Poring over two-pore channel pore mutants.
Ontology highlight
ABSTRACT:
SUBMITTER: Penny CJ
PROVIDER: S-EPMC4876920 | biostudies-literature | 2015 Jun
REPOSITORIES: biostudies-literature
Publications
Poring over two-pore channel pore mutants.
Penny Christopher J CJ Patel Sandip S
Messenger (Los Angeles, Calif. : Print) 20150601 1
Two-pore channels are members of the voltage-gated ion channel superfamily. They localise to the endolysosomal system and are likely targets for the Ca<sup>2+</sup> mobilising messenger NAADP. In this brief review, we relate mutagenesis of the TPC pore to a recently published homology model and discuss how pore mutants are informing us of TPC function. Molecular physiology of these ubiquitous proteins is thus emerging. ...[more]
Similar Datasets
Project description:Twelve sodium-activated potassium channel (KCNT1, Slack) genetic mutants have been identified from severe early-onset epilepsy patients. The changes in biophysical properties of these mutants and the underlying mechanisms causing disease remain elusive. Here, we report that seven of the 12 mutations increase, whereas one mutation decreases, the channel's sodium sensitivity. Two of the mutants exhibit channel over-activity only when the intracellular Na(+) ([Na(+)]i) concentration is ∼80 mM. In contrast, single-channel data reveal that all 12 mutants increase the maximal open probability (Po). We conclude that these mutant channels lead to channel over-activity predominantly by increasing the ability of sodium binding to activate the channel, which is indicated by its maximal Po. The sodium sensitivity of these epilepsy causing mutants probably determines the [Na(+)]i concentration at which these mutants exert their pathological effects.
| S-EPMC4706775 | biostudies-literature
Project description:Intracellular organelles mediate complex cellular functions that often require ion transport across their membranes. Melanosomes are organelles responsible for the synthesis of the major mammalian pigment melanin. Defects in melanin synthesis result in pigmentation defects, visual deficits, and increased susceptibility to skin and eye cancers. Although genes encoding putative melanosomal ion transporters have been identified as key regulators of melanin synthesis, melanosome ion transport and its contribution to pigmentation remain poorly understood. Here we identify two-pore channel 2 (TPC2) as the first reported melanosomal cation conductance by directly patch-clamping skin and eye melanosomes. TPC2 has been implicated in human pigmentation and melanoma, but the molecular mechanism mediating this function was entirely unknown. We demonstrate that the vesicular signaling lipid phosphatidylinositol bisphosphate PI(3,5)P2 modulates TPC2 activity to control melanosomal membrane potential, pH, and regulate pigmentation.
| S-EPMC4882593 | biostudies-literature
Project description:Lysosomal calcium (Ca(2+)) release mediated by NAADP triggers signalling cascades that regulate many cellular processes. The identification of two-pore channel 2 (TPC2) as the NAADP receptor advances our understanding of lysosomal Ca(2+) signalling, yet the lysosome is not amenable to traditional patch-clamp electrophysiology. Previous attempts to record TPC2 single-channel activity put TPC2 outside its native environment, which not reflect TPC2's true physiological properties. To test the feasibility of using nuclear membrane electrophysiology for TPC2 channel characterization, we constructed a stable human TPC2-expressing DT40TKO cell line that lacks endogenous InsP3R and RyR (DT40TKO-hTPC2). Immunostaining revealed hTPC2 expression on the ER and nuclear envelope. Intracellular dialysis of NAADP into Fura-2-loaded DT40TKO-hTPC2 cells elicited cytosolic Ca(2+) transients, suggesting that hTPC2 was functionally active. Using nuclear membrane electrophysiology, we detected a ~220 pS single-channel current activated by NAADP with K(+) as the permeant ion. The detected single-channel recordings displayed a linear current-voltage relationship, were sensitive to Ned-19 inhibition, were biphasically regulated by NAADP concentration, and regulated by PKA phosphorylation. In summary, we developed a cell model for the characterization of the TPC2 channel and the nuclear membrane patch-clamp technique provided an alternative approach to rigorously investigate the electrophysiological properties of TPC2 with minimal manipulation.
