Functional ion channel characterization in the cnidarian nematostella vectensis

Foreman, Katharina Bernadette; Gründer, Stefan (Thesis advisor); Spehr, Marc (Thesis advisor)

Aachen : RWTH Aachen University (2023)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2023


The degenerin/epithelial Na+ channel family (DEG/ENaC) is a superfamily comprised of numerous ion channels with correspondingly diverse functions and expression patterns. It includes acid sensing ion channels (ASICs), which are proton-gated Na+ channels implicated in pain perception, as well as the epithelial Na+ channels (ENaCs), which stay constitutively open and are responsible for Na+ reabsorption. It also includes the FMRFamide-activated Na+ channel (FaNaC) and the more recently discovered Hydra Na+ channel 2/3/5 (HyNaC 2/3/5), which both respond to the binding of peptides to conduct Na+. DEG/ENaCs share a selectivity for Na+ and a sensitivity for the commonly known inhibitor amiloride. Structure is conserved amongst the family members. They usually comprise of two hydrophobic transmembrane domains (TMDs), interconnected by a large extracellular domain. A subunit typically consists of 470-700 amino acids. While they share numerous commonalities, they are also very diverse. DEG/ENaCs assemble as homo- or heteromeric trimers. They are also involved in many different sensory modalities and expressed in a wide range of different organisms and organelles. A novel group of DEG/ENaCs was discovered through phylogenetic analysis in the model organism Nematostella vectensis, the starlet sea anemone. N.vectensis is an anthozoan, part of the cnidaria phylum, considered the most ancient phylum with representatives having an evolved nervous system. Based on this, a collaborative project was formed with the group of Professor Dr. Yehu Moran. We jointly cloned nineteen Nematostella ion channels into Xenopus laevis expression vectors, which we named NeNaC1-24 (Nematostella Na+ channel). The main objective of this work was to shed light on and characterize the function of these NeNaCs using two electrode voltage clamp electrophysiology. We considered these channels to be peptide-gated, for which we tested several peptide candidates, based on previously published work. Additionally, we introduced an amino acid mutation close to TMD2 for each NeNaC, forcing the channels to remain constitutively open. This way we characterized ion channel assembly in a controlled non-physiological manner. We found that all functional ion channels we discovered assemble as homomeric channels. While we did not find any peptide gated ion channels, we determined that NeNaC2 is a proton-gated ion channel, with biphasic current kinetics. It is expressed in Nematostella cnidocytes, the so-called stinging cells the animal uses to capture its prey. This suggests a role in feeding and hunting behavior, conceivably in sensing environmental changes in its direct surroundings. Furthermore, we found NeNaC2 to be inhibited by both amiloride and diminazene, while being more sensitive to diminazene. We also assessed ion selectivity and found NeNaC2 to be moderately selective for Na+ and to be slightly permeable for Ca2+. We also sought to gain insight into the proton binding site of NeNaC2. Mutating H141 in NeNaC2 to an alanine did not abolish current completely, however we did note a reduction in current amplitude. Furthermore, we discovered NeNaC8, a constitutively open ion channel. We characterized NeNaC8 and constitutive currents by manipulating extracellular Ca2+ levels and found it to be similar to bile acid-sensitive ion channel (BASIC), in that it appears to be inhibited by extracellular physiological Ca2+ levels. Sensitivity of NeNaC8 to amiloride and diminazene was also assessed. We also characterized NeNaC14, another acid-sensitive ion channel. Generally showing a low expression level, it is also far less sensitive to protons than NeNaC2. Based on this, we infer H+ is not the physiological ligand for NeNaC14, which still remains to be found.


  • Department of Biology [160000]
  • Chemosensation Laboratory [163310]
  • [512000-2]