Biology subjects

Logothetis, D. E.

Publications and source records attributed to Logothetis, D. E..

6 recordsLinked to original sources

bioRxiv2026

GAT1508, a selective neuronal GIRK1/2 activator, exhibits superior antiepileptic efficacy and synergizes with cannabidiol

G protein-gated inwardly rectifying potassium (GIRK; Kir3) channels are key regulators of neuronal excitability and represent promising therapeutic targets for epilepsy. Here, we characterize GAT1508, a urea-based small-molecule activator selectively targeting neuronal GIRK1/2 heterotetramers, and compare its efficacy with previously described urea compounds ML297 and GiGA1. In a pentylenetetrazole (PTZ)-induced seizure model, GAT1508 demonstrated robust antiseizure activity, outperforming both ML297 and GiGA1 and achieving efficacy comparable to valproic acid. Electrophysiological studies using two-electrode voltage clamp in Xenopus oocytes revealed that GAT1508, unlike ML297 and GiGA1, selectively activates GIRK1/2 channels without significant activation of cardiac GIRK1/4 channels, suggesting reduced risk of cardiovascular side effects. Structural and computational analyses indicate that enhanced stabilization of GAT1508 compared to ML297 within the GIRK1/2 binding pocket promotes both ion permeation and channel gating through coordinated interactions with key residues. Furthermore, GAT1508, like the two other urea drugs, exhibited synergistic activation of GIRK channels when combined with G{beta}{gamma}-mediated pathways and significantly potentiated the antiseizure effects of cannabidiol (CBD) in both hippocampal slice recordings and in vivo seizure models. These findings establish GAT1508 as a potent and selective GIRK1/2 activator and support a novel combination therapeutic strategy for epilepsy based on convergent GIRK channel modulation.

pharmacology and toxicology↗

bioRxiv2026

Asymmetric assembly underlies activation mechanism of neuronal GIRK1/2 channels

G protein-gated inwardly rectifying potassium (GIRK; Kir3) channels are principal effectors of inhibitory GPCR signaling. Here we combine cryo-electron microscopy, single-molecule imaging, electrophysiology and molecular dynamics to define the architecture and activation of neuronal GIRK1/2 channels. GIRK1/2 preferentially assembles as an asymmetric heterotetramer containing one GIRK1 and three GIRK2 subunits. Three PIP2; molecules bound to GIRK2 promote partial cytoplasmic-domain docking, whereas engagement of a fourth PIP2 by GIRK1 completes docking and opens the helix bundle crossing gate. GIRK1 also remodels the electrostatic environment beneath the selectivity filter, where its E141 and D173 residues create a permeation module that enhances K+ conduction. The GIRK1-selective activator ML297 binds at the GIRK1-GIRK2 interface and controls both permeation and gating. These findings establish how asymmetric assembly couples sequential lipid-dependent gating to efficient K+ permeation and GIRK1-dependent pharmacological activation.

biophysics↗

bioRxiv2026

Stoichiometry-dependent gating and structural pharmacology of cardiac GIRK1/4 channels

Cardiac G protein-gated inwardly rectifying potassium-K+ (GIRK) channels, formed by GIRK1/4 heterotetramers or GIRK4 homotetramers, are crucial regulators of heart rate, and their dysfunction is associated with atrial fibrillation. Despite their therapeutic potential as anti-arrhythmic targets, development of selective GIRK modulators has been limited by an incomplete understanding of their structures, gating mechanisms, and pharmacology. Here, we report high-resolution structures of the GIRK4 homotetramer and of GIRK1/4 heterotetramers, revealing that they form in unexpected 1:3 and 2:2 assembly stoichiometries that we corroborate using cell-based single molecule microscopy. Electrophysiological assays and molecular dynamics simulations further show that incorporation of one or two GIRK1 subunits into GIRK4-containing channels progressively opens the helix bundle crossing (HBC) gate and increases basal channel activity. Additional structures of GIRK1/4 complexes with ML297, a small-molecule ligand selective for GIRK1-containing channels, reveal distinct GIRK1-centered intersubunit binding environments. Remarkably, ML297 inhibits GIRK1/4 channels with a 1:3 stoichiometry but potentiates channels with a 2:2 stoichiometry, establishing a direct link between subunit composition, ligand-binding environment, and pharmacological efficacy. Together, these findings provide a structural and mechanistic framework for the rational design of ligands with enhanced selectivity among cardiac and neuronal GIRK channel heterotetramers.

