Raycheva, M. | bioRxiv Science

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↗