Role of the proton channel OTOP1 in taste transduction

PROJECT SUMMARY The broad goal of the proposed experiments is to identify key molecules that allow mammals to detect basic taste stimuli and generate electrical responses. Taste stimuli are detected by taste buds located on the tongue and oral cavity that contain distinct cell types, including the Type III taste receptor cells (TRCs) generally believed to mediate sour taste. Molecular mechanisms of taste reception have been a subject of intense investigation over the last 30 years, with great strides made in identifying receptors for bitter, sweet, umami and sodium taste. The receptor for sour taste was more elusive. In the last two grant periods, we identified Otop1 as a candidate sour receptor, and showed that it encoded a novel proton-selective ion channel (OTOP1), expressed in Type III TRCs. Our results from the last grant period showed that in Otop1 knockout mice (Otop1-/-) responses of isolated taste receptor cells and of the gustatory nerve to acids were strongly attenuated, confirming a key role of OTOP1 in sour sensing. However, we found no change in taste behavior to acid stimuli in Otop1-/- mice. In addition to acid stimuli, Type III TRCs also respond to high concentrations of salts. Of these, ammonium chloride, a breakdown product of amino acids that is a strong taste stimulus, is known to change the pH of the cell cytosol. In the last grant period we showed, quite unexpectedly, that OTOP1 is strongly activated by ammonium chloride, through a mechanism distinct from the pH-gating observed in response to acid stimuli. We also showed that gustatory nerve responses and behavioral aversion to ammonium chloride are attenuated in Otop1-/- mice. These results lead us to hypothesize that OTOP1 functions as a general pH sensor in the gustatory system, where it mediates response to taste stimuli that change extracellular or intracellular pH. We test this hypothesis through three aims. The first aim is focused on identifying pharmacological tools that can be used to activate or inhibit OTOP1 channels using a structured-guided virtual screen and patch clamp recording of heterologously expressed OTOP1 channels. Successful completion of this aim will provide new tools to assess the contribution of OTOP1 to the detection of other taste stimuli under Aims 2 and 3, complementing experiments with Otop1-/- mice. Aims 2 and 3 examine the contribution of OTOP1 to the detection of two stimuli previously shown to be mediated by Type III TRCs, water, and carbonation, using patch clamp recording of HEK-293 cells expressing OTOP1 channels and isolated taste receptor cells (Aim 2) and gustatory nerve recording, swallowing and taste behavior (Aim 3). The successful completion of these aims will lead to a comprehensive understanding of the mechanisms by which Type III taste receptor cells detect a wide range of non-canonical taste stimuli. The identification of taste modifiers may be used to enhance palatability of food, reducing the need to add sweeteners that contribute to the development of diabetes or salts that contribute to hypertension.