Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels comprise a small subfamily of proteins within the superfamily of pore-loop cation channels. In mammals, the HCN channel family comprises four members (HCN1-4) that are expressed in heart and nervous system. The current produced by HCN channels has been known as Ih (or If or Iq). Ih has also been designated as pacemaker current, because it plays a key role in controlling rhythmic activity of cardiac pacemaker cells and spontaneously firing neurons. Extensive studies over the last decade have provided convincing evidence that Ih is also involved in a number of basic physiological processes that are not directly associated with rhythmicity. Examples for these non-pacemaking functions of Ih are the determination of the resting membrane potential, dendritic integration, synaptic transmission, and learning. In this review we summarize recent insights into the structure, function, and cellular regulation of HCN channels. We also discuss in detail the different aspects of HCN channel physiology in the heart and nervous system. To this end, evidence on the role of individual HCN channel types arising from the analysis of HCN knockout mouse models is discussed. Finally, we provide an overview of the impact of HCN channels on the pathogenesis of several diseases and discuss recent attempts to establish HCN channels as drug targets.

Ebolavirus causes sporadic outbreaks of lethal hemorrhagic fever in humans with no currently approved therapy. Cells take up Ebolavirus by macropinocytosis, followed by trafficking through endosomal vesicles. However, few factors controlling endosomal virus movement are known. Here we find that Ebolavirus entry into host cells requires the endosomal calcium channels called two pore channels (TPCs). Disrupting TPC function by gene knockout, small interfering RNAs or small molecule inhibitors halted virus trafficking and prevented infection. Tetrandrine, the most potent small molecule we tested, inhibited infection of human macrophages, the primary target of Ebolavirus in vivo, and also showed therapeutic efficacy in mice. Therefore, TPC proteins play a key role in Ebolavirus infection and may be effective targets for antiviral therapy.

Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels belong to the superfamily of voltage-gated pore loop channels. HCN channels are unique among vertebrate voltage-gated ion channels, in that they have a reverse voltage-dependence that leads to activation upon hyperpolarization. In addition, voltage-dependent opening of these channels is directly regulated by the binding of cAMP. HCN channels are encoded by four genes (HCN1-4) and are widely expressed throughout the heart and the central nervous system. The current flowing through HCN channels, designated I(h) or I(f), plays a key role in the control of cardiac and neuronal rhythmicity ("pacemaker current"). In addition, I(h) contributes to several other neuronal processes, including determination of resting membrane potential, dendritic integration and synaptic transmission. In this review we give an overview on structure, function and regulation of HCN channels. Particular emphasis will be laid on the complex roles of these channels for neuronal function and cardiac rhythmicity.

Second messenger-induced Ca 2+ -release from intracellular stores plays a key role in a multitude of physiological processes. In addition to 1,4,5-inositol trisphosphate (IP 3 ), Ca 2+ , and cyclic ADP ribose (cADPR) that trigger Ca 2+ -release from the endoplasmatic reticulum (ER), nicotinic acid adenine dinucleotide phosphate (NAADP) has been identified as a cellular metabolite that mediates Ca 2+ -release from lysosomal stores. While NAADP-induced Ca 2+ -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 Ca 2+ -release channels. TPCN2 transcripts are widely expressed in the body and encode a lysosomal protein forming homomers. TPCN2 mediates intracellular Ca 2+ -release after activation with lownanomolar 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 Ca 2+ -release is almost completely abolished when the capacity of lysosomes for storing Ca 2+ is pharmacologically blocked. By contrast, TPCN2-specific Ca 2+ -release is unaffected by emptying ER-based Ca 2+ stores. In conclusion, these findings indicate that TPCN2 is a major component of the long-sought lysosomal NAADP-dependent Ca 2+ -release channel.

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.

Organoid models of early tissue development have been produced for the intestine, brain, kidney and other organs, but similar approaches for the heart have been lacking. Here we generate complex, highly structured, three-dimensional heart-forming organoids (HFOs) by embedding human pluripotent stem cell aggregates in Matrigel followed by directed cardiac differentiation via biphasic WNT pathway modulation with small molecules. HFOs are composed of a myocardial layer lined by endocardial-like cells and surrounded by septum-transversum-like anlagen; they further contain spatially and molecularly distinct anterior versus posterior foregut endoderm tissues and a vascular network. The architecture of HFOs closely resembles aspects of early native heart anlagen before heart tube formation, which is known to require an interplay with foregut endoderm development. We apply HFOs to study genetic defects in vitro by demonstrating that NKX2.5-knockout HFOs show a phenotype reminiscent of cardiac malformations previously observed in transgenic mice.

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 coassembly of homologous subunits to heteromeric complexes serves as an important mechanism in generating ion channel diversity. Here, we have studied heteromerization in the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel family. Using a combination of fluorescence confocal microscopy, coimmunoprecipitation, and electrophysiology we found that upon coexpression in HEK293 cells almost all dimeric combinations of HCN channel subunits give rise to the formation of stable channel complexes in the plasma membrane. We also identified HCN1/HCN2 heteromers in mouse brain indicating that heteromeric channels exist in vivo. Surprisingly, HCN2 and HCN3 did not coassemble to heteromeric channels. This finding indicates that heteromerization requires specific structural determinants that are not present in all HCN channel combinations. Using N-glycosidase F we show that native as well as recombinant HCN channels are glycosylated resulting in a 10 -20-kDa shift in the molecular weight. Tunicamycin, an inhibitor of Nlinked glycosylation, blocked surface membrane expression of HCN2. Similarly, a mutant HCN2 channel in which the putative N-glycosylation site in the loop between S5 and the pore helix was replaced by glutamine (HCN2 N380Q ) was not inserted into the plasma membrane and did not yield detectable whole-cell currents. These results indicate that N-linked glycosylation is required for cell surface trafficking of HCN channels. Cotransfection of HCN2 N380Q with HCN4, but not with HCN3, rescued cell surface expression of HCN2 N380Q . Immunoprecipitation revealed that this rescue was due to the formation of a HCN2 N380Q /HCN4 heteromeric channel. Taken together our results indicate that subunit heteromerization and glycosylation are important determinants of the formation of native HCN channels.The hyperpolarization-activated cation current I h (or I f, I q ) plays a key role in the control of important biological processes such as heart beat (1), sleep-wake cycle (2), transduction of sour taste (3), and synaptic plasticity (4). I h is encoded by the hyperpolarization-activated cyclic nucleotide-gated (HCN) 1 channel gene family (for review, see Refs. 5-7). In mammals, the HCN channel family comprises four homologous members (HCN1-4). Structurally, HCN channels belong to the superfamily of voltage-gated cation channels. Like other members of this family HCN channels are supposed to form tetramers with fundamental building blocks consisting of six hydrophobic segments (S1-S6), a positively charged S4 sensor, and an ionconducting hairpin between S5 and S6. In the cytosolic carboxyl terminus each of the four HCN channel subunits carries a cyclic nucleotide-binding site mediating modulation by cAMP. Expression of HCN1-4 cDNAs in heterologous expression systems yields currents with the hallmark properties of I h , namely activation by membrane hyperpolarization, permeation of Na ϩ and K ϩ , shift of the activation curve to more depolarized voltages by intracellular binding of cAMP, and blockage by low mil...