Structural basis for ion selectivity in potassium-selective channelrhodopsins.

KCR channelrhodopsins (K+-selective light-gated ion channels) have received attention as potential inhibitory optogenetic tools but more broadly pose a fundamental mystery regarding how their K+ selectivity is achieved. Here, we present 2.5-2.7 Å cryo-electron microscopy structures of HcKCR1 and HcKCR2 and of a structure-guided mutant with enhanced K+ selectivity. Structural, electrophysiological, computational, spectroscopic, and biochemical analyses reveal a distinctive mechanism for K+ selectivity; rather than forming the symmetrical filter of canonical K+ channels achieving both selectivity and dehydration, instead, three extracellular-vestibule residues within each monomer form a flexible asymmetric selectivity gate, while a distinct dehydration pathway extends intracellularly. Structural comparisons reveal a retinal-binding pocket that induces retinal rotation (accounting for HcKCR1/HcKCR2 spectral differences), and design of corresponding KCR variants with increased K+ selectivity (KALI-1/KALI-2) provides key advantages for optogenetic inhibition in vitro and in vivo. Thus, discovery of a mechanism for ion-channel K+ selectivity also provides a framework for next-generation optogenetics.

Species-Independent Attraction to Biofilms through Electrical Signaling.

Bacteria residing within biofilm communities can coordinate their behavior through cell-to-cell signaling. However, it remains unclear if these signals can also influence the behavior of distant cells that are not part of the community. Using a microfluidic approach, we find that potassium ion channel-mediated electrical signaling generated by a Bacillus subtilis biofilm can attract distant cells. Integration of experiments and mathematical modeling indicates that extracellular potassium emitted from the biofilm alters the membrane potential of distant cells, thereby directing their motility. This electrically mediated attraction appears to be a generic mechanism that enables cross-species interactions, as Pseudomonas aeruginosa cells also become attracted to the electrical signal released by the B. subtilis biofilm. Cells within a biofilm community can thus not only coordinate their own behavior but also influence the behavior of diverse bacteria at a distance through long-range electrical signaling. PAPERCLIP.

Microglia-derived PDGFB promotes neuronal potassium currents to suppress basal sympathetic tonicity and limit hypertension.

Although many studies have addressed the regulatory circuits affecting neuronal activities, local non-synaptic mechanisms that determine neuronal excitability remain unclear. Here, we found that microglia prevented overactivation of pre-sympathetic neurons in the hypothalamic paraventricular nucleus (PVN) at steady state. Microglia constitutively released platelet-derived growth factor (PDGF) B, which signaled via PDGFRα on neuronal cells and promoted their expression of Kv4.3, a key subunit that conducts potassium currents. Ablation of microglia, conditional deletion of microglial PDGFB, or suppression of neuronal PDGFRα expression in the PVN elevated the excitability of pre-sympathetic neurons and sympathetic outflow, resulting in a profound autonomic dysfunction. Disruption of the PDGFBMG-Kv4.3Neuron pathway predisposed mice to develop hypertension, whereas central supplementation of exogenous PDGFB suppressed pressor response when mice were under hypertensive insult. Our results point to a non-immune action of resident microglia in maintaining the balance of sympathetic outflow, which is important in preventing cardiovascular diseases.

A Non-canonical Voltage-Sensing Mechanism Controls Gating in K2P K(+) Channels.

Two-pore domain (K2P) K(+) channels are major regulators of excitability that endow cells with an outwardly rectifying background "leak" conductance. In some K2P channels, strong voltage-dependent activation has been observed, but the mechanism remains unresolved because they lack a canonical voltage-sensing domain. Here, we show voltage-dependent gating is common to most K2P channels and that this voltage sensitivity originates from the movement of three to four ions into the high electric field of an inactive selectivity filter. Overall, this ion-flux gating mechanism generates a one-way "check valve" within the filter because outward movement of K(+) induces filter opening, whereas inward movement promotes inactivation. Furthermore, many physiological stimuli switch off this flux gating mode to convert K2P channels into a leak conductance. These findings provide insight into the functional plasticity of a K(+)-selective filter and also refine our understanding of K2P channels and the mechanisms by which ion channels can sense voltage.

