Novel insights on the physiological roles of the Stomatin-like protein-3 and the Calcium-activated chloride channel TMEM16A in the mouse olfactory epithelium

The possibility to perceive external stimuli from the surrounding environment to correctly interact with it has always represented a leading evolutionary drive for life as we know it today. The ancient sense of smell allows animals to locate food or mates and to escape from predators or hazardous, and it is dramatically important to control animal behaviour and survival. In most mammals, the main olfactory epithelium is the major intranasal system devoted to the discrimination of a plethora of chemical molecules called odorants. The main olfactory epithelium is located within the nasal cavity, and it is a specialized pseudostratified columnar epithelium composed of different types of cells population. The olfactory sensory neurons and the supporting cells are the most abundant cells that detect odorant and preserve tissue integrity, respectively. The mammals’ olfactory sensory neurons are small bipolar neurons with a round cell soma, a long and unbranched axon, and a dendrite that projects toward the apical epithelial portion. From the ending of each dendrite called knobs, several immotile cilia protrude in the mucus layer that covers the olfactory epithelium. In these fine structures take place the olfactory transduction that starts with the binding of an odorant to a specific odorant receptor. This binding switch on the Golf protein that in turn activates the adenylyl cyclase III. This produces an increase of ciliary cAMP that gates the CNG channels allowing the influx of sodium and calcium. The calcium eventually opens the calcium-activated chloride channel TMEM16B resulting into a chloride efflux that further contributes to depolarize the neuron. The supporting cells are tightly packed apically in the main olfactory epithelium above the layer of the olfactory sensory neurons. They have a columnar cell body from which several microvilli protrude in the mucus layer, while a thin basolateral process extends to the basal membrane. Several essential functions for preserving the tissue physiology have been ascribed to supporting cells: endocytosis, metabolism of toxicants, mucus secretion, regulation of the extracellular ionic composition, and phagocytosis of dead cells. By immunohistochemistry, our lab and others have recently reported that supporting cells of the olfactory epithelium express TMEM16A, a calcium-activated chloride channel. The TMEM16A channel is involved in the regulation of the extracellular ionic composition in many other epithelia, and it could play a similar role in the olfactory system. We firstly investigated if the TMEM16A expression is restricted to supporting cells from a specific portion of the main olfactory epithelium. Immunohistochemical results confirmed that TMEM16A is preferentially expressed in supporting cells from a portion of the epithelium close to the respiratory epithelium, although it is also expressed in supporting cells from the dorsal part of the main olfactory epithelium even if with a lower expression rate. Furthermore, I tested the functional expression of the channel performing whole-cell patch-clamp experiments in acute coronal slices of the mouse olfactory epithelium both in the transition and in the dorsal zone. I measured dose-response relations at different intracellular calcium concentrations, tested the ion selectivity of the currents, and used a specific inhibitor of the TMEM16A channel. I found out that supporting cells express calcium-sensitive chloride currents with very similar properties to those of the currents mediated by the native TMEM16A channels. Moreover, the amplitude of the TMEM16A mediated current is significantly smaller in the dorsal zone when compared to that from the transition zone, confirming the immunohistochemical experiments that revealed a higher expression of TMEM16A in supporting cells from the transition part of the epithelium. Moreover, I did not record any current activated by intracellular calcium in supporting cells from TMEM16A KO mice. Altogether, these results demonstrate that the TMEM16A channel is functionally expressed in supporting cells. It has been reported that supporting cells express P2Y purinergic receptors and that they have complex calcium signalling dynamics to a similar extent to that reported in glial cells. For these reasons, I also hypothesized that the TMEM16A channel could be involved in response to physiological stimuli in the olfactory system. Here, I showed that the external application of ATP leads to a PLC-mediated calcium release from the intracellular stores that is sufficient to trigger the activation of TMEM16A mediated currents in supporting cells. Future work will elucidate the physiological role of the TMEM16A channel in the main olfactory epithelium. To conclude, these works provided novel insights into two proteins expressed in the most abundant cell populations of the main olfactory epithelium. STOML-3 is highly expressed in the cilia of olfactory sensory neurons, and TMEM16A is highly expressed in a subpopulation of supporting cells located close to the transition zone with the respiratory epithelium. All in all, the emerging picture from these results is that both STOML-3 and TMEM16A are important candidates for future investigations aimed to elucidate new aspects of the olfactory sensory transduction and the physiology of the olfactory epithelium.