Characterization of HyNaCs - peptide gated DEG/ENaCs from Hydra magnipapillata
Year: 2013
Type: dissertation
Abstract: The DEG/ENaC ion channel family comprises a group of phylogenetically related ion channels found in almost all multicellullar animals (metazoa). Common characteristics of DEG/ENaCs are their selectivity for Na+ and their sensitivity for the inhibitor amiloride. Besides these shared features, the individual DEG/ENaCs display a huge diversity with regard to their expression pattern, stimulus of activation and their physiological function. For example, ASICs are proton-gated channels implied in the perception of pain while ENaCs are constitutively active and facilitate sodium reabsorption across epithelia and FaNaC is the first identified ion channel directly gated by neuropeptides. To obtain a better understanding of the basic properties of this heterogeneous group of ion channels, our group has cloned novel DEG/ENaC subunits from the freshwater polyp Hydra magnipapillata. Hydra belongs to the phylum Cnidaria, which is considered as the most ancient phylum in which a nervous system has evolved. Therefore, the characterisation of DEG/ENaC channels from Hydra will help to identify principal and basic properties, which are shared by all DEG/ENaC members. So far, our group has cloned four DEG/ENaC subunits from Hydra, which were named HyNaC1-4. It was already shown that coinjection of the subunits HyNaC2 and -3 leads to expression of a functional ion channel in Xenopus oocytes that is directly gated by endogenous neuropeptides from Hydra, the HydraRFamides I and II. To date, HyNaCs and related FMRFamide-gated sodium channels (FaNaCs) are the only known peptide-gated ion channels. In this work HyNaC5, a novel DEG/ENaC subunit from Hydra, was investigated. When HyNaC5 was coexpressed together with HyNaC2 and -3, the apparent affinity for the HydraRFamides I and II was increased approximately equal 100-fold. In addition, HyNaC2/3/5 exhibited an increased selectivity for Na+ and was blocked more potently by the inhibitor amiloride than HyNaC2/3. In summary, the results confirm that the subunits HyNaC2, -3, and -5 together form a heterotrimeric, peptide-gated ion channel. Moreover, the increased current amplitudes, the increased apparent affinity for the HydraRFamides and the enhanced sensitivity for the inhibitor amiloride collectively suggest that the ion channel consisting of HyNaC2/3/5 and not HyNaC2/3 is the physiologically relevant channel in Hydra. In the second part of this thesis it was shown that the channel formed by HyNaC2/3/5 is also highly permeably for Ca2+. This finding is surprising, as all known DEG/ENaCs are Ca2+ impermeable or conduct Ca2+ only to a minor extend. Indeed, selectivity for Na+ is a common and name giving feature of DEG/ENaCs. We estimated a relative Ca2+ permeability (PCa/PNa) of approximately equal to 3.8, which places HyNaC2/3/5 into the group of highly Ca2+ conductive channels. In Xenopus oocytes, the influx of Ca2+ through HyNaCs was directly visible by secondary activation of endogenously expressed calcium activated chloride channels (CaCCs). The block of CaCCs revealed the simple, step-like and non-desensitizising current characteristic of HyNaC2/3/5. Moreover, it was shown that Ca2+ also blocks HyNaC2/3/5. The block was voltage-dependent and increased at more positive potentials. Furthermore, a conserved aspartate residue situated at the outer entrance of the ion pore that is essential for both, the high Ca2+ permeability and the Ca2+ block of HyNaC2/3/5 was identified. Substitution of this residue led to a strong reduction of Ca2+ permeability and reduced voltage dependent Ca2+ block. By an in situ hybridisation assay, the expression pattern of hynac2, -3, and -5 was localized at the base of the tentacles in Hydra. The expression overlapped with the previously reported expression of the HydraRFamides I and II. We therefore speculate that HyNaCs – possibly mediated by their permeability for Ca2+ – might play a role in the coordinated movement of the tentacles during feeding in Hydra. Indeed, we could show that application of amiloride, a universal inhibitor of DEG/ENaCs, delays the glutathione induced feeding reaction in Hydra. In addition, the identification of a novel DEG/ENaC with high permeability for Ca2+ from Hydra suggest that selectivity for Na+ was not shared by the common ancestor of all DEG/ENaCs and arose later in evolution.
Source: RWTH Publications (RWTH Aachen)
Authors: Stefan Dürrnagel, Stefan Gründer
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W599755959