. Coulomb repulsion between adjacent ions is found to be the key to high-efficiency K(+) conduction. Crystallographic data are consistent with directly neighboring K(+) ions in the selectivity filter, and our model offers an intuitive explanation for the high throughput rates of K(+) channels.

A molecular switch driving inactivation in the cardiac K+ channel HERG.

K(+) channels control transmembrane action potentials by gating open or closed in response to external stimuli. Inactivation gating, involving a conformational change at the K(+) selectivity filter, has recently been recognized as a major K(+) channel regulatory mechanism. In the K(+) channel hERG, inactivation controls the length of the human cardiac action potential. Mutations impairing hERG inactivation cause life-threatening cardiac arrhythmia, which also occur as undesired side effects of drugs. In this paper, we report atomistic molecular dynamics simulations, complemented by mutational and electrophysiological studies, which suggest that the selectivity filter adopts a collapsed conformation in the inactivated state of hERG. The selectivity filter is gated by an intricate hydrogen bond network around residues S620 and N629. Mutations of this hydrogen bond network are shown to cause inactivation deficiency in electrophysiological measurements. In addition, drug-related conformational changes around the central cavity and pore helix provide a functional mechanism for newly discovered hERG activators.