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.

The retinal L-type Ca 2؉ channel Cav1.4 is distinguished from all other members of the high voltage-activated (HVA) Ca 2؉ channel family by lacking Ca 2؉ -calmodulin-dependent inactivation. In synaptic terminals of photoreceptors and bipolar cells, this feature is essential to translate graded membrane depolarizations into sustained Ca 2؉ influx and tonic glutamate release. The sequences conferring Ca 2؉ -dependent inactivation (CDI) are conserved throughout the HVA calcium channel family, raising the question of how Cav1.4 manages to switch off CDI. Here, we identify an autoinhibitory domain in the distal C terminus of Cav1.4 that serves to abolish CDI. We show that this domain (ICDI, inhibitor of CDI) uncouples the molecular machinery conferring CDI from the inactivation gate by binding to the EF hand motif in the proximal C terminus. Deletion of ICDI completely restores Ca 2؉ -calmodulinmediated CDI in Cav1.4. CDI can be switched off again in the truncated Cav1.4 channel by coexpression of ICDI, indicating that ICDI works as an autonomous unit. Furthermore, we show that in the Cav1.2 L-type Ca 2؉ -channel replacement of the distal C terminus by the corresponding sequence of Cav1.4 is sufficient to block CDI. This finding suggests that autoinhibition of CDI can be introduced principally into other Ca 2؉ channel types. Our data provide a previously undescribed perspective on the regulation of HVA calcium channels by Ca 2؉ .calmodulin ͉ Cav1.4 ͉ retina ͉ Ca channels

Background: ␣-Conotoxin AuIB interacts with ␣3␤4 nAChRs and GABA B receptors, but structural determinants of these interactions are unknown. Results: Using alanine scanning mutagenesis and molecular dynamics, we identified residues crucial for AuIB⅐␣3␤4 nAChR interaction. Conclusion:We identified the key residues that mediate AuIB⅐␣3␤4 nAChR interaction. Significance: Ability to direct ␣-conotoxin binding to nAChRs or GABA B receptors will improve analgesic conopeptides.

Causes for miscarriages and congenital malformations can be genetic, environmental, or a combination of both. Genetic variants, hypoxia, malnutrition, or other factors individually may not affect embryo development, however, they may do so collectively. Biallelic loss-of-function variants in HAAO or KYNU, two genes of the nicotinamide adenine dinucleotide (NAD) synthesis pathway, are causative of congenital malformation and miscarriage in humans and mice. The variants affect normal embryonic development by disrupting the synthesis of NAD, a key factor in multiple biological processes, from its dietary precursor tryptophan, resulting in NAD deficiency. This study demonstrates that congenital malformations caused by NAD deficiency can occur independent of genetic disruption of NAD biosynthesis. C57BL/6J wild-type mice had offspring exhibiting similar malformations when their supply of the NAD precursors tryptophan and vitamin B3 in the diet was restricted during pregnancy. When the dietary undersupply was combined with a maternal heterozygous variant in Haao, which alone does not cause NAD deficiency or malformations, the incidence of embryo loss and malformations was significantly higher, suggesting a gene–environment interaction. Maternal and embryonic NAD levels were deficient. Mild hypoxia as an additional factor exacerbated the embryo outcome. Our data show that NAD deficiency as a cause of embryo loss and congenital malformation is not restricted to the rare cases of biallelic mutations in NAD synthesis pathway genes. Instead, monoallelic genetic variants and environmental factors can result in similar outcomes. The results expand our understanding of the causes of congenital malformations and the importance of sufficient NAD precursor consumption during pregnancy.

No abstract

Background: A class of analgesic ␣-conotoxins potently inhibits N-type calcium channels. Results: The activity of ␣-conotoxins Vc1.1 and RgIA was reduced following knockdown of GABA B receptor expression in sensory neurons and could be reconstituted in HEK 293 cells expressing human GABA B receptors and Ca v 2.2. Conclusion: GABA B receptors are needed for inhibition of Ca v 2.2 by Vc1.1 and RgIA. Significance: These analgesic ␣-conotoxins activate human GABA B receptors.

Birth defects occur in up to 3% of all live births and are the leading cause of infant death. Here we present five individuals from four unrelated families, individuals who share similar phenotypes with disease-causal bi-allelic variants in NADSYN1, encoding NAD synthetase 1, the final enzyme of the nicotinamide adenine dinucleotide (NAD) de novo synthesis pathway. Defects range from the isolated absence of both kidneys to multiple malformations of the vertebrae, heart, limbs, and kidney, and no affected individual survived for more than three months postnatally. NAD is an essential coenzyme for numerous cellular processes. Bi-allelic loss-of-function mutations in genes required for the de novo synthesis of NAD were previously identified in individuals with multiple congenital abnormalities affecting the heart, kidney, vertebrae, and limbs. Functional assessments of NADSYN1 missense variants, through a combination of yeast complementation and enzymatic assays, show impaired enzymatic activity and severely reduced NAD levels. Thus, NADSYN1 represents an additional gene required for NAD synthesis during embryogenesis, and NADSYN1 has bi-allelic missense variants that cause NAD deficiency-dependent malformations. Our findings expand the genotypic spectrum of congenital NAD deficiency disorders and further implicate mutation of additional genes involved in de novo NAD synthesis as potential causes of complex birth defects.