Several essential physiological systems express voltage-gated potassium channels within the KV7 family (comprising KV7.1–7.5), sometimes also co-assembled with auxiliary subunits in the KCNE family (comprising KCNE1–5). An ongoing challenge to KV7 drug development is creating subtype-selective compounds to limit adverse effects. Prior work has shown that the antiepileptic cannabidiol (CBD), a pan-KV7 modulator, inhibits cardiac- and epithelia-associated KV7.1 and KV7.1/KCNE1 channels, while activating neuronal KV7 subtypes (KV7.2–7.5). However, little is known about the binding sites through which CBD mediates inhibitory effects on KV7.1 and KV7.1/KCNE1, limiting insight towards the development of selective KV7 modulators. To address this knowledge gap, we used a combination of the Chai-1 artificial intelligence model (to generate CBD binding site predictions in human KV7.1 and KV7.1/KCNE1 channels), site-directed mutagenesis and electrophysiology of these channels expressed in Xenopus laevis oocytes (to corroborate CBD binding site predictions), and molecular dynamics simulations (to study the biophysical mechanisms underlying CBD binding). We found that CBD binds to two unique sites within KV7.1 and KV7.1/KCNE1. In KV7.1 alone, CBD was bound to an intrasubunit S5–S6 pore domain binding site; referred to as the S5–S6 site. In KV7.1/KCNE1, the addition of the KCNE1 subunit created a novel binding site for CBD, sandwiched between two KV7.1 subunits and one KCNE1 subunit; referred to here as the S6–S5’–E1 site. Molecular dynamics simulations showed that CBD binding to the S6–S5’–E1 KV7.1/KCNE1 site closes off the KV7.1 S5–S6 site. A sequence comparison between KV7 channels revealed key amino acid differences at both the S5–S6 and S6–S5’–E1 sites relative to neuronal KV7s. These support the notion that CBD binds differently in KV7.1 and KV7.1/KCNE1 channels in accordance with its unique inhibitory pharmacological effects on these channels compared to the activating effect in neuronal KV7s. Thus, we provide support for KV7.1 and KV7.1/KCNE1 being inhibited by CBD via distinct binding sites, which can guide future research focused on the rational development of drugs that avoid inhibitory effects on KV7.1 and KV7.1/KCNE1 channels or utilize these sites to modulate channel activity.

Background: G protein-coupled receptors (GPCRs) can signal in the absence of agonists through constitutive activity. This activity can be enhanced by mutations, resulting in receptors known as constitutively active mutants (CAMs). Such receptors are implicated in various physiological and pathophysiological conditions, and also offer significant therapeutic potential. However, the molecular basis of their constitutive activity remains unknown.

Results: To investigate how CAMs affect receptor activation, we employed enhanced sampling simulations to study the dopamine D2 receptor (D2R), a key target in central nervous system therapies. Free energy landscape analyses revealed that CAMs promote a conformational shift favoring an active state similar to the agonist-bound receptor. To then identify novel CAMs, we developed a comprehensive strategy combining structural comparison, in-silico residue scanning, and free energy calculations, validated by luminescence-complementation-based assays. Applied to D2R, this approach uncovered a new single-point CAM, D2R-I48^1.46W, which was functionally validated. Further investigation revealed that this mutation activates allosteric communication pathways primarily involving transmembrane helix 5, particularly Ser194^5.43, underscoring its role in transmitting activation signals to the intracellular domain.

Conclusions: This study elucidates how CAMs reshape the activation landscape of D2R and establishes a broadly applicable computational-experimental framework for discovering constitutively active GPCR variants. These CAMs provide valuable ligand-independent models for probing receptor activation mechanisms at structural, cellular, and physiological levels.

