Experimental approaches to mapping the binding site of RFamides on the peptide-gated Hydra magnipapillata sodium channels (HyNaCs)

Bachmann, Michèle; Spehr, Marc (Thesis advisor); Gründer, Stefan (Thesis advisor)

Aachen : RWTH Aachen University (2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2022


Ion channels of the DEG/ENaC family are involved in a stunning variety of physiological processes. They are characterized by voltage-independent gating, permeability to Na+ and blockage by amiloride. Aside from mechanically stimulated or constitutively open channels, various compounds such as protons and small peptides mediate DEG/ENaC gating. Neuropeptides were long assumed to only serve as ligands in relatively slow metabolic transmission via G-protein coupled receptors. The discovery of FMRFamide-gated sodium channels (FaNaCs) in the snail Helix aspersa, a type of DEG/ENaC activated by FMRFamides, prompted a reorientation of this traditional view. Later, Hydra magnipapillata sodium channels (HyNaCs) were discovered to also be activated by small endogenous neuropeptides called Hydra RFamides. The binding site of RFamide I and II on HyNaCs remains unknown and was explored in this work. For this, the HyNaC2/5/3 heterotrimer in combination with RFamide II was utilized. First, the HyNaC subunits were codon-optimized for heterologous expression in mammalian cells. By changing the native HyNaC gene sequence to codons following the preference of Homo sapiens, we strongly increased total protein expression and current evoked by RFamides on HyNaCs in HEK293 T cells. The unknown stoichiometry of HyNaC subunits in a heterotrimer was elucidated using photo-crosslinking via an unnatural amino acid and revealed an obligate stoichiometry of 2-5-3 in a clockwise orientation. Protein-ligand docking was performed to explore potential binding sites for RFamide II on the heterotrimer. Candidate residues on the thumb domain of HyNaC3 were chosen based on preliminary functional results by the Gründer lab and were used for UV-induced photo-crosslinking. Utilizing various antibodies such as α-FMRFamide, α-RFamide, and purified custom α-RFamide II to detect successful crosslinking via the peptide ligand, we were unable to find a specific signal indicating covalent RFamide-HyNaC complexes. Modification of RFamide II with biotin or a flag-tag at various positions resulted in strongly decreased apparent affinity, after which an alanine scan of the peptide was performed. The scan revealed a strong decrease in binding affinity of RFamide II for every residue except one, position 5 (glycine). Photo-crosslinking using biotinylated peptides also resulted in no specific crosslinking signal on western blot. Lastly, using the purified α-RFamide II antibody in combination with an RFamide II variant that carried an N-terminal cysteine instead of pyroglutamate, which was used for coupling to a carrier during antibody synthesis, did not manage to visualize RFamide II-HyNaC complexes on a western blot. Research elucidating the location of the RFamide binding site on HyNaCs promises to increase understanding of phylogenetic and evolutionary relations between DEG/ENaC family members and help extrapolate results from simpler nervous systems to conserved features in higher animals. Future research using radioactive labelling of RFamide II could lead to insight into the putative binding site on the thumb domain of HyNaC3.