A structurally precise mechanism links an epilepsy-associated KCNC2 potassium channel mutation to interneuron dysfunction.

De novo heterozygous variants in KCNC2 encoding the voltage-gated potassium (K+) channel subunit Kv3.2 are a recently described cause of developmental and epileptic encephalopathy (DEE). A de novo variant in KCNC2 c.374G > A (p.Cys125Tyr) was identified via exome sequencing in a patient with DEE. Relative to wild-type Kv3.2, Kv3.2-p.Cys125Tyr induces K+ currents exhibiting a large hyperpolarizing shift in the voltage dependence of activation, accelerated activation, and delayed deactivation consistent with a relative stabilization of the open conformation, along with increased current density. Leveraging the cryogenic electron microscopy (cryo-EM) structure of Kv3.1, molecular dynamic simulations suggest that a strong π-π stacking interaction between the variant Tyr125 and Tyr156 in the α-6 helix of the T1 domain promotes a relative stabilization of the open conformation of the channel, which underlies the observed gain of function. A multicompartment computational model of a Kv3-expressing parvalbumin-positive cerebral cortex fast-spiking γ-aminobutyric acidergic (GABAergic) interneuron (PV-IN) demonstrates how the Kv3.2-Cys125Tyr variant impairs neuronal excitability and dysregulates inhibition in cerebral cortex circuits to explain the resulting epilepsy.

An outer-pore gate modulates the pharmacology of the TMEM16A channel.

TMEM16A Ca2+-activated chloride channels are involved in multiple cellular functions and are proposed targets for diseases such as hypertension, stroke, and cystic fibrosis. This therapeutic endeavor, however, suffers from paucity of selective and potent modulators. Here, exploiting a synthetic small molecule with a biphasic effect on the TMEM16A channel, anthracene-9-carboxylic acid (A9C), we shed light on sites of the channel amenable for pharmacological intervention. Mutant channels with the intracellular gate constitutively open were generated. These channels were entirely insensitive to extracellular A9C when intracellular Ca2+ was omitted. However, when physiological Ca2+ levels were reestablished, the mutants regained sensitivity to A9C. Thus, intracellular Ca2+ is mandatory for the channel response to an extracellular modulator. The underlying mechanism is a conformational change in the outer pore that enables A9C to enter the pore to reach its binding site. The explanation of this structural rearrangement highlights a critical site for pharmacological intervention and reveals an aspect of Ca2+ gating in the TMEM16A channel.

Transition between conformational states of the TREK-1 K2P channel promoted by interaction with PIP2.

Members of the TREK family of two-pore domain potassium channels are highly sensitive to regulation by membrane lipids, including phosphatidylinositol-4,5-bisphosphate (PIP2). Previous studies have demonstrated that PIP2 increases TREK-1 channel activity; however, the mechanistic understanding of the conformational transitions induced by PIP2 remain unclear. Here, we used coarse-grained molecular dynamics and atomistic molecular dynamics simulations to model the PIP2-binding site on both the up and down state conformations of TREK-1. We also calculated the free energy of PIP2 binding relative to other anionic phospholipids in both conformational states using potential of mean force and free-energy-perturbation calculations. Our results identify state-dependent binding of PIP2 to sites involving the proximal C-terminus, and we show that PIP2 promotes a conformational transition from a down state toward an intermediate that resembles the up state. These results are consistent with functional data for PIP2 regulation, and together provide evidence for a structural mechanism of TREK-1 channel activation by phosphoinositides.

The dynamic interplay of PIP2 and ATP in the regulation of the KATP channel.

ATP-sensitive potassium (KATP ) channels couple the intracellular ATP concentration to insulin secretion. KATP channel activity is inhibited by ATP binding to the Kir6.2 tetramer and activated by phosphatidylinositol 4,5-bisphosphate (PIP2 ). Here, we use molecular dynamics simulation, electrophysiology and fluorescence spectroscopy to show that ATP and PIP2 occupy different binding pockets that share a single amino acid residue, K39. When both ligands are present, simulations suggest that K39 shows a greater preference to co-ordinate with PIP2 than with ATP. They also predict that a neonatal diabetes mutation at K39 (K39R) increases the number of hydrogen bonds formed between K39 and PIP2 , potentially accounting for the reduced ATP inhibition observed in electrophysiological experiments. Our work suggests that PIP2 and ATP interact allosterically to regulate KATP channel activity. KEY POINTS: The KATP channel is activated by the binding of phosphatidylinositol 4,5-bisphosphate (PIP2 ) lipids and inactivated by the binding of ATP. K39 has the potential to bind to both PIP2 and ATP. A mutation to this residue (K39R) results in neonatal diabetes. This study uses patch-clamp fluorometry, electrophysiology and molecular dynamics simulation. We show that PIP2 competes with ATP for K39, and this reduces channel inhibition by ATP. We show that K39R increases channel affinity to PIP2 by increasing the number of hydrogen bonds with PIP2 , when compared with the wild-type K39. This therefore decreases KATP channel inhibition by ATP.

Structural basis of human NOX5 activation.

NADPH oxidase 5 (NOX5) catalyzes the production of superoxide free radicals and regulates physiological processes from sperm motility to cardiac rhythm. Overexpression of NOX5 leads to cancers, diabetes, and cardiovascular diseases. NOX5 is activated by intracellular calcium signaling, but the underlying molecular mechanism of which - in particular, how calcium triggers electron transfer from NADPH to FAD - is still unclear. Here we capture motions of full-length human NOX5 upon calcium binding using single-particle cryogenic electron microscopy (cryo-EM). By combining biochemistry, mutagenesis analyses, and molecular dynamics (MD) simulations, we decode the molecular basis of NOX5 activation and electron transfer. We find that calcium binding to the EF-hand domain increases NADPH dynamics, permitting electron transfer between NADPH and FAD and superoxide production. Our structural findings also uncover a zinc-binding motif that is important for NOX5 stability and enzymatic activity, revealing modulation mechanisms of reactive oxygen species (ROS) production.