molecular biology↗

bioRxiv2026

Ion Channel Nano-Diagnostics for ER+ Breast Cancer

Ion channels are pore-forming transmembrane proteins that allow ions to move down an electrochemical gradient and across the channel pore and regulate many cell functions. Among them, are the G-protein-gated inwardly-rectifying K+ channels 1 (GIRK1) that are ubiquitously expressed with major functions in the brain and heart. Interestingly, significantly higher GIRK1 expression has been found in estrogen receptor positive (ER+) breast cancer patients compared to patients with HER2+ tumors or normal patients, and that was statistically correlated with shorter survival times and metastatic potential. Herein, we report the preparation of [~]4 nm GAT1508-coated poly(ethylene glycol) gold nanoparticle (PEGylated AuNP) biomarker for ER+ breast cancer cell screening through an optical microscope. A urea-based small molecule, GAT1508, with an N-methylpyrazole benzyl group on one side and a bromo-thiophene tail on the other side, has been shown to predominantly bind GIRK1 subunits and specifically activate GIRK1/2 channels. Two derivatives of GAT1508were synthesized and characterized: an ethylamine derivative (GAT1508-EA) with a chain extension from the benzyl ring, and a propylamine derivative (GAT1508-PA) with a chain extension from the pyrazole ring. Electrophysiology (TEVC and whole-cell patch-clump) experiments as well as fluorescence studies (Thallium assay) showed that only GAT1508-PA inhibited GIRK1/2-mediated K+ currents in transfected HEK293GIRK1 cells. Docking studies showed strong binding for the propylamine GAT1508 derivative, both in the amine form (GAT1508-PA) as well as in the amide form (GAT1508-PA-EG2; coupled with PEG as in the AuNPs). GAT1508-PEG-AuNPs (GAT1508-NPs) were synthesized subsequently with [~]65 wt% metal loading. UV-Vis studies revealed the presence of the conjugated ligand at 260 nm. Flow cytometry studies showed binding of Alexa 594-labeled GAT1508-NPs in ER+ MCF-7 breast cancer cells with a strong interaction, while incubation of fixed MCF-7 cells with a GAT1508-NP solution led to optical detection of ER+ breast cancer cells, without the need of fluorescent dyes and additional amplification steps. Detection was not feasible in MDA-MB-231 cells, a triple (-) breast cell line that does not express GIRK1. This is the first study, to our knowledge, that couples nanotechnology with small molecule drug design and electrophysiology to develop ion channel-tracing molecular probes for the detection/screening of ER+ breast cancer.

bioengineering↗

bioRxiv2025

Orthosteric interactions with PIP2 activate TMEM16A channels

TMEM16A channels pass Ca2+-activated Cl- currents that drive a plethora of fundamental physiological processes. TMEM16A channels are activated by a rise in intracellular Ca2+ levels but also require interactions with the signaling phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) to gate open. Although PIP2 is essential for the activity of many types of ion channels, its precise binding site and role in channel gating remain poorly understood in most cases, limiting efforts to study channel dynamics and design targeted modulators. In this study, we identify the PIP2 binding interactions that govern TMEM16A gating and permeation. Using a combination of gating molecular dynamics (GMD) simulations and electrophysiological assays, we reveal how the 4 helix of TMEM16A interacts with both the phosphate headgroups and acyl chains of PIP2 to open an electrostatic ring within the channel and stabilize the extracellular opening of the Cl- conduction pathway. These findings provide key insights into the dynamic role of PIP2 in membrane protein function and shed light on the activation mechanism of TMEM16A. This work establishes a framework for rational targeting of TMEM16A in drug development, with potential therapeutic applications in a variety of diseases.

biophysics↗

bioRxiv2021

PIP2 regulation of TRPC5 channel activation and desensitization

Transient receptor potential canonical type 5 (TRPC5) channels are expressed in the brain and kidney, and have been identified as promising therapeutic targets whose selective inhibition can protect against diseases driven by a leaky kidney filter. They are activated by elevated levels of extracellular Ca2+ or application of lanthanide ions but also by G protein (Gq/11) stimulation. Phosphatidylinositol bis-phosphate (PIP2) hydrolysis leads to protein kinase C- (PKC-) mediated phosphorylation of TRPC5 channels and desensitization of their activity. Even though PIP2 regulation of TRP channels is being widely studied, the roles of PIP2 in maintaining TRPC5 channel activity, the PIP2 involvement in channel stimulation by its hydrolysis product diacyl glycerol (DAG), or the desensitization of activity by DAG-stimulated PKC activity remain unclear. Here, we show that PIP2 controls both the PKC-mediated inhibition of TRPC5 currents as well as the activation by DAG and lanthanides and that it accomplishes this through control of gating rather than channel cell surface density. The mechanistic insights achieved by the present work promise to aid in the development of more selective and precise molecules to block TRPC5 channel activity and illuminate new therapeutic opportunities for targeted therapies for a group of diseases for which there is currently a great unmet need.

physiology↗