ADAM10 is a crucial membrane-bound metalloprotease that regulates cellular physiology by cleaving and releasing membrane-anchored proteins, including adhesion molecules and growth factor precursors, thereby modulating cell signaling, adhesion, and migration. Despite its central role, its activation mechanisms are not fully understood. Here, we model how phosphatidylserine (PS) exposure during apoptosis triggers ADAM10 activation. We confirm that PS externalization is associated with ADAM10-mediated CD43 shedding from the surface of T cells. Intriguingly, ADAM10 activation correlated with loss of ADAM10 monoclonal antibody binding, suggesting a PS-induced conformational change that alters epitope accessibility. To explore this lipid-mediated conformational change of ADAM10, we employed molecular dynamics simulations to map its conformational landscape. Our simulations revealed that in the absence of PS, ADAM10 samples predominantly closed and intermediate states. By contrast, the presence of PS destabilizes the closed conformation, thereby favoring open states. We provide a mechanistic explanation for this PS-induced conformational change, which drives ADAM10 activation and loss of mAb binding through conformational change. These findings offer new insights into the lipid-mediated regulation of ADAM10 and its conformational dynamics.

Hyperpolarization-activated and cyclic nucleotide-gated (HCN) ion channels are members of the cyclic nucleotide-binding family and are crucial for regulating cellular automaticity in many excitable cells. HCN channel activation contributes to pain perception, and propofol, a widely used anesthetic, acts as an analgesic by inhibiting the voltage-dependent activity of HCN channels. However, the molecular determinants of propofol action on HCN channels remain unknown. Here, we use a propofol-analog photoaffinity labeling reagent to identify propofol binding sites in the human HCN1 isoform. Mass spectrometry analyses combined with molecular dynamics simulations show that a binding pocket is formed by extracellularly facing residues in the S3 and S4 transmembrane segments in the resting voltage-sensor conformation. Mutations of residues within the putative binding pocket mitigate or eliminate voltage-dependent modulation of HCN1 currents by propofol. Together, these findings reveal a conformation-specific propofol binding site that underlies voltage-dependent inhibition of HCN currents and provides a framework for identifying highly specific modulators of HCN channel gating.

Cannabidiol (CBD) is an approved antiepileptic with distinct effects on the therapeutically relevant Kv7 channels, it has activating effects on Kv7.2–5 and inhibiting effects on Kv7.1 and the cardiac Kv7.1/KCNE1 channel. Despite a recent CBD bound Kv7.2 structure, the precise mechanism by which it activates Kv7.2–5 channels is unclear. Further, no structures are available for CBD bound to Kv7.1 or indeed any structures at all for Kv7.1/KCNE1, limiting insight into how CBD inhibits these channels. Understanding how CBD has differential effects on the Kv7 ion channels may be key to developing Kv7 subtype selective drugs. We first sought to understand the chemical features of CBD responsible for its effect on Kv7.4, we have recently shown that Kv7.4 is strongly activated by CBD. The binding free energies of chemicals with modified features were calculated using free energy perturbation simulations. We observed that altering the hydroxy groups on the resorcinol ring, reducing the lipophilicity of the aliphatic tail group or changing the orientation/flexibility of the limonene group reduced binding affinity compared to CBD. The modified chemicals were then synthesized and tested through electrophysiologic experiments. Mirroring the simulations, the modified chemicals had a diminished effect on Kv7.4 activation compared to CBD. Switching our focus to Kv7.1 and Kv7.1/KCNE1, we proposed CBD binding modes using docking and molecular dynamics simulations. The binding modes were tested using site directed mutagenesis studies. Our work in progress indicates that CBD binds to the S5 and S6 helices from each subunit in Kv7.1 and from a pair of adjacent subunits in Kv7.1/KCNE1. Collectively, this work furthers our understanding of CBD activity of the Kv7 channels and should aid in drug development initiatives based on the CBD chemical scaffold.

The monocarboxylate transporter (MCT) membrane protein family has 14 human members that perform key cellular functions, such as regulating metabolism. MCT8 and MCT10 have unique cargo specificity, transporting thyroid hormone and, in the case of MCT10, aromatic amino acids. Dysfunctional MCT8 causes the severe Allan-Herndon-Dudley syndrome, yet the (patho)physiology and function of MCT8 and MCT10 are not clearly understood, especially at a structural level. We present the cryoelectron microscopy (cryo-EM) structure of MCT10, displaying the classical major facilitator superfamily fold, caught in an inward-open configuration. Together with cargo docking models, the outward-open MCT10 AlphaFold model and validating functional analysis, cargo specificity and transport principles are proposed. These findings significantly enhance our understanding of the structure and function of MCTs, information that also may be valuable for the development of novel treatments against MCT-related disorders to address global challenges such as diabetes, obesity, and cancer.

