Two-Pore Channels Regulate Expression of Various Receptors and Their Pathway-Related Proteins in Multiple Ways.

Two-pore channels (TPCs) constitute a small family of ion channels within membranes of intracellular acidic compartments, such as endosomes and lysosomes. They were shown to provide transient and locally restricted Ca2+-currents, likely responsible for fusion and/or fission events of endolysosomal membranes and thereby for intracellular vesicle trafficking. Genetic deletion of TPCs not only affects endocytosis, recycling, and degradation of various surface receptors but also uptake and impact of bacterial protein toxins and entry and intracellular processing of some types of viruses. This review points to important examples of these trafficking defects on one part but mainly focuses on the resulting impact of the TPC inactivation on receptor expression and receptor signaling. Thus, a detailed RNA sequencing analysis using TPC1-deficient fibroblasts uncovered a multitude of changes in the expression levels of surface receptors and their pathway-related signaling proteins. We refer to several classes of receptors such as EGF, TGF, and insulin as well as proteins involved in endocytosis.

Two-pore channels affect EGF receptor signaling by receptor trafficking and expression.

Two-pore channels (TPCs) are key components for regulating Ca2+ current from endosomes and lysosomes to the cytosol. This locally restricted Ca2+ current forms the basis for fusion and fission events between endolysosomal membranes and thereby for intracellular trafficking processes. Here, we study the function of TPC1 and TPC2 for uptake, recycling, and degradation of epidermal growth factor receptor (EGFR) using a set of TPC knockout cells. RNA sequencing analysis revealed multiple changes in the expression levels of EGFR pathway-related genes in TPC1-deficient cells. We propose that a prolonged presence of activated EGFRs in endolysosomal signaling platforms, caused by genetic inactivation of TPCs, does not only affect EGFR signaling pathways but also increases de novo synthesis of EGFR. Increased basal phospho-c-Jun levels contribute to the high EGFR expression in TPC-deficient cells. Our data point to a role of TPCs not only as important regulators for the EGFR transportation network but also for EGFR-signaling and expression.

Modulation of voltage-gated CaV2.2 Ca2+ channels by newly identified interaction partners.

Voltage-gated Ca2+ channels are typically integrated in a complex network of protein-protein-interactions, also referred to as Ca2+ channel nanodomains. Amongst the neuronal CaV2 channel family, CaV2.2 is of particular importance due to its general role for signal transmission from the periphery to the central nervous system, but also due to its significance for pain perception. Thus, CaV2.2 is an ideal target candidate to search for pharmacological inhibitors but also for novel modulatory interactors. In this review we summarize the last years findings of our intense screenings and characterization of the six CaV2.2 interaction partners, tetraspanin-13 (TSPAN-13), reticulon 1 (RTN1), member 1 of solute carrier family 38 (SLC38), prostaglandin D2 synthase (PTGDS), transmembrane protein 223 (TMEM223), and transmembrane BAX inhibitor motif 3 (Grina/TMBIM3) containing protein. Each protein shows a unique way of channel modulation as shown by extensive electrophysiological studies. Amongst the newly identified interactors, Grina/TMBIM3 is most striking due to its modulatory effect which is rather comparable to G-protein regulation.

Genetic Inactivation of Two-Pore Channel 1 Impairs Spatial Learning and Memory.

Two-pore channels (TPCs) constitute a small family of cation channels that are localized in membranes of endosomal and lysosomal compartments. Although their roles for vesicular fusion and endolysosomal trafficking have been investigated, our knowledge on their expression pattern and higher order functions in the murine brain is still limited. Western blot analysis indicated a broad expression of TPC1 in the neocortex, cerebellum and hippocampus. In order to investigate the consequences of the genetic inactivation of TPC1, we performed a set of behavioural studies with TPC1-/- mice. TPC1-/- mice were analysed for an altered motor coordination and grip-strength, exploratory drive and anxiety as well as learning and memory. TPC1-/- mice did not show any differences in their exploratory drive or in their anxiety levels. There were also no differences in spontaneous activity or motor performance. However, the Morris water maze test uncovered a deficit in spatial learning and memory in TPC1-/- mice.

Grina/TMBIM3 modulates voltage-gated CaV2.2 Ca2+ channels in a G-protein-like manner.

Grina/TMBIM3 is a poorly characterized transmembrane protein with a broad expression pattern in mammals and with a very ancient origin within eukaryotes. Although initially characterized as an NMDA-receptor associated subunit, there is increasing evidence that Grina/TMBIM3 is involved in the unfolded protein response and controls apoptosis via regulation of Ca2+ homeostasis. Here, we investigate a putative direct interaction of Grina/TMBIM3 with voltage gated Ca2+ channels, in particular with the CaV2.2 α1-subunit and describe its modulatory effects on the current through CaV2.2 N-type channels. Direct interaction was confirmed by co-immunoprecipitation studies and membrane localization was proven. Co-expression of Grina/TMBIM3 with CaV2.2 channels resulted in a significant decrease of the current amplitude and in a slowing of the kinetics of current activation. This effect was accompanied by a significant shift of the voltage dependencies of activation time constants towards more depolarized voltages. Application of a stimulus protocol including a strong depolarizing pulse relieved inhibition of current amplitude by Grina/TMBIM3. When Grina/TMBIM3 was present, inactivation by an action potential-like train of pulses was diminished. Both observations resemble mechanisms that are well-studied modulatory effects of G-protein ßγ subunits on CaV2 channels. The impact of Grina/TMBIM3 and G-protein ßγ subunits are rather comparable with respect to suppression of current amplitude and slowing of activation kinetics. Furthermore, both modulators had the same effect on current inactivation when evoked by an action potential-like train of pulses.

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.

Ablation of Ca(V)2.1 voltage-gated Ca²âº channels in mouse forebrain generates multiple cognitive impairments.

Voltage-gated Ca(V)2.1 (P/Q-type) Ca²âº channels located at the presynaptic membrane are known to control a multitude of Ca²âº-dependent cellular processes such as neurotransmitter release and synaptic plasticity. Our knowledge about their contributions to complex cognitive functions, however, is restricted by the limited adequacy of existing transgenic Ca(V)2.1 mouse models. Global Ca(V)2.1 knock-out mice lacking the α1 subunit Cacna1a gene product exhibit early postnatal lethality which makes them unsuitable to analyse the relevance of Ca(V)2.1 Ca²âº channels for complex behaviour in adult mice. Consequently we established a forebrain specific Ca(V)2.1 knock-out model by crossing mice with a floxed Cacna1a gene with mice expressing Cre-recombinase under the control of the NEX promoter. This novel mouse model enabled us to investigate the contribution of Ca(V)2.1 to complex cognitive functions, particularly learning and memory. Electrophysiological analysis allowed us to test the specificity of our conditional knock-out model and revealed an impaired synaptic transmission at hippocampal glutamatergic synapses. At the behavioural level, the forebrain-specific Ca(V)2.1 knock-out resulted in deficits in spatial learning and reference memory, reduced recognition memory, increased exploratory behaviour and a strong attenuation of circadian rhythmicity. In summary, we present a novel conditional Ca(V)2.1 knock-out model that is most suitable for analysing the in vivo functions of Ca(V)2.1 in the adult murine forebrain.