Channeling Ebola virus entry into the cell: The current outbreak of Ebola virus in West Africa highlights the need for antiviral therapies. One strategy would be to block the Ebola virus's ability to enter host cells. Cells engulf Ebola virus particles, which then traffic into the cell in structures called endosomes. Sakurai et al. now report that the Ebola virus requires calcium channels called two-pore channels (TPCs) in endosomal membranes for successful entry (see the Perspective by Falzarano and Feldmann). The Ebola virus could not enter cells lacking TPCs or cells treated with a TPC inhibitor. Blocking TPCs therapeutically allowed 50% of mice to survive an ordinarily lethal Ebola virus infection. Science , this issue p. 995 ; see also p. 947

Proteins of the mammalian TRP (transient receptor potential) family form a heterogenous group of cation channels important for cellular Ca²⁺ signaling and homeostasis. Here we present the full-length sequence of TRPM3, a member of the melastatin-like subfamily (TRPM) of TRP channels. TRPM3 expression was found in human kidney and brain. HEK293 cells transiently transfected with TRPM3 showed a constitutive Ca²⁺ and Mn²⁺ entry. Whole-cell patch clamp experiments confirmed the spontaneous activity of TRPM3 and revealed permeability ratios P_Ca/P_Na of 1.57 and P_Na/P_Cs of 0.75. In cell-attached patches, spontaneous inward and outward currents were observed. At negative membrane potentials and in the presence of either 140 mm Cs⁺, 140 mm Na⁺, or 100 mm Ca²⁺ in the pipette solution, the single channel conductance levels were 133, 83, and 65 pS, respectively. The Ca²⁺ entry in TRPM3-expressing HEK293 cells increased during treatment with hypotonic extracellular solution. The reduction of extracellular osmolarity was accompanied by cell swelling, suggesting volume-regulated activity of TRPM3. From its function and expression in human kidney, we propose a role of TRPM3 in renal Ca²⁺ homeostasis.

The adult mammalian cochlea lacks regenerative capacity, which is the main reason for the permanence of hearing loss. Vestibular organs, in contrast, replace a small number of lost hair cells. The reason for this difference is unknown. In this work we show isolation of sphere-forming stem cells from the early postnatal organ of Corti, vestibular sensory epithelia, the spiral ganglion, and the stria vascularis. Organ of Corti and vestibular sensory epithelial stem cells give rise to cells that express multiple hair cell markers and express functional ion channels reminiscent of nascent hair cells. Spiral ganglion stem cells display features of neural stem cells and can give rise to neurons and glial cell types. We found that the ability for sphere formation in the mouse cochlea decreases about 100-fold during the second and third postnatal weeks; this decrease is substantially faster than the reduction of stem cells in vestibular organs, which maintain their stem cell population also at older ages. Coincidentally, the relative expression of developmental and progenitor cell markers in the cochlea decreases during the first 3 postnatal weeks, which is in sharp contrast to the vestibular system, where expression of progenitor cell markers remains constant or even increases during this period. Our findings indicate that the lack of regenerative capacity in the adult mammalian cochlea is either a result of an early postnatal loss of stem cells or diminishment of stem cell features of maturing cochlear cells.

Microglial cells are the host macrophages in the central nervous system and respond to brain injury and various neurological diseases. In this process, microglial cells undergo multiple morphological and functional changes from the resting cell toward a fully activated, phagocyting tissue macrophage. In culture, bacterial lipopolysaccharide (LPS) is a frequently used tool to induce this activation. By using calcium-imaging and patch-clamp techniques, we investigated the effect of hydrogen peroxide (H₂O₂), which is released by macrophagic cells themselves, on the intracellular calcium concentration and ion currents in cultured rat microglia. Application of 0.1–5 mM H₂O₂ for several minutes induced small responses in untreated cells but a large calcium influx and cation current in LPS-treated cells. In both untreated and LPS-treated microglia, internal perfusion of ADP-ribose (ADPR) via the patch pipette elicited large cation currents. Both stimuli, H₂O₂ and ADPR, have been reported to activate the recently cloned nonselective cation channel TRPM2. RT-PCR analysis from cultured rat glial and neuronal cells confirmed a strong expression of TRPM2 in rat microglia but not in astrocytes and cerebellar granule cells. In situ hybridizations from mouse brain showed a distribution of TRPM2, which is compatible with the expression in microglial cells. In conclusion, we describe here a novel calcium influx pathway in microglia coupled to hydrogen peroxide and ADPR and provide evidence that this pathway involves TRPM2. The increased sensitivity to H₂O₂ in LPS-stimulated cells suggests a role for TRPM2 in the calcium signaling of activated microglia.

2‐Aminoethoxydiphenyl borate (2‐APB) has been widely used to examine the roles of inositol 1,4,5‐trisphosphate receptors (IP₃Rs) and store‐operated Ca²⁺ entry and is an emerging modulator of cationic channels encoded by transient receptor potential (TRP) genes. Using Ca²⁺‐indicator dye and patch‐clamp recording we first examined the blocking effect of 2‐APB on human TRPC5 channels expressed in HEK‐293 cells. The concentration–response curve has an IC₅₀ of 20 μM and slope close to 1.0, suggesting one 2‐APB molecule binds per channel. The blocking effect is not shared by other Ca²⁺ channel blockers including methoxyverapamil, nifedipine, N‐propargylnitrendipine, or berberine. In whole‐cell and excised membrane patch recordings, 2‐APB acts from the extracellular but not intracellular face of the membrane. Block of TRPC5 by 2‐APB is less at positive voltages, suggesting that it enters the electric field or acts by modulating channel gating. 2‐APB also blocks TRPC6 and TRPM3 expressed in HEK‐293 cells, but not TRPM2. Block of TRP channels by 2‐APB may be relevant to cell proliferation because 2‐APB has a greater inhibitory effect on proliferation in cells overexpressing TRPC5. Our data indicate a specific and functionally important binding site on TRPC5 that enables block by 2‐APB. The site is only available via an extracellular route and the block shows mild voltage‐dependence. British Journal of Pharmacology (2005) 145, 405–414. doi:10.1038/sj.bjp.0706197

Endolysosomal organelles play a key role in trafficking, breakdown and receptor-mediated recycling of different macromolecules such as low-density lipoprotein (LDL)-cholesterol, epithelial growth factor (EGF) or transferrin. Here we examine the role of two-pore channel (TPC) 2, an endolysosomal cation channel, in these processes. Embryonic mouse fibroblasts and hepatocytes lacking TPC2 display a profound impairment of LDL-cholesterol and EGF/EGF-receptor trafficking. Mechanistically, both defects can be attributed to a dysfunction of the endolysosomal degradation pathway most likely on the level of late endosome to lysosome fusion. Importantly, endolysosomal acidification or lysosomal enzyme function are normal in TPC2-deficient cells. TPC2-deficient mice are highly susceptible to hepatic cholesterol overload and liver damage consistent with non-alcoholic fatty liver hepatitis. These findings indicate reduced metabolic reserve of hepatic cholesterol handling. Our results suggest that TPC2 plays a crucial role in trafficking in the endolysosomal degradation pathway and, thus, is potentially involved in the homoeostatic control of many macromolecules and cell metabolites.