From Bench to Biomolecular Simulation: Phospholipid Modulation of Potassium Channels.

Potassium (K+) ion channels are crucial in numerous cellular processes as they hyperpolarise a cell through K+ conductance, returning a cell to its resting potential. K+ channel mutations result in multiple clinical complications such as arrhythmia, neonatal diabetes and migraines. Since 1995, the regulation of K+ channels by phospholipids has been heavily studied using a range of interdisciplinary methods such as cellular electrophysiology, structural biology and computational modelling. As a result, K+ channels are model proteins for the analysis of protein-lipid interactions. In this review, we will focus on the roles of lipids in the regulation of K+ channels, and how atomic-level structures, along with experimental techniques and molecular simulations, have helped guide our understanding of the importance of phospholipid interactions.

Cryo-EM Structures of CusA Reveal a Mechanism of Metal-Ion Export.

Gram-negative bacteria utilize the resistance-nodulation-cell division (RND) superfamily of efflux pumps to expel a variety of toxic compounds from the cell. The Escherichia coli CusA membrane protein, which recognizes and extrudes biocidal Cu(I) and Ag(I) ions, belongs to the heavy-metal efflux (HME) subfamily of RND efflux pumps. We here report four structures of the trimeric CusA heavy-metal efflux pump in the presence of Cu(I) using single-particle cryo-electron microscopy (cryo-EM). We discover that different CusA protomers within the trimer are able to bind Cu(I) ions simultaneously. Our structural data combined with molecular dynamics (MD) simulations allow us to propose a mechanism for ion transport where each CusA protomer functions independently within the trimer.IMPORTANCE The bacterial RND superfamily of efflux pumps mediate resistance to a variety of biocides, including Cu(I) and Ag(I) ions. Here we report four cryo-EM structures of the trimeric CusA pump in the presence of Cu(I). Combined with MD simulations, our data indicate that each CusA protomer within the trimer recognizes and extrudes Cu(I) independently.

Relative Affinities of Protein-Cholesterol Interactions from Equilibrium Molecular Dynamics Simulations.

Specific interactions of lipids with membrane proteins contribute to protein stability and function. Multiple lipid interactions surrounding a membrane protein are often identified in molecular dynamics (MD) simulations and are, increasingly, resolved in cryo-electron microscopy (cryo-EM) densities. Determining the relative importance of specific interaction sites is aided by determination of lipid binding affinities using experimental or simulation methods. Here, we develop a method for determining protein-lipid binding affinities from equilibrium coarse-grained MD simulations using binding saturation curves, designed to mimic experimental protocols. We apply this method to directly obtain affinities for cholesterol binding to multiple sites on a range of membrane proteins and compare our results with free energies obtained from density-based equilibrium methods and with potential of mean force calculations, getting good agreement with respect to the ranking of affinities for different sites. Thus, our binding saturation method provides a robust, high-throughput alternative for determining the relative consequence of individual sites seen in, e.g., cryo-EM derived membrane protein structures surrounded by an array of ancillary lipid densities.

New insights into KATP channel gene mutations and neonatal diabetes mellitus.

The ATP-sensitive potassium channel (KATP channel) couples blood levels of glucose to insulin secretion from pancreatic ß-cells. KATP channel closure triggers a cascade of events that results in insulin release. Metabolically generated changes in the intracellular concentrations of adenosine nucleotides are integral to this regulation, with ATP and ADP closing the channel and MgATP and MgADP increasing channel activity. Activating mutations in the genes encoding either of the two types of KATP channel subunit (Kir6.2 and SUR1) result in neonatal diabetes mellitus, whereas loss-of-function mutations cause hyperinsulinaemic hypoglycaemia of infancy. Sulfonylurea and glinide drugs, which bind to SUR1, close the channel through a pathway independent of ATP and are now the primary therapy for neonatal diabetes mellitus caused by mutations in the genes encoding KATP channel subunits. Insight into the molecular details of drug and nucleotide regulation of channel activity has been illuminated by cryo-electron microscopy structures that reveal the atomic-level organization of the KATP channel complex. Here we review how these structures aid our understanding of how the various mutations in the genes encoding Kir6.2 (KCNJ11) and SUR1 (ABCC8) lead to a reduction in ATP inhibition and thereby neonatal diabetes mellitus. We also provide an update on known mutations and sulfonylurea therapy in neonatal diabetes mellitus.

Evaluating inositol phospholipid interactions with inward rectifier potassium channels and characterising their role in disease.

Membrane proteins are frequently modulated by specific protein-lipid interactions. The activation of human inward rectifying potassium (hKir) channels by phosphoinositides (PI) has been well characterised. Here, we apply a coarse-grained molecular dynamics free-energy perturbation (CG-FEP) protocol to capture the energetics of binding of PI lipids to hKir channels. By using either a single- or multi-step approach, we establish a consistent value for the binding of PIP2 to hKir channels, relative to the binding of the bulk phosphatidylcholine phospholipid. Furthermore, by perturbing amino acid side chains on hKir6.2, we show that the neonatal diabetes mutation E179K increases PIP2 affinity, while the congenital hyperinsulinism mutation K67N results in a reduced affinity. We show good agreement with electrophysiological data where E179K exhibits a reduction in neomycin sensitivity, implying that PIP2 binds more tightly E179K channels. This illustrates the application of CG-FEP to compare affinities between lipid species, and for annotating amino acid residues.