Search

Lysosomes have fundamental physiological roles and have previously been implicated in Parkinson’s disease1–5. However, how extracellular growth factors communicate with intracellular organelles to control lysosomal function is not well understood. Here we report a lysosomal K+ channel complex that is activated by growth factors and gated by protein kinase B (AKT) that we term lysoKGF. LysoKGF consists of a pore-forming protein TMEM175 and AKT: TMEM175 is opened by conformational changes in, but not the catalytic activity of, AKT. The minor allele at rs34311866, a common variant in TMEM175, is associated with an increased risk of developing Parkinson’s disease and reduces channel currents. Reduction in lysoKGF function predisposes neurons to stress-induced damage and accelerates the accumulation of pathological α-synuclein. By contrast, the minor allele at rs3488217—another common variant of TMEM175, which is associated with a decreased risk of developing Parkinson’s disease—produces a gain-of-function in lysoKGF during cell starvation, and enables neuronal resistance to damage. Deficiency in TMEM175 leads to a loss of dopaminergic neurons and impairment in motor function in mice, and a TMEM175 loss-of-function variant is nominally associated with accelerated rates of cognitive and motor decline in humans with Parkinson’s disease. Together, our studies uncover a pathway by which extracellular growth factors regulate intracellular organelle function, and establish a targetable mechanism by which common variants of TMEM175 confer risk for Parkinson’s disease. The identification of a lysosomal K+ channel complex—comprising AKT and the pore-forming TMEM175—provides insights into the mechanisms through which variants of the pore-forming protein affect the development of Parkinson’s disease.

A cryo-electron microscopy structure shows that the mucolipin domain of the lysosomal calcium channel TRPML3 binds phosphatidylinositol-3,5-bisphosphate and gates the channel. Numerous ion channels sit in the membranes of intracellular organelles and are responsible for maintaining concentration gradients and ionic signalling. The transient receptor potential mucolipin (TRPML) channels are Ca(II)-releasing channels that are crucial to endolysosomal function. While TRPML channels regulate physiological processes including membrane trafficking and exocytosis, mutations of TRPML1 cause the lysosomal storage disorder mucolipidosis type IV. Three papers in this issue of Nature report the structure of TRPML channels by cryo-electron microscopy. Seok-Yong Lee and colleagues report the structure of TRPML3, while studies from teams led by Xiaochun Li and Youxing Jiang present the structure of TRPML1. Together, these studies reveal the open and closed states of the TRPML family, indicating the regulatory mechanisms of these channels. As with most TRP channels, TRPML can be gated by specific lipids, and these studies provide insights into substrate binding and channel activation. The modulation of ion channel activity by lipids is increasingly recognized as a fundamental component of cellular signalling. The transient receptor potential mucolipin (TRPML) channel family belongs to the TRP superfamily1,2 and is composed of three members: TRPML1–TRPML3. TRPMLs are the major Ca2+-permeable channels on late endosomes and lysosomes (LEL). They regulate the release of Ca2+ from organelles, which is important for various physiological processes, including organelle trafficking and fusion3. Loss-of-function mutations in the MCOLN1 gene, which encodes TRPML1, cause the neurodegenerative lysosomal storage disorder mucolipidosis type IV, and a gain-of-function mutation (Ala419Pro) in TRPML3 gives rise to the varitint–waddler (Va) mouse phenotype4,5,6. Notably, TRPML channels are activated by the low-abundance and LEL-enriched signalling lipid phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2), whereas other phosphoinositides such as PtdIns(4,5)P2, which is enriched in plasma membranes, inhibit TRPMLs7,8. Conserved basic residues at the N terminus of the channel are important for activation by PtdIns(3,5)P2 and inhibition by PtdIns(4,5)P28. However, owing to a lack of structural information, the mechanism by which TRPML channels recognize PtdIns(3,5)P2 and increase their Ca2+ conductance remains unclear. Here we present the cryo-electron microscopy (cryo-EM) structure of a full-length TRPML3 channel from the common marmoset (Callithrix jacchus) at an overall resolution of 2.9 A. Our structure reveals not only the molecular basis of ion conduction but also the unique architecture of TRPMLs, wherein the voltage sensor-like domain is linked to the pore via a cytosolic domain that we term the mucolipin domain. Combined with functional studies, these data suggest that the mucolipin domain is responsible for PtdIns(3,5)P2 binding and subsequent channel activation, and that it acts as a ‘gating pulley’ for lipid-dependent TRPML gating.