KCNQ1 potassium channels are essential for physiological processes such as cardiac rhythm and intestinal chloride secretion. KCNE family subunits (KCNE1-5) associate with KCNQ1, conferring distinct properties across various tissues. KCNQ1 activation requires membrane depolarization and phosphatidylinositol 4,5-bisphosphate (PIP2) whose cellular levels are controlled by G alpha q-coupled GPCR activation. While modulation of KCNQ1's voltage-dependent activation by KCNE1/3 is well-characterized, their effects on PIP2-dependent gating of KCNQ1 via GPCR signaling remain less understood. Here we resolved structures of KCNQ1-KCNE1 and reassessed the reported KCNQ1-KCNE3 structures with and without PIP2. We revealed that KCNQ1-KCNE1/3 complexes feature two PIP2-binding sites, with KCNE1/3 contributing to a previously overlooked, uncharacterized site involving residues critical for coupling voltage sensor and pore domains. Via this site, KCNE1 and KCNE3 distinctly modulate the PIP2-dependent gating, in addition to the voltage sensitivity, of KCNQ1. Consequently, KCNE3 converts KCNQ1 into a voltage-insensitive PIP2-gated channel governed by GPCR signaling to maintain ion homeostasis in non-excitable cells. KCNE1, by significantly enhancing KCNQ1's PIP2 affinity and resistance to GPCR regulation, forms predominantly voltage-gated channels with KCNQ1 for conducting the slow-delayed rectifier current in excitable cardiac cells. Our study highlights how KCNE1/3 modulates KCNQ1 gating in different cellular contexts, providing insights into tissue-specifically targeting multi-functional channels.

Three proton-sensing G protein-coupled receptors (GPCRs)-GPR4, GPR65, and GPR68-respond to extra- cellular pH to regulate diverse physiology. How protons activate these receptors is poorly understood. We determined cryogenic-electron microscopy (cryo-EM) structures of each receptor to understand the spatial arrangement of proton-sensing residues. Using deep mutational scanning (DMS), we determined the functional importance of every residue in GPR68 activation by generating 9,500 mutants and measuring their effects on signaling and surface expression. Constant-pH molecular dynamics simulations provided insights into the conformational landscape and protonation patterns of key residues. This unbiased approach revealed that, unlike other proton-sensitive channels and receptors, no single site is critical for proton recognition. Instead, a network of titratable residues extends from the extracellular surface to the transmembrane region, converging on canonical motifs to activate proton-sensing GPCRs. Our approach integrating structure, simulations, and unbiased functional interrogation provides a framework for understanding GPCR signaling complexity.

The strict exchange of Na+ for H+ ions across cell membranes is a reaction carried out in almost every cell. Na+/H+ exchangers that perform this task are physiological homodimers, and whilst the ion transporting domain is highly conserved, their dimerization differs. The Na+/H+ exchanger NhaA from Escherichia coli has a weak dimerization interface mediated by a β-hairpin domain and with dimer retention dependent on cardiolipin. Similarly, organellar Na+/H+ exchangers NHE6, NHE7 and NHE9 also contain β-hairpin domains and recent analysis of Equus caballus NHE9 indicated PIP2 lipids could bind at the dimer interface. However, structural validation of the predicted lipid-mediated oligomerization has been lacking. Here, we report cryo-EM structures of E. coli NhaA and E. caballus NHE9 in complex with cardiolipin and phosphatidylinositol-3,5-bisphosphate PI(3,5)P2 lipids binding at their respective dimer interfaces. We further show how the endosomal specific PI(3,5)P2 lipid stabilizes the NHE9 homodimer and enhances transport activity. Indeed, we show that NHE9 is active in endosomes, but not at the plasma membrane where the PI(3,5)P2 lipid is absent. Thus, specific lipids can regulate Na+/H+ exchange activity by stabilizing dimerization in response to either cell specific cues or upon trafficking to their correct membrane location.