A Treatment to Eliminate SARS-CoV-2 Replication in Human Airway Epithelial Cells Is Safe for Inhalation as an Aerosol in Healthy Human Subjects.

BACKGROUND: Low airway surface pH is associated with many airway diseases, impairs antimicrobial host defense, and worsens airway inflammation. Inhaled Optate is designed to safely raise airway surface pH and is well tolerated in humans. Raising intracellular pH partially prevents activation of SARS-CoV-2 in primary normal human airway epithelial (NHAE) cells, decreasing viral replication by several mechanisms. METHODS: We grew primary NHAE cells from healthy subjects, infected them with SARS-CoV-2 (isolate USA-WA1/2020), and used clinical Optate at concentrations used in humans in vivo to determine whether Optate would prevent viral infection and replication. Cells were pretreated with Optate or placebo prior to infection (multiplicity of infection = 1), and viral replication was determined with plaque assay and nucleocapsid (N) protein levels. Healthy human subjects also inhaled Optate as part of a Phase 2a safety trial. RESULTS: Optate almost completely prevented viral replication at each time point between 24 h and 120 h, relative to placebo, on both plaque assay and N protein expression (P < .001). Mechanistically, Optate inhibited expression of major endosomal trafficking genes and raised NHAE intracellular pH. Optate had no effect on NHAE cell viability at any time point. Inhaled Optate was well tolerated in 10 normal subjects, with no change in lung function, vital signs, or oxygenation. CONCLUSIONS: Inhaled Optate may be well suited for a clinical trial in patients with pulmonary SARS-CoV-2 infection. However, it is vitally important for patient safety that formulations designed for inhalation with regard to pH, isotonicity, and osmolality be used. An inhalational treatment that safely prevents SARS-CoV-2 viral replication could be helpful for treating patients with pulmonary SARS-CoV-2 infection.

Cleavage of TRPM7 releases the kinase domain from the ion channel and regulates its participation in Fas-induced apoptosis.

Transient receptor potential melastatin-like 7 (TRPM7) is a channel protein that also contains a regulatory serine-threonine kinase domain. Here, we find that Trpm7-/- T cells are deficient in Fas-receptor-induced apoptosis and that TRPM7 channel activity participates in the apoptotic process and is regulated by caspase-dependent cleavage. This function of TRPM7 is dependent on its function as a channel, but not as a kinase. TRPM7 is cleaved by caspases at D1510, disassociating the carboxy-terminal kinase domain from the pore without disrupting the phosphotransferase activity of the released kinase but substantially increasing TRPM7 ion channel activity. Furthermore, we show that TRPM7 regulates endocytic compartmentalization of the Fas receptor after receptor stimulation, an important process for apoptotic signaling through Fas receptors. These findings raise the possibility that other members of the TRP channel superfamily are also regulated by caspase-mediated cleavage, with wide-ranging implications for cell death and differentiation.

Voltage- and temperature-dependent activation of TRPV3 channels is potentiated by receptor-mediated PI(4,5)P2 hydrolysis.

TRPV3 is a thermosensitive channel that is robustly expressed in skin keratinocytes and activated by innocuous thermal heating, membrane depolarization, and chemical agonists such as 2-aminoethyoxy diphenylborinate, carvacrol, and camphor. TRPV3 modulates sensory thermotransduction, hair growth, and susceptibility to dermatitis in rodents, but the molecular mechanisms responsible for controlling TRPV3 channel activity in keratinocytes remain elusive. We show here that receptor-mediated breakdown of the membrane lipid phosphatidylinositol (4,5) bisphosphate (PI(4,5)P(2)) regulates the activity of both native TRPV3 channels in primary human skin keratinocytes and expressed TRPV3 in a HEK-293-derived cell line stably expressing muscarinic M(1)-type acetylcholine receptors. Stimulation of PI(4,5)P(2) hydrolysis or pharmacological inhibition of PI 4 kinase to block PI(4,5)P(2) synthesis potentiates TRPV3 currents by causing a negative shift in the voltage dependence of channel opening, increasing the proportion of voltage-independent current and causing thermal activation to occur at cooler temperatures. The activity of single TRPV3 channels in excised patches is potentiated by PI(4,5)P(2) depletion and selectively decreased by PI(4,5)P(2) compared with related phosphatidylinositol phosphates. Neutralizing mutations of basic residues in the TRP domain abrogate the effect of PI(4,5)P(2) on channel function, suggesting that PI(4,5)P(2) directly interacts with a specific protein motif to reduce TRPV3 channel open probability. PI(4,5)P(2)-dependent modulation of TRPV3 activity represents an attractive mechanism for acute regulation of keratinocyte signaling cascades that control cell proliferation and the release of autocrine and paracrine factors.

