The tetramerization domain potentiates Kv4 channel function by suppressing closed-state inactivation.

A-type Kv4 potassium channels undergo a conformational change toward a nonconductive state at negative membrane potentials, a dynamic process known as pre-open closed states or closed-state inactivation (CSI). CSI causes inhibition of channel activity without the prerequisite of channel opening, thus providing a dynamic regulation of neuronal excitability, dendritic signal integration, and synaptic plasticity at resting. However, the structural determinants underlying Kv4 CSI remain largely unknown. We recently showed that the auxiliary KChIP4a subunit contains an N-terminal Kv4 inhibitory domain (KID) that directly interacts with Kv4.3 channels to enhance CSI. In this study, we utilized the KChIP4a KID to probe key structural elements underlying Kv4 CSI. Using fluorescence resonance energy transfer two-hybrid mapping and bimolecular fluorescence complementation-based screening combined with electrophysiology, we identified the intracellular tetramerization (T1) domain that functions to suppress CSI and serves as a receptor for the binding of KID. Disrupting the Kv4.3 T1-T1 interaction interface by mutating C110A within the C3H1 motif of T1 domain facilitated CSI and ablated the KID-mediated enhancement of CSI. Furthermore, replacing the Kv4.3 T1 domain with the T1 domain from Kv1.4 (without the C3H1 motif) or Kv2.1 (with the C3H1 motif) resulted in channels functioning with enhanced or suppressed CSI, respectively. Taken together, our findings reveal a novel (to our knowledge) role of the T1 domain in suppressing Kv4 CSI, and that KChIP4a KID directly interacts with the T1 domain to facilitate Kv4.3 CSI, thus leading to inhibition of channel function.

A TRPV4 channel C-terminal folding recognition domain critical for trafficking and function.

The Ca(2+)-permeable transient receptor potential vanilloid subtype 4 (TRPV4) channel mediates crucial physiological functions, such as calcium signaling, temperature sensing, and maintaining cell volume and energy homeostasis. Noticeably, most disease-causing genetic mutations are concentrated in the cytoplasmic domains. In the present study, we focused on the role of the TRPV4 C terminus in modulating protein folding, trafficking, and activity. By examining a series of C-terminal deletions, we identified a 20-amino acid distal region covering residues 838-857 that is critical for channel folding, maturation, and trafficking. Surface biotinylation, confocal imaging, and fluorescence-based calcium influx assay demonstrated that mutant proteins missing this region were trapped in the endoplasmic reticulum and unglycosylated, leading to accelerated degradation and loss of channel activity. Rosetta de novo structural modeling indicated that residues 838-857 assume a defined conformation, with Gly(849) and Pro(851) located at critical positions. Patch clamp recordings confirmed that lowering the temperature from 37 to 30 °C rescued channel activity of folding-defective mutants. Moreover, biochemical tests demonstrated that, in addition to participating in C-C interaction, the C terminus also interacts with the N terminus. Taken together, our findings indicate that the C-terminal region of TRPV4 is critical for channel protein folding and maturation, and the short distal segment plays an essential role in this process. Therefore, selectively disrupting the folding-sensitive region may present therapeutic potential for treating overactive TRPV4-mediated diseases, such as pain and skeletal dysplasias.

Auxiliary KChIP4a suppresses A-type K+ current through endoplasmic reticulum (ER) retention and promoting closed-state inactivation of Kv4 channels.

In the brain and heart, auxiliary Kv channel-interacting proteins (KChIPs) co-assemble with pore-forming Kv4 α-subunits to form a native K(+) channel complex and regulate the expression and gating properties of Kv4 currents. Among the KChIP1-4 members, KChIP4a exhibits a unique N terminus that is known to suppress Kv4 function, but the underlying mechanism of Kv4 inhibition remains unknown. Using a combination of confocal imaging, surface biotinylation, and electrophysiological recordings, we identified a novel endoplasmic reticulum (ER) retention motif, consisting of six hydrophobic and aliphatic residues, 12-17 (LIVIVL), within the KChIP4a N-terminal KID, that functions to reduce surface expression of Kv4-KChIP complexes. This ER retention capacity is transferable and depends on its flanking location. In addition, adjacent to the ER retention motif, the residues 19-21 (VKL motif) directly promote closed-state inactivation of Kv4.3, thus leading to an inhibition of channel current. Taken together, our findings demonstrate that KChIP4a suppresses A-type Kv4 current via ER retention and enhancement of Kv4 closed-state inactivation.