Hexokinase Is an Innate Immune Receptor for the Detection of Bacterial Peptidoglycan.

Degradation of Gram-positive bacterial cell wall peptidoglycan in macrophage and dendritic cell phagosomes leads to activation of the NLRP3 inflammasome, a cytosolic complex that regulates processing and secretion of interleukin (IL)-1ß and IL-18. While many inflammatory responses to peptidoglycan are mediated by detection of its muramyl dipeptide component in the cytosol by NOD2, we report here that NLRP3 inflammasome activation is caused by release of N-acetylglucosamine that is detected in the cytosol by the glycolytic enzyme hexokinase. Inhibition of hexokinase by N-acetylglucosamine causes its dissociation from mitochondria outer membranes, and we found that this is sufficient to activate the NLRP3 inflammasome. In addition, we observed that glycolytic inhibitors and metabolic conditions affecting hexokinase function and localization induce inflammasome activation. While previous studies have demonstrated that signaling by pattern recognition receptors can regulate metabolic processes, this study shows that a metabolic enzyme can act as a pattern recognition receptor. PAPERCLIP.

A Glial K/Cl Transporter Controls Neuronal Receptive Ending Shape by Chloride Inhibition of an rGC.

Neurons receive input from the outside world or from other neurons through neuronal receptive endings (NREs). Glia envelop NREs to create specialized microenvironments; however, glial functions at these sites are poorly understood. Here, we report a molecular mechanism by which glia control NRE shape and associated animal behavior. The C. elegans AMsh glial cell ensheathes the NREs of 12 neurons, including the thermosensory neuron AFD. KCC-3, a K/Cl transporter, localizes specifically to a glial microdomain surrounding AFD receptive ending microvilli, where it regulates K(+) and Cl(-) levels. We find that Cl(-) ions function as direct inhibitors of an NRE-localized receptor-guanylyl-cyclase, GCY-8, which synthesizes cyclic guanosine monophosphate (cGMP). High cGMP mediates the effects of glial KCC-3 on AFD shape by antagonizing the actin regulator WSP-1/NWASP. Components of this pathway are broadly expressed throughout the nervous system, suggesting that ionic regulation of the NRE microenvironment may be a conserved mechanism by which glia control neuron shape and function.

A Potassium-Dependent Oxygen Sensing Pathway Regulates Plant Root Hydraulics.

Aerobic organisms survive low oxygen (O2) through activation of diverse molecular, metabolic, and physiological responses. In most plants, root water permeability (in other words, hydraulic conductivity, Lpr) is downregulated under O2 deficiency. Here, we used a quantitative genetics approach in Arabidopsis to clone Hydraulic Conductivity of Root 1 (HCR1), a Raf-like MAPKKK that negatively controls Lpr. HCR1 accumulates and is functional under combined O2 limitation and potassium (K(+)) sufficiency. HCR1 regulates Lpr and hypoxia responsive genes, through the control of RAP2.12, a key transcriptional regulator of the core anaerobic response. A substantial variation of HCR1 in regulating Lpr is observed at the Arabidopsis species level. Thus, by combinatorially integrating two soil signals, K(+) and O2 availability, HCR1 modulates the resilience of plants to multiple flooding scenarios.

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.

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.

Spontaneous Activity of Cochlear Hair Cells Triggered by Fluid Secretion Mechanism in Adjacent Support Cells.

Spontaneous electrical activity of neurons in developing sensory systems promotes their maturation and proper connectivity. In the auditory system, spontaneous activity of cochlear inner hair cells (IHCs) is initiated by the release of ATP from glia-like inner supporting cells (ISCs), facilitating maturation of central pathways before hearing onset. Here, we find that ATP stimulates purinergic autoreceptors in ISCs, triggering Cl(-) efflux and osmotic cell shrinkage by opening TMEM16A Ca(2+)-activated Cl(-) channels. Release of Cl(-) from ISCs also forces K(+) efflux, causing transient depolarization of IHCs near ATP release sites. Genetic deletion of TMEM16A markedly reduces the spontaneous activity of IHCs and spiral ganglion neurons in the developing cochlea and prevents ATP-dependent shrinkage of supporting cells. These results indicate that supporting cells in the developing cochlea have adapted a pathway used for fluid secretion in other organs to induce periodic excitation of hair cells.