An aqueous H+ permeation pathway in the voltage-gated proton channel Hv1.

Hv1 voltage-gated proton channels mediate rapid and selective transmembrane H(+) flux and are gated by both voltage and pH gradients. Selective H(+) transfer in membrane proteins is commonly achieved by Grotthuss proton 'hopping' in chains of ionizable amino acid side chains and intraprotein water molecules. To identify whether ionizable residues are required for proton permeation in Hv1, we neutralized candidate residues and measured expressed voltage-gated H(+) currents. Unexpectedly, charge neutralization was insufficient to abrogate either the Hv1 conductance or coupling of pH gradient and voltage-dependent activation. Molecular dynamics simulations revealed water molecules in the central crevice of Hv1 model structures but not in homologous voltage-sensor domain (VSD) structures. Our results indicate that Hv1 most likely forms an internal water wire for selective proton transfer and that interactions between water molecules and S4 arginines may underlie coupling between voltage- and pH-gradient sensing.

Hv1 proton channels are required for high-level NADPH oxidase-dependent superoxide production during the phagocyte respiratory burst.

Granulocytes generate a "respiratory burst" of NADPH oxidase-dependent superoxide anion (O(2)(-*)) production that is required for efficient clearance of bacterial pathogens. Hv1 mediates a voltage-gated H(+) channel activity that is proposed to serve a charge-balancing role in granulocytic phagocytes such as neutrophils and eosinophils. Using mice in which the gene encoding Hv1 is replaced by beta-Geo reporter protein sequence, we show that Hv1 expression is required for measurable voltage-gated H(+) current in unstimulated phagocytes. O(2)(-*) production is substantially reduced in the absence of Hv1, suggesting that Hv1 contributes a majority of the charge compensation required for optimal NADPH oxidase activity. Despite significant reduction in superoxide production, Hv1(-/-) mice are able to clear several types of bacterial infections.

All four CatSper ion channel proteins are required for male fertility and sperm cell hyperactivated motility.

Mammalian spermatozoa become motile at ejaculation, but before they can fertilize the egg, they must acquire more thrust to penetrate the cumulus and zona pellucida. The forceful asymmetric motion of hyperactivated spermatozoa requires Ca2+ entry into the sperm tail by an alkalinization-activated voltage-sensitive Ca2+-selective current (ICatSper). Hyperactivation requires CatSper1 and CatSper2 putative ion channel genes, but the function of two other related genes (CatSper3 and CatSper4) is not known. Here we show that targeted disruption of murine CatSper3 or CatSper4 also abrogated ICatSper, sperm cell hyperactivated motility and male fertility but did not affect spermatogenesis or initial motility. Direct protein interactions among CatSpers, the sperm specificity of these proteins, and loss of ICatSper in each of the four CatSper-/- mice indicate that CatSpers are highly specialized flagellar proteins.

A voltage-gated proton-selective channel lacking the pore domain.

Voltage changes across the cell membrane control the gating of many cation-selective ion channels. Conserved from bacteria to humans, the voltage-gated-ligand superfamily of ion channels are encoded as polypeptide chains of six transmembrane-spanning segments (S1-S6). S1-S4 functions as a self-contained voltage-sensing domain (VSD), in essence a positively charged lever that moves in response to voltage changes. The VSD 'ligand' transmits force via a linker to the S5-S6 pore domain 'receptor', thereby opening or closing the channel. The ascidian VSD protein Ci-VSP gates a phosphatase activity rather than a channel pore, indicating that VSDs function independently of ion channels. Here we describe a mammalian VSD protein (H(V)1) that lacks a discernible pore domain but is sufficient for expression of a voltage-sensitive proton-selective ion channel activity. H(v)1 currents are activated at depolarizing voltages, sensitive to the transmembrane pH gradient, H+-selective, and Zn2+-sensitive. Mutagenesis of H(v)1 identified three arginine residues in S4 that regulate channel gating and two histidine residues that are required for extracellular inhibition of H(v)1 by Zn2+. H(v)1 is expressed in immune tissues and manifests the characteristic properties of native proton conductances (G(vH+)). In phagocytic leukocytes, G(vH+) are required to support the oxidative burst that underlies microbial killing by the innate immune system. The data presented here identify H(v)1 as a long-sought voltage-gated H+ channel and establish H(v)1 as the founding member of a family of mammalian VSD proteins.

An introduction to TRP channels.

