The two-pore channel TPC1 is required for efficient protein processing through early and recycling endosomes

Two-pore channels (TPCs) are localized in endo-lysosomal compartments and assumed to play an important role for vesicular fusion and endosomal trafficking. Recently, it has been shown that both TPC1 and 2 were required for host cell entry and pathogenicity of Ebola viruses. Here, we investigate the cellular function of TPC1 using protein toxins as model substrates for distinct endosomal processing routes. Toxin uptake and activation through early endosomes but not processing through other compartments were reduced in TPC1 knockout cells. Detailed co-localization studies with subcellular markers confirmed predominant localization of TPC1 to early and recycling endosomes. Proteomic analysis of native TPC1 channels finally identified direct interaction with a distinct set of syntaxins involved in fusion of intracellular vesicles. Together, our results demonstrate a general role of TPC1 for uptake and processing of proteins in early and recycling endosomes, likely by providing high local Ca2+ concentrations required for SNARE-mediated vesicle fusion

Agonist-mediated switching of ion selectivity in TPC2 differentially promotes lysosomal function

Ion selectivity is a defining feature of a given ion channel and is considered immutable. Here we show that ion selectivity of the lysosomal ion channel TPC2, which is hotly debated (Calcraft et al., 2009; Guo et al., 2017; Jha et al., 2014; Ruas et al., 2015; Wang et al., 2012), depends on the activating ligand. A high-throughput screen identified two structurally distinct TPC2 agonists. One of these evoked robust Ca2+-signals and non-selective cation currents, the other weaker Ca2+-signals and Na+-selective currents. These properties were mirrored by the Ca2+-mobilizing messenger, NAADP and the phosphoinositide, PI(3,5)P2, respectively. Agonist action was differentially inhibited by mutation of a single TPC2 residue and coupled to opposing changes in lysosomal pH and exocytosis. Our findings resolve conflicting reports on the permeability and gating properties of TPC2 and they establish a new paradigm whereby a single ion channel mediates distinct, functionally-relevant ionic signatures on demand

The two-pore channel TPCN2 mediates NAADP-dependent Ca2+-release from lysosomal stores

Second messenger-induced Ca2+-release from intracellular stores plays a key role in a multitude of physiological processes. In addition to 1,4,5-inositol trisphosphate (IP3), Ca2+, and cyclic ADP ribose (cADPR) that trigger Ca2+-release from the endoplasmatic reticulum (ER), nicotinic acid adenine dinucleotide phosphate (NAADP) has been identified as a cellular metabolite that mediates Ca2+-release from lysosomal stores. While NAADP-induced Ca2+-release has been found in many tissues and cell types, the molecular identity of the channel(s) conferring this release remained elusive so far. Here, we show that TPCN2, a novel member of the two-pore cation channel family, displays the basic properties of native NAADP-dependent Ca2+-release channels. TPCN2 transcripts are widely expressed in the body and encode a lysosomal protein forming homomers. TPCN2 mediates intracellular Ca2+-release after activation with low-nanomolar concentrations of NAADP while it is desensitized by micromolar concentrations of this second messenger and is insensitive to the NAADP analog nicotinamide adenine dinucleotide phosphate (NADP). Furthermore, TPCN2-mediated Ca2+-release is almost completely abolished when the capacity of lysosomes for storing Ca2+ is pharmacologically blocked. By contrast, TPCN2-specific Ca2+-release is unaffected by emptying ER-based Ca2+ stores. In conclusion, these findings indicate that TPCN2 is a major component of the long-sought lysosomal NAADP-dependent Ca2+-release channel

The cGMP-Dependent Protein Kinase II Is an Inhibitory Modulator of the Hyperpolarization-Activated HCN2 Channel

Opening of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is facilitated by direct binding of cyclic nucleotides to a cyclic nucleotide-binding domain (CNBD) in the C-terminus. Here, we show for the first time that in the HCN2 channel cGMP can also exert an inhibitory effect on gating via cGMP-dependent protein kinase II (cGKII)-mediated phosphorylation. Using coimmunoprecipitation and immunohistochemistry we demonstrate that cGKII and HCN2 interact and colocalize with each other upon heterologous expression as well as in native mouse brain. We identify the proximal C-terminus of HCN2 as binding region of cGKII and show that cGKII phosphorylates HCN2 at a specific serine residue (S641) in the C-terminal end of the CNBD. The cGKII shifts the voltage-dependence of HCN2 activation to 2–5 mV more negative voltages and, hence, counteracts the stimulatory effect of cGMP on gating. The inhibitory cGMP effect can be either abolished by mutation of the phosphorylation site in HCN2 or by impairing the catalytic domain of cGKII. By contrast, the inhibitory effect is preserved in a HCN2 mutant carrying a CNBD deficient for cGMP binding. Our data suggest that bidirectional regulation of HCN2 gating by cGMP contributes to cellular fine-tuning of HCN channel activity

Measurement of the top quark mass using the matrix element technique in dilepton final states

We present a measurement of the top quark mass in pp¯ collisions at a center-of-mass energy of 1.96 TeV at the Fermilab Tevatron collider. The data were collected by the D0 experiment corresponding to an integrated luminosity of 9.7  fb−1. The matrix element technique is applied to tt¯ events in the final state containing leptons (electrons or muons) with high transverse momenta and at least two jets. The calibration of the jet energy scale determined in the lepton+jets final state of tt¯ decays is applied to jet energies. This correction provides a substantial reduction in systematic uncertainties. We obtain a top quark mass of mt=173.93±1.84  GeV

HCN4 subunit expression in fast-spiking interneurons of the rat spinal cord and hippocampus