Astrocyte regulation of synaptic behavior.

Astrocytes regulate multiple aspects of neuronal and synaptic function from development through to adulthood. Instead of addressing each function independently, this review provides a comprehensive overview of the different ways astrocytes modulate neuronal synaptic function throughout life, with a particular focus on recent findings in each area. It includes the emerging functions of astrocytes, such as a role in synapse formation, as well as more established roles, including the uptake and recycling of neurotransmitters. This broad approach covers the many ways astrocytes and neurons constantly interact to maintain the correct functioning of the brain. It is important to consider all of these diverse functions of astrocytes when investigating how astrocyte-neuron interactions regulate synaptic behavior to appreciate the complexity of these ongoing interactions.

Regulation of the Renal NaCl Cotransporter and Its Role in Potassium Homeostasis.

Daily dietary potassium (K+) intake may be as large as the extracellular K+ pool. To avoid acute hyperkalemia, rapid removal of K+ from the extracellular space is essential. This is achieved by translocating K+ into cells and increasing urinary K+ excretion. Emerging data now indicate that the renal thiazide-sensitive NaCl cotransporter (NCC) is critically involved in this homeostatic kaliuretic response. This suggests that the early distal convoluted tubule (DCT) is a K+ sensor that can modify sodium (Na+) delivery to downstream segments to promote or limit K+ secretion. K+ sensing is mediated by the basolateral K+ channels Kir4.1/5.1, a capacity that the DCT likely shares with other nephron segments. Thus, next to K+-induced aldosterone secretion, K+ sensing by renal epithelial cells represents a second feedback mechanism to control K+ balance. NCC's role in K+ homeostasis has both physiological and pathophysiological implications. During hypovolemia, NCC activation by the renin-angiotensin system stimulates Na+ reabsorption while preventing K+ secretion. Conversely, NCC inactivation by high dietary K+ intake maximizes kaliuresis and limits Na+ retention, despite high aldosterone levels. NCC activation by a low-K+ diet contributes to salt-sensitive hypertension. K+-induced natriuresis through NCC offers a novel explanation for the antihypertensive effects of a high-K+ diet. A possible role for K+ in chronic kidney disease is also emerging, as epidemiological data reveal associations between higher urinary K+ excretion and improved renal outcomes. This comprehensive review will embed these novel insights on NCC regulation into existing concepts of K+ homeostasis in health and disease.

The Physiology of the Gastric Parietal Cell.

Parietal cells are responsible for gastric acid secretion, which aids in the digestion of food, absorption of minerals, and control of harmful bacteria. However, a fine balance of activators and inhibitors of parietal cell-mediated acid secretion is required to ensure proper digestion of food, while preventing damage to the gastric and duodenal mucosa. As a result, parietal cell secretion is highly regulated through numerous mechanisms including the vagus nerve, gastrin, histamine, ghrelin, somatostatin, glucagon-like peptide 1, and other agonists and antagonists. The tight regulation of parietal cells ensures the proper secretion of HCl. The H+-K+-ATPase enzyme expressed in parietal cells regulates the exchange of cytoplasmic H+ for extracellular K+. The H+ secreted into the gastric lumen by the H+-K+-ATPase combines with luminal Cl- to form gastric acid, HCl. Inhibition of the H+-K+-ATPase is the most efficacious method of preventing harmful gastric acid secretion. Proton pump inhibitors and potassium competitive acid blockers are widely used therapeutically to inhibit acid secretion. Stimulated delivery of the H+-K+-ATPase to the parietal cell apical surface requires the fusion of intracellular tubulovesicles with the overlying secretory canaliculus, a process that represents the most prominent example of apical membrane recycling. In addition to their unique ability to secrete gastric acid, parietal cells also play an important role in gastric mucosal homeostasis through the secretion of multiple growth factor molecules. The gastric parietal cell therefore plays multiple roles in gastric secretion and protection as well as coordination of physiological repair.

Phosphatidic acid-regulated SOS2 controls sodium and potassium homeostasis in Arabidopsis under salt stress.