The aim of this review is to provide a basic framework for understanding the function of mammalian transient receptor potential (TRP) channels, particularly as they have been elucidated in heterologous expression systems. Mammalian TRP channel proteins form six-transmembrane (6-TM) cation-permeable channels that may be grouped into six subfamilies on the basis of amino acid sequence homology (TRPC, TRPV, TRPM, TRPA, TRPP, and TRPML). Selected functional properties of TRP channels from each subfamily are summarized in this review. Although a single defining characteristic of TRP channel function has not yet emerged, TRP channels may be generally described as calcium-permeable cation channels with polymodal activation properties. By integrating multiple concomitant stimuli and coupling their activity to downstream cellular signal amplification via calcium permeation and membrane depolarization, TRP channels appear well adapted to function in cellular sensation. Our review of recent literature implicating TRP channels in neuronal growth cone steering suggests that TRPs may function more widely in cellular guidance and chemotaxis. The TRP channel gene family and its nomenclature, the encoded proteins and alternatively spliced variants, and the rapidly expanding pharmacology of TRP channels are summarized in online supplemental material.

Rapid vesicular translocation and insertion of TRP channels.

The broadly expressed transient receptor potential (TRP) family of ion channels are permeant to cations, most resulting in increased intracellular calcium. However, their regulation and gating is not well understood. Here, we report that growth factor stimulation initiates the rapid translocation of the transient receptor potential ion channel, TRPC5, from vesicles held in reserve just under the plasma membrane. This process, which we term 'rapid vesicular insertion of TRP' (RiVIT), dramatically increases membrane-associated TRPC5 channels and functional TRPC5 current, resulting in tight spatial-temporal control of these Ca(2+)-permeant nonselective channels. Epidermal growth factor (EGF)-induced incorporation of functional TRP channels requires phosphatidylinositide 3-kinase (PI(3)K), the Rho GTPase Rac1 and phosphatidylinositol 4-phosphate 5-kinase (PIP(5)K alpha). The increase in TRPC5 availability affects neurite extension rates in cultured hippocampal neurons, and may be a general mechanism for initiating Ca(2+) influx and cell morphological changes in response to stimuli.

TRPV3 is a calcium-permeable temperature-sensitive cation channel.

Transient receptor potential (TRP) proteins are cation-selective channels that function in processes as diverse as sensation and vasoregulation. Mammalian TRP channels that are gated by heat and capsaicin (>43 degrees C; TRPV1 (ref. 1)), noxious heat (>52 degrees C; TRPV2 (ref. 2)), and cooling (< 22 degrees C; TRPM8 (refs 3, 4)) have been cloned; however, little is known about the molecular determinants of temperature sensing in the range between approximately 22 degrees C and 40 degrees C. Here we have identified a member of the vanilloid channel family, human TRPV3 (hTRPV3) that is expressed in skin, tongue, dorsal root ganglion, trigeminal ganglion, spinal cord and brain. Increasing temperature from 22 degrees C to 40 degrees C in mammalian cells transfected with hTRPV3 elevated intracellular calcium by activating a nonselective cationic conductance. As in published recordings from sensory neurons, the current was steeply dependent on temperature, sensitized with repeated heating, and displayed a marked hysteresis on heating and cooling. On the basis of these properties, we propose that hTRPV3 is thermosensitive in the physiological range of temperatures between TRPM8 and TRPV1.

Serotonin transporter function and pharmacology are sensitive to expression level: evidence for an endogenous regulatory factor.

We express mammalian serotonin transporters (SERTs) in Xenopus oocytes by cRNA injection and measure 5-hydroxytryptamine (5-HT) transport and 5-HT-induced current at varying expression levels. Transport and current both increase sigmoidally with the amount of cRNA injected, but current requires approximately 5-fold more cRNA to elicit a half-maximal response. Western blots of SERT protein demonstrate that current, but not transport, correlates linearly with the amount of SERT on the plasma membrane. In oocytes co-injected with wild-type SERT and an inactive SERT mutant, transport is similar to SERT alone, but current is attenuated. The charge/transport ratio reports the differential sensitivity of transport and current to increasing SERT cRNA injection and mutant co-expression. Manipulations that alter the charge/transport ratio also perturb substrate and inhibitor recognition. 5-HT, d-amphetamine, cocaine, and paroxetine inhibit transport more potently at lower expression levels; however, 5-HT potency for induction of current is similar at high and low expression. Moreover, the apparent potency of cRNA for transport depends on 5-HT concentration. We postulate that SERT interacts allosterically with an endogenous factor of limited abundance to alter substrate and inhibitor potency and the balance of 5-HT transport and channel-like activity.