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= Two-pore channels = Two-pore channels (TPCs) are eukaryotic intracellular voltage-gated and ligand gated cation selective ion channels found to be expressed in both plant vacuoles and animal acidic organelles. These organelles consist of endosomes and lysosomes. TPCs are formed from two transmembrane non-equivalent tandem Shaker-like, pore-forming subunits, dimerized to form quasi-tetramers. TPCs regulate sodium and calcium ion conductance, intravasicular pH, and trafficking excitability. Activation of TPCs is induced by a decrease in transmembrane potential, or an increase in calcium concentrations in the cytosol. Inhibition may be caused by low pH of the lumen and low calcium concentration. Regulation by phosphorylation can open the pore in both plants and animals. The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to mediate calcium release from these acidic organelles through TPCs. These TPC2 are NAADP-gated calcium release channels where NAADP antagonists can block TPC currents. It is possible that NAADP interaction may be with and associate protein of TPCs. Some key roles of TPCs to consider include calcium dependent responses in muscle contraction, hormone secretion, fertilization, and differentiation. Disorders linked to TPCs include membrane trafficking, Parkinson’s disease, Ebola, and fatty liver.

Basic features
Two-pore channels (TPCs) are eukaryotic voltage-gated and ligand-gated cation channels. TPCs have two non-equivalent tandem pore forming subunits. These subunits come together to form a quasi-tetrameric channel. TPCs are found in organelle membranes of vacuolar and lysosomes. The integrate chain is in membrane potential, second messenger molecules, and phosphorylation, to regulate cellular ion homeostasis, intravasicular pH, and endo-lysosomal trafficking. Plant TPC1 is a nonselective channel, whereas TPC1 in humans is sodium selective. TPC2 conducts sodium ions, calcium ions and possibly hydrogen ions.

Structure and domains
At the mouth of the TPC pore, there are four residues with negative charges that can interact with ions that pass through. This site is too wide to select ions. Below the group of negative charges is the selectivity filter which is largely hydrophobic. There are two non-identical Shaker-like pore forming subunits. Subunit 1 consists of the VSD1 domain and subunit 2 consists of the VSD2 domain. The two subunit domains are separated by an EF-hand domain. Each of the two subunits are built from 12 transmembrane helices. The two central pore domains are combined together from the voltage sensing domains, VSD1 and VSD2. Both the N-terminal domain (NTD) and C-terminal domain (CTD) extend out on the cytosolic side, along with the EF-hand domain in the center that extends into the cytoplasm. The EF-hand domain extends into the cytosol and is positioned between domain 1 and 2. Here it can be activated by cytosolic calcium. Voltage sensing domain 2, VSD2 is voltage sensitive active and can be inhibited by calcium in the lumen. This is a conformation change from the activation state to the inactive state. Two rings of hydrophobic residues seal the pore cavity from the cytoplasm; this results in forming the pore gate. To regulate ion conductance, voltage sensors, selectivity filter, and the gate work together in a coordinated manner to open and close TPCs.

The VSD2 domain contains a normal voltage sensing motif, arginine residues R1, R2 and R3 and alpha helix S10, in respect to other voltage-gated ion channels structures, but this domain adopts a distinct conformation in the resting state of a voltage sensor. Luminal calcium acts as a TPC1 inhibitor, preventing ion conductance. There are two calcium binding sites for VRD2 on the luminal side. The first site does not affect the channel. Site 2, composed of residues in VSD2 and the pore domain, inhibits the channel by shifting the voltage dependence to more positive voltages.

TPCs are also phosphorylation-gated channels. Sites of phosphorylation are at the N-terminal and C-terminal domains. These terminals are positioned to provide allosteric change in order to be activated by calcium from the cytosol.

Human and plant TPCs are multi-modal for conductance. The mechanism for channel opening is likely contributed to a combination of calcium concentrations, voltage, and phosphoregulation integration, in order to govern the conduction of ions through TPCs.

Biological roles (function/dysfunction)
Two-pore channels were analyzed by using cell biological methods, endolysosomal patch clamp techniques, and a variety of other methods to study their functions. From these, it was suggested that TPCs have some power in controlling the luminal pH in endolysosomal vesicles. When TPC2 expression is decreased or knocked out, there is a resultant elevation in production of melanin and thus melanosomal pH, and when TPC2 expression is increased, there is less production of melanin.