The maintenance of sodium/potassium (Na+ /K+ ) homeostasis in plant cells is essential for salt tolerance. Plants export excess Na+ out of cells mainly through the Salt Overly Sensitive (SOS) pathway, activated by a calcium signal; however, it is unknown whether other signals regulate the SOS pathway and how K+ uptake is regulated under salt stress. Phosphatidic acid (PA) is emerging as a lipid signaling molecule that modulates cellular processes in development and the response to stimuli. Here, we show that PA binds to the residue Lys57 in SOS2, a core member of the SOS pathway, under salt stress, promoting the activity and plasma membrane localization of SOS2, which activates the Na+ /H+ antiporter SOS1 to promote the Na+ efflux. In addition, we reveal that PA promotes the phosphorylation of SOS3-like calcium-binding protein 8 (SCaBP8) by SOS2 under salt stress, which attenuates the SCaBP8-mediated inhibition of Arabidopsis K+ transporter 1 (AKT1), an inward-rectifying K+ channel. These findings suggest that PA regulates the SOS pathway and AKT1 activity under salt stress, promoting Na+ efflux and K+ influx to maintain Na+ /K+ homeostasis.

Gasdermin D Restrains Type I Interferon Response to Cytosolic DNA by Disrupting Ionic Homeostasis.

Inflammasome-activated caspase-1 cleaves gasdermin D to unmask its pore-forming activity, the predominant consequence of which is pyroptosis. Here, we report an additional biological role for gasdermin D in limiting cytosolic DNA surveillance. Cytosolic DNA is sensed by Aim2 and cyclic GMP-AMP synthase (cGAS) leading to inflammasome and type I interferon responses, respectively. We found that gasdermin D activated by the Aim2 inflammasome suppressed cGAS-driven type I interferon response to cytosolic DNA and Francisella novicida in macrophages. Similarly, interferon-ß (IFN-ß) response to F. novicida infection was elevated in gasdermin D-deficient mice. Gasdermin D-mediated negative regulation of IFN-ß occurred in a pyroptosis-, interleukin-1 (IL-1)-, and IL-18-independent manner. Mechanistically, gasdermin D depleted intracellular potassium (K+) via membrane pores, and this K+ efflux was necessary and sufficient to inhibit cGAS-dependent IFN-ß response. Thus, our findings have uncovered an additional interferon regulatory module involving gasdermin D and K+ efflux.

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.

Mechanistic basis for potassium efflux-driven activation of the human NLRP1 inflammasome.

Nigericin, an ionophore derived from Streptomyces hygroscopicus, is arguably the most commonly used tool compound to study the NLRP3 inflammasome. Recent findings, however, showed that nigericin also activates the NLRP1 inflammasome in human keratinocytes. In this study, we resolve the mechanistic basis of nigericin-driven NLRP1 inflammasome activation. In multiple nonhematopoietic cell types, nigericin rapidly and specifically inhibits the elongation stage of the ribosome cycle by depleting cytosolic potassium ions. This activates the ribotoxic stress response (RSR) sensor kinase ZAKα, p38, and JNK, as well as the hyperphosphorylation of the NLRP1 linker domain. As a result, nigericin-induced pyroptosis in human keratinocytes is blocked by extracellular potassium supplementation, ZAKα knockout, or pharmacologic inhibitors of ZAKα and p38 kinase activities. By surveying a panel of ionophores, we show that electroneutrality of ion movement is essential to activate ZAKα-driven RSR and a greater extent of K+ depletion is necessary to activate ZAKα-NLRP1 than NLRP3. These findings resolve the mechanism by which nigericin activates NLRP1 in nonhematopoietic cell types and demonstrate an unexpected connection between RSR, perturbations of potassium ion flux, and innate immunity.

c-di-AMP determines the hierarchical organization of bacterial RCK proteins.