Distinct roles for two Caenorhabditis elegans acid-sensing ion channels in an ultradian clock.

Biological clocks are fundamental to an organism's health, controlling periodicity of behaviour and metabolism. Here, we identify two acid-sensing ion channels, with very different proton sensing properties, and describe their role in an ultradian clock, the defecation motor program (DMP) of the nematode Caenorhabditis elegans. An ACD-5-containing channel, on the apical membrane of the intestinal epithelium, is essential for maintenance of luminal acidity, and thus the rhythmic oscillations in lumen pH. In contrast, the second channel, composed of FLR-1, ACD-3 and/or DEL-5, located on the basolateral membrane, controls the intracellular Ca2+ wave and forms a core component of the master oscillator that controls the timing and rhythmicity of the DMP. flr-1 and acd-3/del-5 mutants show severe developmental and metabolic defects. We thus directly link the proton-sensing properties of these channels to their physiological roles in pH regulation and Ca2+ signalling, the generation of an ultradian oscillator, and its metabolic consequences.

Biological clocks regulate a myriad of processes that occur periodically, from sleeping and waking to how cells use nutrients and energy. One such clock is the one that controls intestinal movements and defecation in the nematode worm Caenorhabditis elegans, which consists of three muscle contractions occurring every 50 seconds. This rhythm is controlled by calcium and proton signalling in the cells of the intestine. The cells of the nematode intestine form a tube, through which gut contents pass. The inside of the tube is acidic, but acidity also plays a role on the outer face of the intestinal tube. In this area, nutrients are distributed and signals are conveyed to other tissues, such as muscles. In fact, acid ­ in the form of protons ­ secreted from the intestinal cells stimulates the muscles that contract in the biological clock that controls the worms' defecation. However, it is poorly understood how the worms control the release of these protons. Kaulich et al. identified two ion channels on the membranes of intestinal cells that become inhibited when the levels of acid surrounding them are high. These channels play distinct roles in controlling the contractions that move the contents of the roundworms' intestines along. The first channel contains a protein called ACD-5, and it is in the membrane of the intestinal cells that faces the inside of the intestinal tube. The second channel is formed by three proteins: FLR-1, ACD-3 and DEL-5. This channel is found on the other side of the intestinal cells, the region where nutrients are distributed and signals are conveyed to the rest of the body. To determine the role of each channel, Kaulich et al. genetically engineered the worms so they would not make the proteins that make up the channels, and imaged the live nematodes to see the effects of removing each channel. The inside of the intestines of worms lacking the ACD-5 containing channel was less acidic than that of normal worms, and the timing of the contractions that control defecation was also slightly altered. Removing the second channel (the one formed by three different proteins), however, had more dramatic effects: the worms were thin, developed more slowly, had less fat tissue and defecated very irregularly. Kaulich et al. imaged live worms to show that the second channel plays a major role in regulating oscillations in acidity both inside and outside cells, as well as controlling calcium levels. This demonstrates that this channel is responsible for the rhythmicity in the contractions that control defecation in the nematodes. Their findings provide important insights towards better understanding proton signalling and the role of acid-sensing ion channels in cellular contexts and biological clocks.

Screening strategy and time points for newborn hearing re-screening with high risk factors.

Objective: To compare and analyze the pass rate and screening strategy of hearing rescreening for newborns with high risk factors. Methods: Retrospective chart review of high-risk newborns who failed their initial newborn hearing screen and subsequently underwent secondary hearing tests from June 2011 to June 2018 in Guangzhou Women and Children's Medical Center were performed. Results: Eight hundred and sixty-eight newborns with high risk factors were included in the study. The 57-70 days (83.5%) and 71-84 days (83.4%) group had the highest pass rate compared with 42-56 days (75.8%) and < 42 days (68.3%) group. As for different screening strategies, the pass rate of OAE(otoacoustic emissions), AABR (auto auditory brainstem response) and OAE + AABR was the highest in 57-70 days group and 71-84 days group, respectively. The OAE + AABR had the lowest pass rate compared to the other two modalities. When the pass rate was compared as different risk factors, the 57-70 days and 71-84 days group also had the highest pass rate compared with 42-56 days and < 42 days group and the pass rate had no significant differences among various risk factors group. Conclusion: Our results showed that all the pass rate of OAE, AABR and OAE + AABR was the highest in 57-70 days group and 71-84 days group with significant difference, suggesting that the delayed screening time (>57 days) may increase the re-screening pass rate and reduce anxiety of parents, which is of great significance for clinical work.

Modulating serine palmitoyltransferase-deoxysphingolipid axis in cancer therapy.