The sodium-leak channel NALCN forms a subthreshold sodium conductance that controls the resting membrane potentials of neurons. The auxiliary subunits of the channel and their functions in mammals are largely unknown. In this study, we demonstrate that two large proteins UNC80 and UNC79 are subunits of the NALCN complex. UNC80 knockout mice are neonatal lethal. The C-terminus of UNC80 contains a domain that interacts with UNC79 and overcomes a soma-retention signal to achieve dendritic localization. UNC80 lacking this domain, as found in human patients, still supports whole-cell NALCN currents but lacks dendritic localization. Our results establish the subunit composition of the NALCN complex, uncover the inter-subunit interaction domains, reveal the functional significance of regulation of dendritic membrane potential by the sodium-leak channel complex, and provide evidence supporting that genetic variations found in individuals with intellectual disability are the causes for the phenotype observed in patients., The sodium-leak channel NALCN controls the resting membrane potentials of neurons. Here, the authors identified two subunits of NALCN, UNC80 and UNC79. Domains in UNC80, which are mutated in individuals with intellectual disability, interact to achieve the dendritic localization of NALCN complex.

The earliest intracellular signals that occur after T cell activation are local, subsecond Ca2+ microdomains. Here, we identified a Ca2+ entry component involved in Ca2+ microdomain formation in both unstimulated and stimulated T cells. In unstimulated T cells, spontaneously generated small Ca2+ microdomains required ORAI1, STIM1, and STIM2. Super-resolution microscopy of unstimulated T cells identified a circular subplasmalemmal region with a diameter of about 300 nm with preformed patches of colocalized ORAI1, ryanodine receptors (RYRs), and STIM1. Preformed complexes of STIM1 and ORAI1 in unstimulated cells were confirmed by coimmunoprecipitation and Forster resonance energy transfer studies. Furthermore, within the first second after T cell receptor (TCR) stimulation, the number of Ca2+ microdomains increased in the subplasmalemmal space, an effect that required ORAI1, STIM2, RYR1, and the Ca2+ mobilizing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate). These results indicate that preformed clusters of STIM and ORAI1 enable local Ca2+ entry events in unstimulated cells. Upon TCR activation, NAADP-evoked Ca2+ release through RYR1, in coordination with Ca2+ entry through ORAI1 and STIM, rapidly increases the number of Ca2+ microdomains, thereby initiating spread of Ca2+ signals deeper into the cytoplasm to promote full T cell activation.

Spinal projection neurons convey nociceptive signals to multiple brain regions including the parabrachial (PB) nucleus, which contributes to the emotional valence of pain perception. Despite the clear importance of projection neurons to pain processing, our understanding of the factors that shape their intrinsic membrane excitability remains limited. Here, we investigate a potential role for the Na leak channel NALCN in regulating the activity of spino-PB neurons in the developing rodent. Pharmacological reduction of NALCN current (INALCN), or the genetic deletion of NALCN channels, significantly reduced the intrinsic excitability of lamina I spino-PB neurons. In addition, substance P (SP) activated INALCN in ascending projection neurons through downstream Src kinase signaling, and the knockout of NALCN prevented SP-evoked action potential discharge in this neuronal population. These results identify, for the first time, NALCN as a strong regulator of neuronal activity within central pain circuits and also elucidate an additional ionic mechanism by which SP can modulate spinal nociceptive processing. Collectively, these findings indicate that the level of NALCN conductance within spino-PB neurons tightly governs ascending nociceptive transmission to the brain and thereby potentially influences pain perception.

Interstitial cells of Cajal (ICCs) are the pacemaking cells in the gastrointestinal muscles that generate the rhythmic oscillations in membrane potential known as slow waves. ICCs also mediate or transduce inputs from the enteric nervous system. Substance P (SubP) is a member of the family of mammalian tachykinin peptides that are predominantly released by enteric neurons. This study assessed the relationship of Na(+)-leak channel (NALCN) in the SubP-induced depolarization in pacemaking activity in the gastrointestinal tract. The patch-clamp technique for whole-cell recording was used in cultured cluster and single ICCs. Electrophysiological and pharmacological properties of SubP in ICC pacemaking activity were similar to those of NALCN. Reverse-transcription polymerase chain reaction, Western blotting, and immunohistochemistry all showed abundant and localized expression of NALCN messenger RNA and protein in mouse small intestine. NALCN is involved in the SubP-induced depolarization of intestinal pacemaking activity. The protein is a potential target for pharmacological treatment of motor disorders of the gut.