Hyperpolarisation-activated (Ih) currents are considered important for dendritic integration, synaptic transmission, setting membrane potential and rhythmic action potential (AP) discharge in neurons of the central nervous system. Hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels underlie these currents and are composed of homo- and hetero-tetramers of HCN channel subunits (HCN1–4), which confer distinct biophysical properties on the channel. Despite understanding the structure–function relationships of HCN channels with different subunit stoichiometry, our knowledge of their expression in defined neuronal populations remains limited. Recently, we have shownthat HCN subunit expression is a feature of a specific population of dorsal horn interneurons that exhibit high-frequency AP discharge. Here we expand on this observation and use neuroanatomical markers to first identify well-characterised neuronal populations in the lumbar spinal cord and hippocampus and subsequently determine whether HCN4 expression correlates with high-frequency AP discharge in these populations. In the spinal cord, HCN4 is expressed in several putative inhibitory interneuron populations including parvalbumin (PV)-expressing islet cells (84.1%; SD: ±2.87), in addition to all putative Renshaw cells and Ia inhibitory interneurons. Similarly, virtually all PVexpressing cells in the hippocampal CA1 subfield (93.5%;±3.40) and the dentate gyrus (90.9%; ±6.38) also express HCN4. This HCN4 expression profile in inhibitory interneurons mirrors both the prevalence of Ih sub-threshold currents and high-frequency AP discharge. Our findings indicate that HCN4 subunits are expressed in several populations of spinal and hippocampal interneurons, which are known to express both Ih sub-threshold currents and exhibit high-frequency AP discharge. As HCN channel function plays a critical role in pain perception, learning and memory,and sleep as well as the pathogenesis of several neurologicaldiseases, these findings provide important insights into the identity and neurochemical status of cells that could underlie such conditions

Population diversity and function of hyperpolarization-activated current in olfactory bulb mitral cells

Although neurons are known to exhibit a broad array of intrinsic properties that impact critically on the computations they perform, very few studies have quantified such biophysical diversity and its functional consequences. Using in vivo and in vitro whole-cell recordings here we show that mitral cells are extremely heterogeneous in their expression of a rebound depolarization (sag) at hyperpolarized potentials that is mediated by a ZD7288-sensitive current with properties typical of hyperpolarization-activated cyclic nucleotide gated (HCN) channels. The variability in sag expression reflects a functionally diverse population of mitral cells. For example, those cells with large amplitude sag exhibit more membrane noise, a lower rheobase and fire action potentials more regularly than cells where sag is absent. Thus, cell-to-cell variability in sag potential amplitude reflects diversity in the integrative properties of mitral cells that ensures a broad dynamic range for odor representation across these principal neurons

Modified Cav1.4 Expression in the Cacna1fnob2 Mouse Due to Alternative Splicing of an ETn Inserted in Exon 2

The Cacna1fnob2 mouse is reported to be a naturally occurring null mutation for the Cav1.4 calcium channel gene and the phenotype of this mouse is not identical to that of the targeted gene knockout model. We found two mRNA species in the Cacna1fnob2 mouse: approximately 90% of the mRNA represents a transcript with an in-frame stop codon within exon 2 of CACNA1F, while approximately 10% of the mRNA represents a transcript in which alternative splicing within the ETn element has removed the stop codon. This latter mRNA codes for full length Cav1.4 protein, detectable by Western blot analysis that is predicted to differ from wild type Cav1.4 protein in a region of approximately 22 amino acids in the N-terminal portion of the protein. Electrophysiological analysis with either mouse Cav1.4wt or Cav1.4nob2 cDNA revealed that the alternatively spliced protein does not differ from wild type with respect to activation and inactivation characteristics; however, while the wild type N-terminus interacted with filamin proteins in a biochemical pull-down experiment, the alternatively spliced N-terminus did not. The Cacna1fnob2 mouse electroretinogram displayed reduced b-wave and oscillatory potential amplitudes, and the retina was morphologically disorganized, with substantial reduction in thickness of the outer plexiform layer and sprouting of bipolar cell dendrites ectopically into the outer nuclear layer. Nevertheless, the spatial contrast sensitivity (optokinetic response) of Cacna1fnob2 mice was generally similar to that of wild type mice. These results suggest the Cacna1fnob2 mouse is not a CACNA1F knockout model. Rather, alternative splicing within the ETn element can lead to full-length Cav1.4 protein, albeit at reduced levels, and the functional Cav1.4 mutant may be incapable of interacting with cytoskeletal filamin proteins. These changes, do not alter the ability of the Cacna1fnob2 mouse to detect and follow moving sine-wave gratings compared to their wild type counterparts

Hyperpolarization-Activated Current (Ih) in Ganglion-Cell Photoreceptors

Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin and serve as the primary retinal drivers of non-image-forming visual functions such as circadian photoentrainment, the pupillary light reflex, and suppression of melatonin production in the pineal. Past electrophysiological studies of these cells have focused on their intrinsic photosensitivity and synaptic inputs. Much less is known about their voltage-gated channels and how these might shape their output to non-image-forming visual centers. Here, we show that rat ipRGCs retrolabeled from the suprachiasmatic nucleus (SCN) express a hyperpolarization-activated inwardly-rectifying current (Ih). This current is blocked by the known Ih blockers ZD7288 and extracellular cesium. As in other systems, including other retinal ganglion cells, Ih in ipRGCs is characterized by slow kinetics and a slightly greater permeability for K+ than for Na+. Unlike in other systems, however, Ih in ipRGCs apparently does not actively contribute to resting membrane potential. We also explore non-specific effects of the common Ih blocker ZD7288 on rebound depolarization and evoked spiking and discuss possible functional roles of Ih in non-image-forming vision. This study is the first to characterize Ih in a well-defined population of retinal ganglion cells, namely SCN-projecting ipRGCs