A schematic illustration is given regarding serine restriction on tumor growth. Once the cellular abundance of serine decreased or alanine accumulated, the serine palmitoyltransferase (SPT) alternatively conjugates alanine and palmitoyl-CoA to form 3-keto-intermediates, which is rapidly converted to 1-deoxysphinganine and further metabolized to 1-deoxydihydroceramide (1-DeoxyDHCER) and 1-deoxyceramide (1-DeoxyDHCER), so that to exert cytotoxicity for tumor suppression.

The Caenorhabditis elegans tmc-1 is involved in egg-laying inhibition in response to harsh touch.

The conserved family of Transmembrane channel-like (TMC) proteins has attracted significant interest since two members appear to be key components of the mammalian hair cell mechanotransducer involved in hearing. C. elegans expresses two TMC proteins, TMC-1 and TMC-2. TMC-1 is widely expressed in in both muscles and the nervous system. This wide expression pattern suggests that TMC-1 might serve different functions in the various neurons. TMC-1 has previously been shown to function in neurons, playing a role in chemosensation in the ASH neurons and mechanosensation in OLQ neurons, further supporting this hypothesis. tmc-1 is expressed in the high-threshold mechanosensory neuron, ALA. We show that tmc-1 mutants show defects in the ALA-dependent inhibition of egg-laying in response to a harsh mechanical stimulus.

Photosensitive and Photoswitchable TRPA1 Agonists Optically Control Pain through Channel Desensitization.

Transient receptor potential ankyrin 1 (TRPA1) channel, as a nonselective ligand-gated cation channel robustly in dorsal root ganglion sensory neurons, is implicated in sensing noxious stimuli and nociceptive signaling. However, small-molecule tools targeting TRPA1 lack temporal and spatial resolution, limiting their use for validation of TRPA1 as a therapeutic target for pain. In our previous work, we found that 4,4'-(diazene-1,2-diyl)dianiline (AB1) is a photoswitchable TRPA1 agonist, but the poor water solubility and activity hinder its further development. Here, we report a series of specific and potent azobenzene-derived photoswitchable TRPA1 agonists (series 1 and 2) that enable optical control of the TRPA1 channel. Two representative compounds 1g and 2c can alleviate capsaicin-induced pain in the cheek model of mice through channel desensitization but not in TRPA1 knockout mice. Taken together, our findings demonstrate that photoswitchable TRPA1 agonists can be used as pharmacological tools for study of pain signaling.

Disrupting phosphorylation of Tyr-1070 at GluN2B selectively produces resilience to depression-like behaviors.

Drugs targeting N-methyl-D-aspartate receptors (NMDARs) have been approved to treat major depressive disorder (MDD); however, the presence of undesirable psychotomimetic and cognitive side effects may limit their utility. In this study, we show that the phosphorylation levels of the GluN2B subunit at tyrosine (Y) 1070 increase in mice after both acute and chronic restraint stress (CRS) exposure. Preventing GluN2B-Y1070 phosphorylation via Y1070F mutation knockin produces effects similar to those of antidepressants but does not affect cognitive or anxiety-related behaviors in subject mice. Mechanistically, the Y1070F mutation selectively reduces non-synaptic NMDAR currents and increases the number of excitatory synapses in the layer 5 pyramidal neurons of medial prefrontal cortex (mPFC) but not in the hippocampus. Altogether, our study identifies phosphorylation levels of GluN2B-Y1070 in the mPFC as a dynamic, master switch guarding depressive behaviors, suggesting that disrupting the Y1070 phosphorylation of GluN2B subunit has the potential for developing new antidepressants.

Metabolism in tumor microenvironment: Implications for cancer immunotherapy.

Tumor microenvironment is a special environment for tumor survival, which is characterized by hypoxia, acidity, nutrient deficiency, and immunosuppression. The environment consists of the vasculature, immune cells, extracellular matrix, and proteins or metabolic molecules. A large number of recent studies have shown that not only tumor cells but also the immune cells in the tumor microenvironment have undergone metabolic reprogramming, which is closely related to tumor drug resistance and malignant progression. Tumor immunotherapy based on T cells gives patients new hope, but faces the dilemma of low response rate. New strategies sensitizing cancer immunotherapy are urgently needed. Metabolic reprogramming can directly affect the biological activity of tumor cells and also regulate the differentiation and activation of immune cells. The authors aim to review the characteristics of tumor microenvironment, the metabolic changes of tumor-associated immune cells, and the regulatory role of metabolic reprogramming in cancer immunotherapy.