Extracellular K+, Na+, and Ca2+ ions all influence the resting membrane potential of the neuron. However, the mechanisms by which extracellular Na+ and Ca2+ regulate basal neuronal excitability are not well understood. Recent findings suggest that NALCN, in association with UNC79 and UNC80, contributes a basal Na+ leak conductance in neurons. Mutations in Nalcn, Unc79, or Unc80 lead to severe phenotypes that include neonatal lethality and disruption in rhythmic behaviors. This review discusses the properties of the NALCN complex, its regulation, and its contribution to neuronal function and animal behavior.

During mammalian fertilization, the contact between sperm and egg triggers increases in intracellular Ca2+ concentration ([Ca2++]i) in sperm. Voltage-gated Ca2+ channels (CaVs) are believed to mediate the initial phase of [Ca2+]i increases in sperm induced by egg coat (zona pellucida [ZP]) glycoproteins, while store depletion-activated Ca2+ entry is thought to mediate the sustained phase. Using patch-clamp recording and Ca2+ imaging, we show herein that CaV channel currents, while found in spermatogenic cells, are not detectable in epididymal sperm and are not essential for the ZP-induced [Ca2+]i changes. Instead, CATSPER channels localized in the distal portion of sperm (the principal piece) are required for the ZP-induced [Ca2+]i increases. Furthermore, the ZP-induced [Ca2+]i increase starts from the sperm tail and propagates toward the head.

SummaryCircadian clocks regulate membrane excitability in master pacemaker neurons to control daily rhythms of sleep and wake. Here, we find that two distinctly timed electrical drives collaborate to impose rhythmicity on Drosophila clock neurons. In the morning, a voltage-independent sodium conductance via the NA/NALCN ion channel depolarizes these neurons. This current is driven by the rhythmic expression of NCA localization factor-1, linking the molecular clock to ion channel function. In the evening, basal potassium currents peak to silence clock neurons. Remarkably, daily antiphase cycles of sodium and potassium currents also drive mouse clock neuron rhythms. Thus, we reveal an evolutionarily ancient strategy for the neural mechanisms that govern daily sleep and wake.

By mutagenesis, we demonstrated that the palmitoylation of the membrane-proximal Cys396 and Cys399of CD4, and the association of CD4 with Lck contribute to the enrichment of CD4 in lipid rafts. Ab cross-linking of CD4 induces an extensive membrane patching on the T cell surface, which is related to lipid raft aggregation. The lipid raft localization of CD4 is critical for CD4 to induce the aggregation of lipid rafts. The localization of CD4 in lipid rafts also correlates to the ability of CD4 to enhance receptor tyrosine phosphorylation. Thus, our data suggest that CD4-induced aggregation of lipid rafts may play an additional role in CD4 signaling besides its adhesion to MHC molecules and association with Lck.

Action potentials (APs) are fundamental cellular electrical signals. The genesis of short APs lasting milliseconds is well understood. Ultra-long APs (ulAPs) lasting seconds to minutes also occur in eukaryotic organisms, but their biological functions and mechanisms of generation are largely unknown. Here, we identify TPC3, a previously uncharacterized member of the two-pore channel protein family, as a new voltage-gated Na(+) channel (NaV) that generates ulAPs, and that establishes membrane potential bistability. Unlike the rapidly inactivating NaVs that generate short APs in neurons, TPC3 has a high activation threshold, activates slowly and does not inactivate-three properties that help generate long-lasting APs and guard the membrane against unintended perturbation. In amphibian oocytes, TPC3 forms a channel similar to channels induced by depolarization and sperm entry into eggs. TPC3 homologues are present in plants and animals, and they may be important for cellular processes and behaviours associated with prolonged membrane depolarization.

Dihydropyridine receptors in vertebrate skeletal muscle serve a dual role: as voltage sensors for excitation-contraction coupling and as voltage-activated calcium channels. Although they were the first of six classes of calcium channels to be cloned, skeletal muscle dihydropyridine receptors remain the only ones not functionally expressed as calcium channels in Xenopus oocytes, leading to the hypothesis that an interacting component is missing. Using beta1b, an isoform previously found in brain, we have for the first time reconstituted skeletal muscle calcium channel function in Xenopus oocytes. We show that this beta subunit is necessary for functional expression and that the alpha2delta subunit significantly enhances the expressed current. The majority of the alpha1 subunit in skeletal muscle is a truncated form. Here we show that both the full-length and truncated forms produce functional calcium channels in Xenopus oocytes, but the truncated form gives significantly larger currents. In addition, we show that the beta1b transcript is expressed in rat skeletal muscle, although at a much lower level than the abundant beta1a isoform.

