The transient receptor potential A1 (TRPA1) channel is the molecular target for environmental irritants and pungent chemicals, such as cinnamaldehyde and mustard oil. Extracellular Ca 2؉ is a key regulator of TRPA1 activity, both potentiating and subsequently inactivating it. In this report, we provide evidence that the effect of extracellular Ca 2؉ on these processes is indirect and can be entirely attributed to entry through TRPA1 and subsequent elevation of intracellular calcium. Specifically, we found that in a pore mutant of TRPA1, D918A, in which Ca 2؉ permeability was greatly reduced, extracellular Ca 2؉ produced neither potentiation nor inactivation. Both processes were restored by reducing intracellular Ca 2؉ buffering, which allowed intracellular Ca 2؉ levels to become elevated upon entry through D918A channels. Application of Ca 2؉ to the cytosolic face of excised patches was sufficient to produce both potentiation and inactivation of TRPA1 channels. Moreover, in whole cell recordings, elevation of intracellular Ca 2؉ by UV uncaging of 1-(4,5-dimethoxy-2-nitrophenyl)-EDTA-potentiated TRPA1 currents. In addition, our data show that potentiation and inactivation are independent processes. TRPA1 currents could be inactivated by Mg 2؉ , Ba 2؉ , and Ca 2؉ but potentiated only by Ba 2؉ and Ca 2؉ . Saturating activation by cinnamaldehyde or mustard oil occluded potentiation but did not interfere with inactivation. Last, neither process was affected by mutation of a putative intracellular Ca 2؉ -binding EF-hand motif. In conclusion, we have further clarified the mechanisms of potentiation and inactivation of TRPA1 using the D918A pore mutant, an important tool for investigating the contribution of Ca 2؉ influx through TRPA1 to nociceptive signaling. Members of the transient receptor potential (TRP)2 family of ion channels that are expressed by sensory neurons in dorsal root and trigeminal ganglia serve as sensors for temperature and noxious stimuli (1, 2). Of these, TRPA1 is one of the key sensors for pungent chemicals and environmental irritants and is essential for behavioral responses of mice to conditions that evoke inflammatory pain (3-7). Inflammatory mediators, such as bradykinin, bind to G protein-coupled receptors on nociceptors, initiating a second messenger signaling cascade that leads to Ca 2ϩ influx mediated in part by the opening of Ca 2ϩ -permeable TRPA1 channels (5,8,9). TRPA1 is also activated directly by a wide range of chemicals that cause painful sensations, including food additives, such as mustard oil (MO), cinnamaldehyde (Cin), onion, raw garlic, and thyme; environmental irritants, such as formaldehyde and acrolein (a component of automobile exhaust); and products of oxidative stress (4, 8, 10 -16). Many of these chemicals activate TRPA1 by binding covalently to reactive cysteine residues in the amino terminus of the protein (17, 18), producing a modification of the channel that can last for more than 1 h and which leads to persistent activation of TRPA1 currents (18,19). Ca 2ϩ plays at...

SUMMARYInfluenza A virus hemagglutinin (HA) changes conformation and drives membrane fusion of viral and endosomal membrane at low pH. Membrane fusion proceeds through an intermediate called hemifusion1,2. For viral fusion the hemifusion structures are not determined3. Here, influenza virus-like particles (VLP)4 carrying wild-type (WT) HA or HA hemifusion mutant G1S5 and liposome mixtures were studied at low pH by Volta phase plate (VPP) cryo-electron tomography (cET) which improves signal-to-noise ratio close to focus. We determined two distinct hemifusion structures: a hemifusion diaphragm (HD) and a novel structure termed lipidic junction. Liposomes with lipidic junctions were ruptured with membrane edges stabilized by HA. The rupture frequency and HD diameter were not affected by G1S mutation, but decreased when the cholesterol level in the liposomes was close to physiological concentrations. We propose that HA induces merger between the viral and a target membrane by one of two independent pathways: rupture-insertion pathway leading to lipidic junction and hemifusion-stalk pathway leading to fusion pore. The latter is relevant under the conditions of influenza virus infection of cells. Cholesterol concentration functions as a pathway switch due to its negative spontaneous curvature in the target bilayer as determined by continuum analysis.

Five tastes have been identified, each of which is transduced by a separate set of taste cells. Of these sour, which is associated with acid stimuli, is the least understood. Genetic ablation experiments have established that sour is detected by a subset of taste cells that express the TRP channel PKD2L1 and its partner PKD1L3, however the mechanisms by which this subset of cells detects acids remain unclear. Previous efforts to understand sour taste transduction have been hindered because sour responsive cells represent only a small fraction of cells in a taste bud, and numerous ion channels with no role in sour sensing are sensitive to acidic pH. To identify acidsensitive conductances unique to sour cells, we created genetically modified mice in which sour cells were marked by expression of YFP under the control of the PKD2L1 promoter. To measure responses to sour stimuli we developed a method in which suction electrode recording is combined with UV photolysis of NPE-caged proton. Using these methods, we report that responses to sour stimuli are not mediated by Na + permeable channels as previously thought, but instead are mediated by a proton conductance specific to PKD2L1-expressing taste cells. This conductance is sufficient to drive action potential firing in response to acid stimuli, is enriched in the apical membrane of PKD2L1-expressing taste cells and is not affected by targeted deletion of the PKD1L3 gene. We conclude that, during sour transduction, protons enter through an apical proton conductance to directly depolarize the taste cell membrane.gustatory | mouse | TRPM5 | acetic acid | HCl

The lipid phosphatidylinositol 4,5-bisphosphate (PIP2) forms nanoscopic clusters in cell plasma membranes; however, the processes determining PIP2 mobility and thus its spatial patterns are not fully understood. Using super-resolution imaging of living cells, we find that PIP2 is tightly colocalized with and modulated by overexpression of the influenza viral protein hemagglutinin (HA). Within and near clusters, HA and PIP2 follow a similar spatial dependence, which can be described by an HA-dependent potential gradient; PIP2 molecules move as if they are attracted to the center of clusters by a radial force of 0.079 5 0.002 pN in HAb2 cells. The measured clustering and dynamics of PIP2 are inconsistent with the unmodified forms of the raft, tether, and fence models. Rather, we found that the spatial PIP2 distributions and how they change in time are explained via a novel, to our knowledge, dynamic mechanism: a radial gradient of PIP2 binding sites that are themselves mobile. This model may be useful for understanding other biological membrane domains whose distributions display gradients in density while maintaining their mobility.