Biology subjects

Schmalzing, G.

Publications and source records attributed to Schmalzing, G..

5 recordsLinked to original sources

bioRxiv2026

hNav1.5α forms an antiparallel intracellular homodimer but is incorporated into the plasma membrane as a monomer

Electrophysiological studies have long treated the cardiac voltage-gated sodium channel Nav1.5 (SCN5A) as a monomeric pore-forming unit, consistent with all available cryo-EM structures, which show only monomeric architectures. In contrast, biochemical studies -- cross-linking, single-molecule pulldown, and native electrophoresis -- have reported [~]500 kDa Nav1.5 homodimers with coupled gating. To reconcile this apparent discrepancy, we combined selective labeling of the total (metabolic [35S]methionine) and plasma-membrane (membrane-impermeant IRDye 800CW) pools of hNav1.5 with high-resolution clear native electrophoresis (hrCNE) in Xenopus laevis oocytes. Total hNav1.5 migrated predominantly as a homodimer that dissociated into monomers upon denaturation, whereas surface-labeled hNav1.5 migrated exclusively as a monomer, confirming mature, Golgi-processed glycosylation by Endo H/PNGase F digestion. This monomer-dimer distribution was unaffected by co-expression with hNav{beta}1-{beta}4 subunits. Using an orthogonal SpyCatcher/SpyTag covalent tagging strategy, we captured the intracellular homodimer as an irreversible [~]500 kDa complex, and engineered TEV protease cleavage sites revealed that the two protomers associate in a previously unrecognized antiparallel, cyclic arrangement. AlphaFold2-Multimer confidently predicted a monomeric hNav1.5 fold but failed to generate a high-confidence homodimer interface, indicating that this arrangement is not strongly sequence-encoded. Together, our data resolve the electrophysiology-biochemistry discrepancy: hNav1.5 assembles as an antiparallel homodimer in intracellular compartments, likely subject to quality control, but is delivered to the plasma membrane -- the physiologically conducting compartment -- exclusively as a monomer, irrespective of {beta}-subunit association.

biochemistry↗

bioRxiv2025

Functional analysis of ligand-gated chloride channels in a cnidarian sheds light on the evolution of inhibitory signalling

{psi}-aminobutyric acid (GABA) is the predominant inhibitory transmitter in the vertebrate nervous system. Fast inhibitory signalling is mediated by type A GABA receptors (GABAARs), which form pentameric ligand-gated ion channels. While GABA is also present in plants and prokaryotes, it is unknown when it was first used for fast neuronal transmission. Cnidaria represent a sister group to all Bilateria and possess a variety of putative GABAARs, none of which has been functionally characterized. In this study, we surveyed putative inhibitory ion channel receptors from the model cnidarians Nematostella and Hydra. Phylogenetic analysis revealed a surprising complexity of these receptors. The majority formed a cnidarian-specific radiation with some receptors forming a basal clade. We functionally analyzed seven putative Nematostella GABAARs of this radiation and found that none was activated by GABA or glycine, whereas three were activated by glutamate. Using site-directed mutagenesis, we identified a lysine residue in the canonical ligand-binding pocket that is important for activation by glutamate. Our results identified a group of inhibitory ion channel receptors in Cnidaria that uses glutamate as a ligand. Moreover, they suggest that inhibitory ion channel receptors in Cnidaria massively diversified, which may have been instrumental in the evolution of complex behaviors and sensory processing by the cnidarian nervous system. This work lays the foundation for understanding the diversity and evolution of inhibitory receptors in Cnidaria and the evolution of inhibitory signalling in animal nervous systems.

neuroscience↗

bioRxiv2024

Two serial filters determine P2X7R cation selectivity, Ser342 in the central pore and lateral acidic residues at the cytoplasmic interface

The human P2X7R (hP2X7R) is a homotrimeric cell surface receptor gated by extracellular ATP4- with two transmembrane helices per subunit, TM1 and TM2. A ring of three S342 residues, one from each pore-forming TM2 helix, located halfway across the membrane bilayer, functions to close and open the gate in the apo and ATP4- bound open states, respectively. The hP2X7R is selective for small inorganic cations, but can also conduct larger organic cations such as Tris+. Here, we show by voltage-clamp electrophysiology in Xenopus laevis oocytes that mutation of S342 residues to positively charged lysines decreases the selectivity for Na+ over Tris+, but maintains cation selectivity. Deep in the membrane, laterally below the S342 ring are nine acidic residues arranged as an isosceles triangle consisting of residues E14, D352, and D356 on each side, which do not move significantly during gating. When the E14K mutation is combined with lysine substitutions of D352 and/or D356, cation selectivity is lost and permeation of the small anion Cl- is allowed. Lysine substitutions of S342 together with D352 or E14 plus D356 in the acidic triangle convert the hP2X7R mutant to a fully Cl--selective ATP4--gated receptor. We conclude that the ion selectivity of wild-type hP2X7R is determined by two sequential filters in one single pathway: (1) a primary size filter, S342, in the membrane center and (2) three cation filters lateral to the channel axis, one per subunit interface, consisting of a total of nine acidic residues at the cytoplasmic interface. SignificancePore size and electrostatic interactions are key to the permeation selectivity of ion channels. Previous cysteine scanning mutagenesis identified a tri-serine-342 ring located halfway across the membrane as the gate and selectivity filter of the P2X7 receptor channel, accessible from the inside to cationic but not anionic reagents. Consistent with a downstream cation filter, we could now switch P2X7R from cation to anion selectivity by lysine substitution of acidic residues at the cytoplasmic interface. Our data show that two sequential selectivity filters control the cation selectivity of the P2X7R channel, a dynamic tri-serine-342 size filter and three conformationally static cation filters of three acidic residues each. We propose that the ion selectivity of P2X receptors involves the mechanism described here.