International audience; Survival in the wild requires organismal adaptations to the availability of nutrients. Endosomes and lysosomes are key intracellular organelles that couple nutrition and metabolic status to cellular responses, but how they detect cytosolic ATP levels is not well understood. Here, we identify an endolysosomal ATP-sensitive Na+ channel (lysoNaATP). The channel is a complex formed by two-pore channels (TPC1 and TPC2), ion channels previously thought to be gated by nicotinic acid adenine dinucleotide phosphate (NAADP), and the mammalian target of rapamycin (mTOR). The channel complex detects nutrient status, becomes constitutively open upon nutrient removal and mTOR translocation off the lysosomal membrane, and controls the lysosome’s membrane potential, pH stability, and amino acid homeostasis. Mutant mice lacking lysoNaATP have much reduced exercise endurance after fasting. Thus, TPCs make up an ion channel family that couples the cell’s metabolic state to endolysosomal function and are crucial for physical endurance during food restriction.

Lysosomes, traditionally believed to be the terminal “recycle center” for biological “garbage”, are now known to play indispensable roles in membrane trafficking and intracellular signaling pathways. The multiple functions require the establishment of lysosomal acidic lumen, and this pH regulation has been shown to be tightly coupled with ionic (H+, Ca2+, Mg2+, Na+, K+, and Cl−) flux/homeostasis of the lysosome. Moreover, recent studies have suggested that the ion fluxes may directly regulate lysosomal dynamics. PI(3,5)P2 is an endolysosome-specific phosphoinositide that regulates ion homeostasis and membrane trafficking of endolysosomes via poorly understood mechanisms; human mutations of PI(3,5)P2-metabolizing enzymes cause muscle and neurodegenerative diseases. Here we measure that sodium is the predominant cation in the lysosome using atomic absorption, indicating a large Na+ concentration gradient is present across the lysosomal membrane. By the lysosome patch-clamp technique, we report that PI(3,5)P2 specifically activates two novel Na+-selective channels in the lysosome. Their identification provides insight into divalent cation selectivity, as well as to the mechanisms used by membranous lipids to directly regulate ion flux resulting in rapid changes in membrane potential and the fusogenic potential of intracellular organelles. We are currently investigating their physiological functions in detail.

The molecular mechanisms underlying Ca2+entry into sperm are now much more well defined thanks to direct recordings of mature sperm cells. This article reviews the function of a sperm-specific ion channel, CatSper. CatSpers have a clearly defined function in sperm's hyperactivated motility and are essential for male fertility. We propose that physiological stimuli such as zona pellucida and cyclic nucleotides induce Ca2+entry through CatSper channels instead of acting on CaVand CNG channels as previously thought.

The transition from quiescence of the sperm in the epididymis to capacitation in the female reproductive tract depends on alkalinization of the intracellular milieu. A recent study underscores the role of a particular proton channel in the process of alkalinization.

TheDmca1Dgene encodes aDrosophilacalcium channel α1subunit. We describe the first functional characterization of a mutation in this gene. This α1subunit mediates the dihydropyridine-sensitive calcium channel current in larval muscle but does not contribute to the amiloride-sensitive current in that tissue. A mutation, which changes a highly conserved Cys to Tyr in transmembrane domain IS1, identifies a residue important for channel function not only inDrosophilamuscle but also in mammalian cardiac channels. In both cases, mutations in this Cys residue slow channel activation and reduce expressed currents. Amino acid substitutions at this Cys position in the cardiac α1subunit show that the size of the side chain, rather than its ability to form disulfide bonds, affects channel activation.

Several neurotransmitters act through G-protein coupled receptors (GPCR) to evoke a “slow” excitation of neurons. These include peptides, such as substance P (SP) and neurotensin (NT), as well as acetylcholine and noradrenaline. Unlike the fast (∼ ms) ionotropic actions of small molecule neurotransmitters, the slow excitation is poorly understood at the molecular level, but can be mainly attributed to suppressing K+ currents and/or activating a non-selective cation channel. Several K+ channels, including members in the KV7 subfamily, are inhibited by the neurotransmitters in a G protein-dependent fashion. The molecular identity of the cation channel has yet to be determined; similarly how the channel is activated and its relative contribution to neuronal excitability induced by the neuropeptides are unknown. We show that, in the hippocampal and ventral tegmental area neurons, SP and NT activate the voltage-independent Na+-leak channel NALCN, a unique member in the 4×6TM channel family that also includes the voltage-gated Ca2+ (CaV) and Na+ (NaV) channels. The activation by SP through NK1R (a GPCR receptor for SP) is not blocked by the non-hydrolyzable GTP or GDP analogs. These findings identify NALCN as the cation channel activated by SP receptor, and suggest that a G protein-independent mechanism is involved in the coupling from receptor to channel.