Agonist-specific voltage-dependent gating of lysosomal two-pore Na+ channels (original) (raw)
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Agonist-mediated switching of ion selectivity in TPC2 differentially promotes lysosomal function
eLife
Ion selectivity is a defining feature of a given ion channel and is considered immutable. Here we show that ion selectivity of the lysosomal ion channel TPC2, which is hotly debated (Calcraft et al., 2009; Guo et al., 2017; Jha et al., 2014; Ruas et al., 2015; Wang et al., 2012), depends on the activating ligand. A high-throughput screen identified two structurally distinct TPC2 agonists. One of these evoked robust Ca2+-signals and non-selective cation currents, the other weaker Ca2+-signals and Na+-selective currents. These properties were mirrored by the Ca2+-mobilizing messenger, NAADP and the phosphoinositide, PI(3,5)P2, respectively. Agonist action was differentially inhibited by mutation of a single TPC2 residue and coupled to opposing changes in lysosomal pH and exocytosis. Our findings resolve conflicting reports on the permeability and gating properties of TPC2 and they establish a new paradigm whereby a single ion channel mediates distinct, functionally-relevant ionic sign...
Cell, 2012
Mammalian two-pore channel proteins (TPC1, TPC2; TPCN1, TPCN2) encode ion channels in intracellular endosomes and lysosomes and were proposed to mediate endolysosomal calcium release triggered by the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). By directly recording TPCs in endolysosomes from wild-type and TPC double-knockout mice, here we show that, in contrast to previous conclusions, TPCs are in fact sodium-selective channels activated by PI(3,5)P 2 and are not activated by NAADP. Moreover, the primary endolysosomal ion is Na + , not K + , as had been previously assumed. These findings suggest that the organellar membrane potential may undergo large regulatory changes and may explain the specificity of PI(3,5)P 2 in regulating the fusogenic potential of intracellular organelles.
Electrophysiology of Endolysosomal Two-Pore Channels: A Current Account
Cells
Two-pore channels TPC1 and TPC2 are ubiquitously expressed pathophysiologically relevant proteins that reside on endolysosomal vesicles. Here, we review the electrophysiology of these channels. Direct macroscopic recordings of recombinant TPCs expressed in enlarged lysosomes in mammalian cells or vacuoles in plants and yeast demonstrate gating by the Ca2+-mobilizing messenger NAADP and/or the lipid PI(3,5)P2. TPC currents are regulated by H+, Ca2+, and Mg2+ (luminal and/or cytosolic), as well as protein kinases, and they are impacted by single-nucleotide polymorphisms linked to pigmentation. Bisbenzylisoquinoline alkaloids, flavonoids, and several approved drugs demonstrably block channel activity. Endogenous TPC currents have been recorded from a number of primary cell types and cell lines. Many of the properties of endolysosomal TPCs are recapitulated upon rerouting channels to the cell surface, allowing more facile recording through conventional electrophysiological means. Single...
Structural mechanisms of phospholipid activation of the human TPC2 channel
eLife
Mammalian two-pore channels (TPCs) regulate the physiological functions of the endolysosome. Here we present cryo-EM structures of human TPC2 (HsTPC2), a phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2)-activated, Na+ selective channel, in the ligand-bound and apo states. The apo structure captures the closed conformation, while the ligand-bound form features the channel in both open and closed conformations. Combined with functional analysis, these structures provide insights into the mechanism of PI(3,5)P2-regulated gating of TPC2, which is distinct from that of TPC1. Specifically, the endolysosome-specific PI(3,5)P2 binds at the first 6-TM and activates the channel - independently of the membrane potential - by inducing a structural change at the pore-lining inner helix (IS6), which forms a continuous helix in the open state but breaks into two segments at Gly317 in the closed state. Additionally, structural comparison to the voltage-dependent TPC1 structure allowed us to identi...
TPCs: Endolysosomal channels for Ca2+ mobilization from acidic organelles triggered by NAADP
FEBS Letters, 2010
Two-pore channels (TPCs or TPCNs) are novel members of the large superfamily of voltage-gated cation channels with slightly higher sequence homology to the pore-forming subunits of voltagegated Ca 2+ and Na + channels than most other members. Recent studies demonstrate that TPCs locate to endosomes and lysosomes and form Ca 2+ release channels that respond to activation by the Ca 2+ mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). With multiple endolysosomal targeted NAADP receptors now identified, important new insights into the regulation of endolysosomal function in health and disease will therefore be unveiled.