biophysics↗

bioRxiv2022

Diclofenac and other Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) are Competitive Antagonists of the human P2X3 Receptor

The P2X3 receptor (P2X3R), an ATP-gated non-selective cation channel of the P2X receptor family, is expressed in sensory neurons and involved in nociception. P2X3R inhibition was shown to reduce chronic and neuropathic pain. In a previous screening of 2000 approved drugs, natural products and bioactive substances, various non-steroidal anti-inflammatory drugs (NSAIDs) were found to inhibit P2X3R-mediated currents. To investigate whether the inhibition of P2X receptors contributes to the analgesic effect of NSAIDs, we characterized the potency and selectivity of various NSAIDs at P2X3R and other P2XR subtypes using two-electrode voltage clamp electrophysiology. We identified diclofenac as a hP2X3R and hP2X2/3R antagonist with micromolar potency (with IC50 values of 138.2 {micro}M and 76.7 {micro}M, respectively). A weaker inhibition of hP2X1R, hP2X4R and hP2X7R by diclofenac was determined. Flufenamic acid (FFA) proved to inhibit hP2X3R, rP2X3R and hP2X7R (IC50 values of 221{micro}M, 264.1{micro}M and [~] 900{micro}M, respectively), questioning its widespread use as a nonselective ion channel blocker, when P2XR-mediated currents are under study. Inhibition of the hP2X3R or hP2X2/3R by diclofenac could be overcome by prolonged ATP-application or increasing concentrations of the agonist ,{beta}-meATP, respectively, indicating competition of diclofenac and the agonists. Molecular dynamics simulation showed that diclofenac largely overlaps with ATP bound to the open state of the hP2X3R. Our results strongly support a competitive antagonism through which diclofenac, by interacting with residues of the ATP-binding site, left flipper, and dorsal fin domains inhibits gating of P2X3R by conformational fixation of the left flipper and dorsal fin domains. In summary, we demonstrate the inhibition of the human P2X3 receptor by various NSAIDs. Diclofenac proved to be the most effective antagonist with a strong inhibition of hP2X3R and hP2X2/3R and a weaker inhibition of hP2X1R, hP2X4R and hP2X7R. Considering their involvement in nociception, inhibition of hP2X3R and hP2X2/3R by micromolar concentrations of diclofenac may contribute to the analgesic effect as well as the side effect of taste disturbances of diclofenac and represent an additional mode of action besides the well-known high potency COX inhibition.

pharmacology and toxicology↗

bioRxiv2019

Uncoupling sodium channel dimers rescues phenotype of pain-linked Nav1.7 mutation

The voltage-gated sodium channel Nav1.7 is essential for an adequate perception of painful stimuli. Its mutations cause various pain syndromes in human patients. The hNav1.7/A1632E mutation induces symptoms of erythromelalgia and paroxysmal extreme pain disorder (PEPD), and its main gating change is a strongly enhanced persistent current.\n\nUsing molecular simulations, we demonstrate that the disease causing persistent current of hNav1.7/A1632E is due to impaired binding of the IFM motif, thus affecting proper function of the recently proposed allosteric fast inactivation mechanism. By using native polyacrylamide gel electrophoresis (PAGE) gels, we show that hNav1.7 dimerizes. The disease-linked persistent current depends on the channels functional dimerization status: Using difopein, a 14-3-3 inhibitor known to uncouple dimerization of hNav1.5, we detect a significant decrease in hNav1.7/A1632E induced persistent currents.\n\nOur work identifies that functional uncoupling of hNav1.7/A1632E dimers rescues the pain-causing molecular phenotype by interferes with an allosteric fast inactivation mechanism, which we link for the first time to channel dimerization. Our work supports the concept of sodium channel dimerization and reveals its relevance to human pain syndromes.

neuroscience↗