In bacteria, intracellular K+ is involved in the regulation of membrane potential, cytosolic pH, and cell turgor as well as in spore germination, environmental adaptation, cell-to-cell communication in biofilms, antibiotic sensitivity, and infectivity. The second messenger cyclic-di-AMP (c-di-AMP) has a central role in modulating the intracellular K+ concentration in many bacterial species, controlling transcription and function of K+ channels and transporters. However, our understanding of how this regulatory network responds to c-di-AMP remains poor. We used the RCK (Regulator of Conductance of K+) proteins that control the activity of Ktr channels in Bacillus subtilis as a model system to analyze the regulatory function of c-di-AMP with a combination of in vivo and in vitro functional and structural characterization. We determined that the two RCK proteins (KtrA and KtrC) are neither physiologically redundant or functionally equivalent. KtrC is the physiologically dominant RCK protein in the regulation of Ktr channel activity. In explaining this hierarchical organization, we found that, unlike KtrA, KtrC is very sensitive to c-di-AMP inactivation and lack of c-di-AMP regulation results in RCK protein toxicity, most likely due to unregulated K+ flux. We also found that KtrC can assemble with KtrA, conferring c-di-AMP regulation to the functional KtrA/KtrC heteromers and potentially compensating KtrA toxicity. Altogether, we propose that the central role of c-di-AMP in the control of the K+ machinery, by modulating protein levels through gene transcription and by regulating protein activity, has determined the evolutionary selection of KtrC as the dominant RCK protein, shaping the hierarchical organization of regulatory components of the K+ machinery.

Mycobacterium tuberculosis response to cholesterol is integrated with environmental pH and potassium levels via a lipid metabolism regulator.

Successful colonization of the host requires Mycobacterium tuberculosis (Mtb) to sense and respond coordinately to disparate environmental cues during infection and adapt its physiology. However, how Mtb response to environmental cues and the availability of key carbon sources may be integrated is poorly understood. Here, by exploiting a reporter-based genetic screen, we have unexpectedly found that overexpression of transcription factors involved in Mtb lipid metabolism altered the dampening effect of low environmental potassium concentrations ([K+]) on the pH response of Mtb. Cholesterol is a major carbon source for Mtb during infection, and transcriptional analyses revealed that Mtb response to acidic pH was augmented in the presence of cholesterol and vice versa. Strikingly, deletion of the putative lipid regulator mce3R had little effect on Mtb transcriptional response to acidic pH or cholesterol individually, but resulted specifically in loss of cholesterol response augmentation in the simultaneous presence of acidic pH. Similarly, while mce3R deletion had little effect on Mtb response to low environmental [K+] alone, augmentation of the low [K+] response by the simultaneous presence of cholesterol was lost in the mutant. Finally, a mce3R deletion mutant was attenuated for growth in foamy macrophages and for colonization in a murine infection model that recapitulates caseous necrotic lesions and the presence of foamy macrophages. These findings reveal the critical coordination between Mtb response to environmental cues and cholesterol, a vital carbon source, and establishes Mce3R as a transcription factor that crucially serves to integrate these signals.

Identification of the potassium-binding site in serotonin transporter.

Clearance of serotonin (5-hydroxytryptamine, 5-HT) from the synaptic cleft after neuronal signaling is mediated by serotonin transporter (SERT), which couples this process to the movement of a Na+ ion down its chemical gradient. After release of 5-HT and Na+ into the cytoplasm, the transporter faces a rate-limiting challenge of resetting its conformation to be primed again for 5-HT and Na+ binding. Early studies of vesicles containing native SERT revealed that K+ gradients can provide an additional driving force, via K+ antiport. Moreover, under appropriate conditions, a H+ ion can replace K+. Intracellular K+ accelerates the resetting step. Structural studies of SERT have identified two binding sites for Na+ ions, but the K+ site remains enigmatic. Here, we show that K+ antiport can drive substrate accumulation into vesicles containing SERT extracted from a heterologous expression system, allowing us to study the residues responsible for K+ binding. To identify candidate binding residues, we examine many cation binding configurations using molecular dynamics simulations, predicting that K+ binds to the so-called Na2 site. Site-directed mutagenesis of residues in this site can eliminate the ability of both K+ and H+ to drive 5-HT accumulation into vesicles and, in patch clamp recordings, prevent the acceleration of turnover rates and the formation of a channel-like state by K+ or H+. In conclusion, the Na2 site plays a pivotal role in orchestrating the sequential binding of Na+ and then K+ (or H+) ions to facilitate 5-HT uptake in SERT.