TMEM175 Is an Organelle K(+) Channel Regulating Lysosomal Function.

Potassium is the most abundant ion to face both plasma and organelle membranes. Extensive research over the past seven decades has characterized how K(+) permeates the plasma membrane to control fundamental processes such as secretion, neuronal communication, and heartbeat. However, how K(+) permeates organelles such as lysosomes and endosomes is unknown. Here, we directly recorded organelle K(+) conductance and discovered a major K(+)-selective channel KEL on endosomes and lysosomes. KEL is formed by TMEM175, a protein with unknown function. Unlike any of the ∼80 plasma membrane K(+) channels, TMEM175 has two repeats of 6-transmembrane-spanning segments and has no GYG K(+) channel sequence signature-containing, pore-forming P loop. Lysosomes lacking TMEM175 exhibit no K(+) conductance, have a markedly depolarized ΔΨ and little sensitivity to changes in [K(+)], and have compromised luminal pH stability and abnormal fusion with autophagosomes during autophagy. Thus, TMEM175 comprises a K(+) channel that underlies the molecular mechanism of lysosomal K(+) permeability.

A Conserved Bicycle Model for Circadian Clock Control of Membrane Excitability.

Circadian 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.

mTOR regulates lysosomal ATP-sensitive two-pore Na(+) channels to adapt to metabolic state.

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 (lysoNa(ATP)). 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 lysoNa(ATP) 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.

A growth-factor-activated lysosomal K+ channel regulates Parkinson's pathology.

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.

Biallelic Mutations in UNC80 Cause Persistent Hypotonia, Encephalopathy, Growth Retardation, and Severe Intellectual Disability.

Ion channel proteins are required for both the establishment of resting membrane potentials and the generation of action potentials. Hundreds of mutations in genes encoding voltage-gated ion channels responsible for action potential generation have been found to cause severe neurological diseases. In contrast, the roles of voltage-independent "leak" channels, important for the establishment and maintenance of resting membrane potentials upon which action potentials are generated, are not well established in human disease. UNC80 is a large component of the NALCN sodium-leak channel complex that regulates the basal excitability of the nervous system. Loss-of-function mutations of NALCN cause infantile hypotonia with psychomotor retardation and characteristic facies (IHPRF). We report four individuals from three unrelated families who have homozygous missense or compound heterozygous truncating mutations in UNC80 and persistent hypotonia, encephalopathy, growth failure, and severe intellectual disability. Compared to control cells, HEK293T cells transfected with an expression plasmid containing the c.5098C>T (p.Pro1700Ser) UNC80 mutation found in one individual showed markedly decreased NALCN channel currents. Our findings demonstrate the fundamental significance of UNC80 and basal ionic conductance to human health.

The effect of phosphate accumulation on metal ion homeostasis in Saccharomyces cerevisiae.

Much of what is currently understood about the cell biology of metals involves their interactions with proteins. By comparison, little is known about interactions of metals with intracellular inorganic compounds such as phosphate. Here we examined the role of phosphate in metal metabolism in vivo by genetically perturbing the phosphate content of Saccharomyces cerevisiae cells. Yeast pho80 mutants cannot sense phosphate and have lost control of phosphate uptake, storage, and metabolism. We report here that pho80 mutants specifically elevate cytosolic and nonvacuolar levels of phosphate and this in turn causes a wide range of metal homeostasis defects. Intracellular levels of the hard-metal cations sodium and calcium increase dramatically, and cells become susceptible to toxicity from the transition metals manganese, cobalt, zinc, and copper. Disruptions in phosphate control also elicit an iron starvation response, as pho80 mutants were seen to upregulate iron transport genes. The iron-responsive transcription factor Aft1p appears activated in cells with high phosphate content in spite of normal intracellular iron levels. The high phosphate content of pho80 mutants can be lowered by mutating Pho4p, the transcription factor for phosphate uptake and storage genes. Such lowering of phosphate content by pho4 mutations reversed the high calcium and sodium content of pho80 mutants and prevented the iron starvation response. However, pho4 mutations only partially reversed toxicity from heavy metals, representing a novel outcome of phosphate dysregulation. Overall, these studies underscore the importance of maintaining a charge balance in the cell; a disruption in phosphate metabolism can dramatically impact on metal homeostasis.

Intellectual disability-associated UNC80 mutations reveal inter-subunit interaction and dendritic function of the NALCN channel complex.

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.

A non-inactivating high-voltage-activated two-pore Na⁺ channel that supports ultra-long action potentials and membrane bistability.

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.