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.