Two-pore channels (TPCs) are eukaryotic intracellular voltage-gated and ligand gated cation selective ion channels. There are two known paralogs in the human genome, TPC1s and TPC2s. In humans, TPC1s are sodium selective and TPC2s conduct sodium ions, calcium ions and possibly hydrogen ions. Plant TPC1s are non-selective channels. Expression of TPCs are found 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. Quasi-tetramers appear very similar to tetramers, but are not quite the same. Some key roles of TPCs include calcium dependent responses in muscle contraction(s), hormone secretion, fertilization, and differentiation. Disorders linked to TPCs include membrane trafficking, Parkinson's disease, Ebola, and fatty liver. As implied by their name, TPC channels possess two pores and were named for their two Shaker-like repeats, which each have a pore domain. This contrasts with two-pore-domain potassium channels, which confusingly have only one pore and were named for the fact that each subunit has two P (pore) domains in its primary sequence.
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 these channels. Some of these ions are sodium, calcium, and NAADP. Present knowledge of TPCs has come from experiments done on mice and plants, especially Arabidopsis thaliana. Additionally, because of the localization of these channels in mammals, it is difficult to use electrophysiological recordings on them. Therefore, these TPC 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 in order to make conclusions about mammalian TPC functions. Specifically, scientists were able to conclude that human TPC are predominantly voltage-dependent sodium channels, and that PI(3,5)P2, an endolysosome-specific phosphoinositide (PIP), is a direct activator of TPC channels while NAADP is actually not an activator as it was once previously assumed to be.
Structure and domains At the mouth of the TPC pore, there are four amino acid 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 voltage sensing domain 1 (VSD1) and subunit 2 consists of the voltage sensing domain 2 (VSD2). The two subunit domains are separated by an EF-hand domain that has a calcium ion binding motif. This binding motif can facilitate channel activation by cytosolic calcium ions. Each of the two subunits are built from 12 transmembrane helices. The two central pore domains are combined 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, positioned between VSD1 and VSD2, where it can be activated by cytosolic calcium. The VSD2 domain 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. Voltage sensors, selectivity filter, and the gate work together in a coordinated manner to open and close TPCs for regulation of ion conductance. 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 VSD2 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. Activation of TPCs is induced by a decrease in transmembrane potential, or by an increase in calcium concentrations in the cytosol. Low pH of the lumen and low calcium concentration could cause inhibition of these channels. TPCs are also phosphorylation-gated channels in both animals as well as plants. 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.
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