| S-EPMC4738322 | biostudies-literature
Project description:Two-pore channels, or two-pore Na+ channels (TPCs), contain two homologous domains, each containing a functional unit typical of voltage-dependent cation channels. Each domain is considered to be responsible for either phosphoinositide (PI) binding or voltage sensing. Among the three members of the TPC family, TPC1 and TPC2 are activated by PI(3,5)P2, while TPC3 has been thought not to be affected by any PIs. Here, we report that TPC3 is sensitive to PI(3,4)P2 and PI(3,5)P2, but not to PI(4,5)P2, and that the extremely slow increase in TPC3 currents induced by depolarization in Xenopus oocytes is due to the production of PI(3,4)P2 Similarly to TPC1, the cluster of basic amino acid residues in domain I is critical for PI sensitivity, but with a slight variation that may allow TPC3 to be sensitive to both PI(3,4)P2 and PI(3,5)P2 We also found that TPC3 has a unique PI-dependent modulation mechanism of voltage dependence, which is achieved by a specific bridging interaction between domain I and domain II. Taken together, these findings show that TPC3 is a unique member of the TPC family that senses PIs and displays a strong coupling between PI binding and voltage-dependent gating.
| S-EPMC6683669 | biostudies-literature
Project description:TASK2 is a member of the two-pore domain K(+) channel family that plays a role in acid-base homeostasis; TASK2 knockout animals have plasma electrolyte patterns typical of the human clinical condition of renal tubular acidosis. It is expressed preferentially in epithelia, including the proximal tubules of the kidney. In common with the other TASK channels, TASK2 is sensitive to changes in extracellular pH, although the molecular mechanism of such pH sensing is not understood. We have examined the role of charged residues in the extracellular domains in pH sensing using a mutational approach. Mutant channels were expressed in CHO cells and studied by whole-cell and single-channel patch clamp. Neutralization of no single amino acid in isolation gave complete loss of pH sensitivity. However, the combined removal of five charged amino acids in the large extracellular loop linking the first transmembrane and pore domains, the M1-P1 loop, resulted in an essentially pH-insensitive channel, stabilized in the open state. Wild-type channels contain two such loops, but a concatemeric construct, comprised of one wild-type subunit and one containing the five mutations, was fully pH-sensitive, indicating that only one M1-P1 loop is required to yield a fully pH-sensitive channel, demonstrating a regulatory role of this distinctive structure in two-pore domain K(+) channels. Thus, pH sensing in TASK2 channels is conferred by the combined action of several charged residues in the large extracellular M1-P1 loop.
| S-EPMC1276079 | biostudies-literature
Project description:Two-pore channels (TPCs) are Ca2+-permeable ion channels localised to the endo-lysosomal system where they regulate trafficking of various cargoes including viruses. As a result, TPCs are emerging as important drug targets. However, their pharmacology is ill-defined. There are no approved drugs to target them. And their mechanism of ligand activation is largely unknown. Here, we identify a number of FDA-approved drugs as TPC pore blockers. Using a model of the pore of human TPC2 based on recent structures of mammalian TPCs, we virtually screened a database of ~1500 approved drugs. Because TPCs have recently emerged as novel host factors for Ebola virus entry, we reasoned that Ebola virus entry inhibitors may exert their effects through inhibition of TPCs. Cross-referencing hits from the TPC virtual screen with two recent high throughput anti-Ebola screens yielded approved drugs targeting dopamine and estrogen receptors as common hits. These compounds inhibited endogenous NAADP-evoked Ca2+ release from sea urchin egg homogenates, NAADP-mediated channel activity of TPC2 re-routed to the plasma membrane, and PI(3,5)P2-mediated channel activity of TPC2 expressed in enlarged lysosomes. Mechanistically, single channel analyses showed that the drugs reduced mean open time consistent with a direct action on the pore. Functionally, drug potency in blocking TPC2 activity correlated with inhibition of Ebola virus-like particle entry. Our results expand TPC pharmacology through the identification of approved drugs as novel blockers, support a role for TPCs in Ebola virus entry, and provide insight into the mechanisms underlying channel regulation. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
| S-EPMC7114365 | biostudies-literature
Project description:Within the Caenorhabditis elegans genome there exist at least 42 genes encoding TWK (two-P domain K(+)) channels, potassium channel subunits that contain two pore regions and four transmembrane domains. We now report the first functional characterization of a TWK channel from C. elegans. Although potassium channels have been reported to be activated by a variety of factors, TWK-18 currents increase dramatically with increases in temperature. Two mutant alleles of the twk-18 gene confer uncoordinated movement and paralysis in C. elegans. Expression of wild-type and mutant TWK-18 channels in Xenopus oocytes showed that mutant channels express much larger potassium currents than wild-type channels. Promoter-green fluorescent protein fusion experiments indicate that TWK-18 is expressed in body wall muscle. Our genetic and physiological data suggest that the movement defects observed in mutant twk-18 animals may be explained by an increased activity of the mutant TWK-18 channels.
