Search

The sense of taste starts with activation of receptor cells in taste buds by chemical stimuli which then communicate this signal via innervating oral sensory neurons to the CNS. The cell bodies of oral sensory neurons reside in the geniculate ganglion (GG) and nodose/petrosal/jugular ganglion. The geniculate ganglion contains two main neuronal populations: BRN3A+ somatosensory neurons that innervate the pinna and PHOX2B+ sensory neurons that innervate the oral cavity. While much is known about the different taste bud cell subtypes, considerably less is known about the molecular identities of PHOX2B+ sensory subpopulations. In the GG, as many as 12 different subpopulations have been predicted from electrophysiological studies, while transcriptional identities exist for only 3 to 6. Importantly, the cell fate pathways that diversify PHOX2B+ oral sensory neurons into these subpopulations are unknown. The transcription factor EGR4 was identified as being highly expressed in GG neurons. EGR4 deletion causes GG oral sensory neurons to lose their expression of PHOX2B and other oral sensory genes and up-regulate BRN3A. This is followed by a loss of chemosensory innervation of taste buds, a loss of type II taste cells responsive to bitter, sweet, and umami stimuli, and a concomitant increase in type I glial-like taste bud cells. These deficits culminate in a loss of nerve responses to sweet and umami taste qualities. Taken together, we identify a critical role of EGR4 in cell fate specification and maintenance of subpopulations of GG neurons, which in turn maintain the appropriate sweet and umami taste receptor cells.

STIM proteins are sensors of endoplasmic reticulum (ER) luminal Ca 2+ changes and rapidly translocate into near plasma membrane (PM) junctions to activate Ca 2+ entry through the Orai family of highly Ca 2+ -selective “store-operated” channels (SOCs). Dissecting the STIM–Orai coupling process is restricted by the abstruse nature of the ER–PM junctional domain. To overcome this problem, we studied coupling by using STIM chimera and cytoplasmic C-terminal domains of STIM1 and STIM2 (S1ct and S2ct) and identifying a fundamental action of the powerful SOC modifier, 2-aminoethoxydiphenyl borate (2-APB), the mechanism of which has eluded recent scrutiny. We reveal that 2-APB induces profound, rapid, and direct interactions between S1ct or S2ct and Orai1, effecting full Ca 2+ release-activated Ca 2+ (CRAC) current activation. The short 235-505 S1ct coiled-coil region was sufficient for functional Orai1 coupling. YFP-tagged S1ct or S2ct fragments cleared from the cytosol seconds after 2-APB addition, binding avidly to Orai1-CFP with a rapid increase in FRET and transiently increasing CRAC current 200-fold above basal levels. Functional S1ct–Orai1 coupling occurred in STIM1/STIM2 −/− DT40 chicken B cells, indicating ct fragments operate independently of native STIM proteins. The 2-APB-induced S1ct–Orai1 and S2-ct–Orai1 complexes undergo rapid reorganization into discrete colocalized PM clusters, which remain stable for >100 s, well beyond CRAC activation and subsequent deactivation. In addition to defining 2-APB's action, the locked STIMct–Orai complex provides a potentially useful probe to structurally examine coupling.

The TRP family of ion channels transduce an extensive range of chemical and physical signals. TRPC6 is a receptor-activated nonselective cation channel expressed widely in vascular smooth muscle and other cell types. We report here that TRPC6 is also a sensor of mechanically and osmotically induced membrane stretch. Pressure-induced activation of TRPC6 was independent of phospholipase C. The stretch responses were blocked by the tarantula peptide, GsMTx-4, known to specifically inhibit mechanosensitive channels by modifying the external lipid-channel boundary. The GsMTx-4 peptide also blocked the activation of TRPC6 channels by either receptor-induced PLC activation or by direct application of diacylglycerol. The effects of the peptide on both stretch- and diacylglycerol-mediated TRPC6 activation indicate that the mechanical and chemical lipid sensing by the channel has a common molecular mechanism that may involve lateral-lipid tension. The mechanosensing properties of TRPC6 channels highly expressed in smooth muscle cells are likely to play a key role in regulating myogenic tone in vascular tissue.

Receptor-induced Ca 2+ signals are key to the function of all cells and involve release of Ca 2+ from endoplasmic reticulum (ER) stores, triggering Ca 2+ entry through plasma membrane (PM) “store-operated channels” (SOCs). The identity of SOCs and their coupling to store depletion remain molecular and mechanistic mysteries. The single transmembrane-spanning Ca 2+ -binding protein, STIM1, is necessary in this coupling process and is proposed to function as an ER Ca 2+ sensor to provide the trigger for SOC activation. Here we reveal that, in addition to being an ER Ca 2+ sensor, STIM1 functions within the PM to control operation of the Ca 2+ entry channel itself. Increased expression levels of STIM1 correlate with a gain in function of Ca 2+ release-activated Ca 2+ (CRAC) channel activity. Point mutation of the N-terminal EF hand transforms the CRAC channel current ( I CRAC ) into a constitutively active, Ca 2+ store-independent mode. Mutants in the EF hand and cytoplasmic C terminus of STIM1 alter operational parameters of CRAC channels, including pharmacological profile and inactivation properties. Last, Ab externally applied to the STIM1 N-terminal EF hand blocks both I CRAC in hematopoietic cells and SOC-mediated Ca 2+ entry in HEK293 cells, revealing that STIM1 has an important functional presence within the PM. The results reveal that, in addition to being an ER Ca 2+ sensor, STIM1 functions within the PM to exert control over the operation of SOCs. As a cell surface signaling protein, STIM1 represents a key pharmacological target to control fundamental Ca 2+ -regulated processes including secretion, contraction, metabolism, cell division, and apoptosis.