Voltage-gated sodium channels and their roles in drug action
Current Anaesthesia and Critical Care, 2005
The voltage-gated ion channels (VGICs) represent a superfamily of glycoprotein molecules that are able to form membrane spanning channels that 'gate' in response to changes in membrane potential. This regulates ion selective fluxes on the microsecond-millisecond timescale. They are crucially involved in regulating activity in excitable cells of the CNS and in skeletal muscle fibres. VGICs are directly involved in control of cellular resting potential, neurotransmitter release, determination of spike firing threshold and initiation, propagation and shaping of action potentials. They represent a major target for local anaesthetic, anti-convulsant/analgesic and anti-arrhythmic drugs.
Elucidating the Structural Basis of the Intracellular pH Sensing Mechanism of TASK-2 K2P Channels
International Journal of Molecular Sciences, 2020
Two-pore domain potassium (K2P) channels maintain the cell’s background conductance by stabilizing the resting membrane potential. They assemble as dimers possessing four transmembrane helices in each subunit. K2P channels were crystallized in “up” and “down” states. The movements of the pore-lining transmembrane TM4 helix produce the aperture or closure of side fenestrations that connect the lipid membrane with the central cavity. When the TM4 helix is in the up-state, the fenestrations are closed, while they are open in the down-state. It is thought that the fenestration states are related to the activity of K2P channels and the opening of the channels preferentially occurs from the up-state. TASK-2, a member of the TALK subfamily of K2P channels, is opened by intracellular alkalization leading the deprotonation of the K245 residue at the end of the TM4 helix. This charge neutralization of K245 could be sensitive or coupled to the fenestration state. Here, we describe the relationship between the states of the intramembrane fenestrations and K245 residue in TASK-2 channel. By using molecular modeling and simulations, we show that the protonated state of K245 (K245+) favors the open fenestration state and, symmetrically, that the open fenestration state favors the protonated state of the lysine residue. We show that the channel can be completely blocked by Prozac, which is known to induce fenestration opening in TREK-2. K245 protonation and fenestration aperture have an additive effect on the conductance of the channel. The opening of the fenestrations with K245+ increases the entrance of lipids into the selectivity filter, blocking the channel. At the same time, the protonation of K245 introduces electrostatic potential energy barriers to ion entrance. We computed the free energy profiles of ion penetration into the channel in different fenestration and K245 protonation states, to show that the effects of the two transformations are summed up, leading to maximum channel blocking. Estimated rates of ion transport are in qualitative agreement with experimental results and support the hypothesis that the most important barrier for ion transport under K245+ and open fenestration conditions is the entrance of the ions into the channel.
Voltage-gated sodium channels as therapeutic targets
Drug Discovery Today, 2000
Voltage-gated sodium channels (VGSCs) play a central role in the generation and propagation of action potentials in neurons and other cells. VGSC modulators have their origins in empirical pharmacology and are being used as local anaesthetics, antiarrhythmics, analgesics and antiepileptics, and for other disorders. However, the identification of a multigene family of VGSCs, along with tools to study the different subtypes in pathophysiology, is now providing a rational basis for selective intervention. Recent advances have addressed the technical challenges of expressing and assaying these complex proteins, enabling the correlation of empirical pharmacology to subtypes and the screening of individual subtypes for novel inhibitors with increased potency and selectivity.
A molecular view of the function and pharmacology of acid-sensing ion channels
Pharmacological Research, 2020
The pH in the different tissues and organs of our body is kept within tight limits. Local pH changes occur, however, temporarily under physiological conditions, as for example in synapses during neuronal activity. In pathological situations, such as in ischemia, inflammation, and tumor growth, long-lasting acidification develops. Acid-sensing ion channels (ASICs) are low pH-activated Na +-permeable ion channels that are widely expressed in the central and peripheral nervous systems. ASICs act as pH sensors, leading to This review article was published as S. Vullo, S. Kellenberger, A molecular view of the function and pharmacology of acid-sensing ion channels, Pharmacol Res (2019),