| S-EPMC6772866 | biostudies-literature
Project description:Two-pore segment channel 2 (TPC2) is a ubiquitously expressed, lysosomally targeted ion channel that aids in terminating autophagy and is inhibited upon its association with mechanistic target of rapamycin (mTOR). It is controversial whether TPC2 mediates lysosomal Ca2+ release or selectively conducts Na+ and whether the binding of nicotinic acid adenine dinucleotide phosphate (NAADP) or phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] is required for the activity of this ion channel. We show that TPC2 is required for intracellular Ca2+ signaling in response to NAADP or to mTOR inhibition by rapamycin. In pulmonary arterial myocytes, rapamycin and NAADP evoked global Ca2+ transients that were blocked by depletion of lysosomal Ca2+ stores. Preincubation of cells with high concentrations of rapamycin resulted in desensitization and blocked NAADP-evoked Ca2+ signals. Moreover, rapamycin and NAADP did not evoke discernable Ca2+ transients in myocytes derived from Tpcn2 knockout mice, which showed normal responses to other Ca2+-mobilizing signals. In HEK293 cells stably overexpressing human TPC2, shRNA-mediated knockdown of mTOR blocked rapamycin- and NAADP-evoked Ca2+ signals. Confocal imaging of a genetically encoded Ca2+ indicator fused to TPC2 demonstrated that rapamycin-evoked Ca2+ signals localized to lysosomes and were in close proximity to TPC2. Therefore, inactivation of mTOR may activate TPC2 and consequently lysosomal Ca2+ release.
| S-EPMC6055479 | biostudies-literature
Project description:Voltage-gated ion channels endow membranes with excitability and the means to propagate action potentials that form the basis of all neuronal signaling. We determined the structure of a voltage-gated sodium channel, two-pore channel 3 (TPC3), which generates ultralong action potentials. TPC3 is distinguished by activation only at extreme membrane depolarization (V50 ∼ +75 mV), in contrast to other TPCs and NaV channels that activate between -20 and 0 mV. We present electrophysiological evidence that TPC3 voltage activation depends only on voltage sensing domain 2 (VSD2) and that each of the three gating arginines in VSD2 reduces the activation threshold. The structure presents a chemical basis for sodium selectivity, and a constricted gate suggests a closed pore consistent with extreme voltage dependence. The structure, confirmed by our electrophysiology, illustrates the configuration of a bona fide resting state voltage sensor, observed without the need for any inhibitory ligand, and independent of any chemical or mutagenic alteration.
| S-EPMC6995003 | biostudies-literature
Project description:Multi-domain voltage-gated ion channels appear to have evolved through sequential rounds of intragenic duplication from a primordial one-domain precursor. Whereas modularity within one-domain symmetrical channels is established, little is known about the roles of individual regions within more complex asymmetrical channels where the domains have undergone substantial divergence. Here we isolated and characterised both of the divergent pore regions from human TPC2, a two-domain channel that holds a key intermediate position in the evolution of voltage-gated ion channels. In HeLa cells, each pore localised to the ER and caused Ca2+ depletion, whereas an ER-targeted pore mutated at a residue that inactivates full-length TPC2 did not. Additionally, one of the pores expressed at high levels in E. coli. When purified, it formed a stable, folded tetramer. Liposomes reconstituted with the pore supported Ca2+ and Na+ uptake that was inhibited by known blockers of full-length channels. Computational modelling of the pore corroborated cationic permeability and drug interaction. Therefore, despite divergence, both pores are constitutively active in the absence of their partners and retain several properties of the wild-type pore. Such symmetrical 'pore-only' proteins derived from divergent channel domains may therefore provide tractable tools for probing the functional architecture of complex ion channels.
| S-EPMC5150636 | biostudies-literature