Wesseling, Matthijs et al. (2019) International Worm Meeting "Expressing human epithelial Na channel (ENaC) subunits in C. elegans to model human salt taste."

Hoorn, Ewout, Jansen, Gert, Wesseling, Matthijs

[International Worm Meeting, 2019]

Human salt taste is one of the main drivers of dietary salt intake and directly correlates with blood pressure. The average western diet contains a high amount of salt and this contributes to various pathologies including kidney diseases and cardiovascular problems. Salt sensitivity and preference vary among people, but how this is regulated molecularly is unclear. The main salt sensor in the human taste buds is the epithelial sodium channel (ENaC), a heterotrimeric channel consisting of an ?, ? and ? subunit. It is unclear how ENaC activity in the taste buds is regulated. In the kidney, the activity of ENaC is regulated by proteases. Cleavage of the ? subunit by furin and the ? subunit by furin and other 'channel activating' proteases such as prostasin, kallikrein and plasmin have been shown to increase the open-probability of the channel, whereas protease inhibitors reduce ENaC activity. It is unclear if this mechanism also applies to the regulation of ENaC in salt taste, where proteases and protease inhibitors could be supplied in saliva. We study the molecular mechanisms of salt taste in C. elegans. C. elegans is attracted to NaCl concentrations between 0.1 and 200 mM and avoids higher NaCl concentrations. Low NaCl concentrations are mainly sensed by the ASE sensory neurons and high concentrations by the ASH nociceptive neurons. Thus far, there are no indications that ENaC channels play a role. We aim to generate a humanized NaCl-taste worm model that expresses all three human ENaC subunits in the ASH cells. To achieve this, we will use a two-step approach using CRISPR/Cas9 induced homology directed repair for each subunit. First, we will introduce sra-6::gfp in one of three different loci on different chromosomes. These strains will allow us to confirm proper expression in the ASH neurons. Subsequently, we will replace GFP for an ENaC subunit. Expression of each ENaC subunit and localization within the cell will be analyzed using immunofluorescence experiments. We expect that, should the human ENaC channel be functional, the ENaC expressing animals will be less attracted or even repelled by salt. To reduce interference of the C. elegans NaCl-taste machinery, we will use che-1 mutant animals, that lack functional ASE neurons. This 'humanized' model system will subsequently be used to study the possible role that proteolytic processing of ENaC may have in salt detection.

van Vuuren, Laura et al. (2021) International Worm Meeting "Expressing human epithelial Na channel subunits in C. elegans to model human salt taste"

van Vuuren, Laura, Jansen, Gert, Hoorn, Ewout

[International Worm Meeting, 2021]

Human salt taste is one of the main drivers of dietary salt intake and directly correlates with blood pressure. Salt sensitivity and preference vary among people, but the underlying molecular mechanism of this variation is unknown. Many studies have shown that the epithelial sodium channel (ENaC) is the main salt sensor involved in salt taste in rodents and humans. In the kidney, the activity of ENaC is regulated by proteases. Recently it was shown that the salivary proteome is different in salt sensitive and salt insensitive tasters. We hypothesize that salivary proteases regulate ENaC open-probability and salt taste. We use C. elegans as model to study human salt taste. C. elegans is attracted to NaCl concentrations up to 200 mM and avoid higher NaCl concentrations. Low NaCl concentrations are mainly sensed by the ASE neurons and high concentrations by the ASH nociceptive neurons. Thus far, there are no indications that ENaC channels play a role. We will generate a humanized NaCl-taste worm model that expresses all three human ENaC subunits in the ASH cells. We use a two-step approach using CRISPR/Cas9 induced homology directed repair for each subunit. We first introduced a 3.7 kb sra-6::gfp construct in a save harbor locus on chromosome I. This strain showed proper GFP expression in the ASH neurons. Subsequently, we introduced the SCNN1A gene, encoding the ENaC alpha subunit, after the sra-6 promoter and fused in frame with GFP. In these animals we see weak GFP expression in the cell bodies of the ASH neurons. We are currently in the process of integrating sra-6::mScarlet on chromosome II, where we will subsequently introduce the human SCNN1B gene, and sra-6::tagBFP2 on chromosome IV, for subsequent introduction of the SCNN1C gene. We expect that the ENaC expressing animals will be less attracted or even repelled by salt. To reduce interference of the C. elegans NaCl-taste machinery, we will use che-1 mutant animals, that lack functional ASE neurons. This 'humanized' model system will subsequently be used to study the possible role that proteolytic processing of ENaC may have in salt detection.