TPCs also are involved in nutrient detection as they become active constitutively on identifying the status of the nutrients. This is done by direct communication between the TPCs and mammalian/mechanistic targets of rapamycin (mTORs), which are associated with detecting levels of oxygen, nutrients, and energy in the cells and thus help with regulation of metabolism. This is how the TPCs play a role in this physiological regulation through this interaction.

With the knockdown of these channels, we find that several various ailments can occur, from metabolic and general infectious diseases to even cancer. The pathological conditions due to this lacking of TPCs are covered in the following sections.

Membrane Trafficking
TPCs play an integral role in membrane trafficking pathways. They are sectioned in endosomes and lysosomes, especially functioning in endo-lysosomal fusions. TPC trafficking activity has been noted to be conserved; but modifying TPCs affects transportation in the endocytotic pathway. The exact roles of TCPs are specific to cell type and context. These channels are permeable to calcium, making them function as Ca2+ ion channels. When stimulated by NAADP – a second messenger for TPCs –, calcium is released into the cytosol. The influx of calcium is what regulates the fusion between the endosome and lysosomes and what mediates trafficking events. When the function of TCPs are lost, substrates accumulate creating congestion. When the function of TCPs are increased, the lysosome becomes enlarged – which logically relates to increased fusion events with the endosome to lysosome.

Parkinson's disease
One implication of membrane trafficking dysfunction leads to Parkinson’s disease. Mutations to LRRK2 enzyme alter autophagy dependent upon NAADP and TPC2. The mutation increases the amount of Ca2+ flow through TPC2 by NAADP evoked signals. This increase in signaling leads to an increased size of the lysosomes due to the increased rate and amount of fusion. The lysosome, therefore, is not able to break down components the way it should, leading to the disease. As TPC2 plays a vital role in this specific mechanism of Parkinson’s disease development, it may potentially be a therapeutic target.

Ebola
The Ebolavirus takes advantage of host cell endocytotic membrane trafficking, leaving TPCs as a potential drug target. Ebolavirus enter cells through micropinocytosis with endosomal vesicles. After entrance into the endosomal vesicle, Ebolavirus membrane fuses with the endosomal membrane to release the viral contents into the cytosol before the endosome can fuse with the lysosome. For the movement of the virus in endosomes, Ca2+ is necessary. As NAADP regulates maturation of endosomes by the calcium release through TCPs, normal functioning of TCPs allows the Ebolavirus to escape. Therefore, when TCPs are not functioning, the Ebolavirus cannot escape before the fusion of the endosome with the lysosome. In fact, when mice are treated with tetradine the infection is inhibited. This is because tetradine blocks TPC functioning of calcium release and thus, the Ebolaviruses is stuck within the endosomal network destined to be degraded by the lysosome.

Fatty liver
TPCs have been implicated in fatty liver diseases, such as NAFLD and NASH. As TPC2 is a cation channel for endocytotic membrane trafficking, TPCs contribute in trafficking LDL molecules for their breakdown and recycling. This primarily occurs within the liver. The degradation pathway causes LDL to end up in endosomes and lysosomes – where TPCs are located. The TPC mechanism once again allows the influx of calcium for the fusion of the endosomes and lysosomes (where LDL is degraded). When TPCs are not present, or are not functioning properly, the degradation pathway is defected trafficking. Without the fusion event LDL accumulates in liver cells. The loss of TPCs have been found to be a cause of the yellow coloration of liver, an expression of fatKty liver which indicates liver damage.

History and discovery
Although much is left to be discovered about TPC function, they have been extensively studied thus far. Many questions have been raised about the specific function of TPC channels, as well as the ions and molecules that appear to be most closely affiliated with channels such as sodium, calcium, and NAADP. Present knowledge of TPCs has come from experiments done on mice and plants, especially Arabidopsis thaliana. Because of the localization of these channels in mammals, it is difficult to use electrophysiological recordings. Therefore, these channels have to be expressed in alternative compartments or organelles of the cell, such as plant vacuoles to be studied using the electrophysiological methods – especially the patch clamp technique. In order to clearly visualize the plant vacuoles, scientists have relied on fluorescent microscopy in their experiments. Using these techniques, scientists have been able to collect significant qualitative data to make conclusions about mammalian TPC functions. Specifically, scientists were able to conclude that human TPC are voltage-dependent (predominantly)sodium channels, that PI(3,5)P2, an endolysosome-specific phosphoinositide (PIP), is a direct activator of TPC channels and that NAADP is actually not an activator, which was once previously assumed to be.