Ankyrin Is An Intracellular Tether for TMC Mechanotransduction Channels.

Mechanotransduction channels have been proposed as force sensors in various physiological processes, such as hearing and touch. In particular, TMC1 has been shown to constitute the pore of hair cell mechanotransduction channels, but little is known about how force is sensed by TMC channels. Here, we identify UNC-44/ankyrin as an essential component of the TMC-1 mechanotransduction channel complex in the sensory cilia of Caenorhabditis elegans mechanoreceptor neurons. Ankyrin binds indirectly to TMC-1 via evolutionarily conserved CIB proteins, which are required for TMC-1-mediated mechanosensation in C. elegans OLQ neurons and body wall muscles. Mechanosensory activity conferred by ectopically expressed TMCs in mechanoinsensitive neurons depends on both ankyrin and CIB proteins, indicating that the ankyrin-CIB subcomplex is required for TMC mechanosensitivity. Our work indicates that ankyrin is a long-sought intracellular tether that transmits force to TMC mechanotransduction channels.

Correction: Lymphopenia predicts disease severity of COVID-19: a descriptive and predictive study.

[This corrects the article DOI: 10.1038/s41392-020-0148-4.].

Inhibition of Hsp70 Suppresses Neuronal Hyperexcitability and Attenuates Epilepsy by Enhancing A-Type Potassium Current.

The heat shock protein 70 (Hsp70) is upregulated in response to stress and has been implicated as a stress marker in temporal lobe epilepsy (TLE). However, whether Hsp70 plays a pathologic or protective role in TLE remains unclear. Here we report a deleterious role of Hsp70 in kainic acid (KA)-induced seizures. Hsp70 expression is upregulated in a KA model of TLE, and silencing or inhibition of Hsp70 suppresses neuronal hyperexcitability and attenuates acute or chronic epilepsy by enhancing A-type potassium current in hippocampal neurons. Hsp70 upregulation leads to proteosomal degradation of Kv4-KChIP4a channel complexes primarily encoding neuronal A-type current. Furthermore, Hsp70 directly binds to the N terminus of auxiliary KChIP4a and targets Kv4-KChIP4a complexes to proteasome. Taken together, our findings reveal a role of Hsp70 in the pathogenesis of epilepsy through degradation of Kv4-KChIP4a complexes, and pharmacological inhibition of Hsp70 may represent therapeutic potential for epilepsy or hyperexcitability-related neurological disorders.

CIB2 interacts with TMC1 and TMC2 and is essential for mechanotransduction in auditory hair cells.

Inner ear hair cells detect sound through deflection of stereocilia, the microvilli-like projections that are arranged in rows of graded heights. Calcium and integrin-binding protein 2 is essential for hearing and localizes to stereocilia, but its exact function is unknown. Here, we have characterized two mutant mouse lines, one lacking calcium and integrin-binding protein 2 and one carrying a human deafness-related Cib2 mutation, and show that both are deaf and exhibit no mechanotransduction in auditory hair cells, despite the presence of tip links that gate the mechanotransducer channels. In addition, mechanotransducing shorter row stereocilia overgrow in hair cell bundles of both Cib2 mutants. Furthermore, we report that calcium and integrin-binding protein 2 binds to the components of the hair cell mechanotransduction complex, TMC1 and TMC2, and these interactions are disrupted by deafness-causing Cib2 mutations. We conclude that calcium and integrin-binding protein 2 is required for normal operation of the mechanotransducer channels and is involved in limiting the growth of transducing stereocilia.Inner ear hair cells detect sound through deflection of stereocilia that harbor mechanically-gated channels. Here the authors show that protein responsible for Usher syndrome, CIB2, interacts with these channels and is essential for their function and hearing in mice.

Antimicrobial peptides from human platelets.

Platelets share structural and functional similarities with granulocytes known to participate in antimicrobial host defense. To evaluate the potential antimicrobial activities of platelet proteins, normal human platelets were stimulated with human thrombin in vitro. Components of the stimulated-platelet supernatants were purified to homogeneity by reversed-phase high-performance liquid chromatography. Purified peptides with inhibitory activity against Escherichia coli ML35 in an agar diffusion antimicrobial assay were characterized by mass spectrometry, amino acid analysis, and sequence determination. These analyses enabled the identification of seven thrombin-releasable antimicrobial peptides from human platelets: platelet factor 4 (PF-4), RANTES, connective tissue activating peptide 3 (CTAP-3), platelet basic protein, thymosin beta-4 (Tbeta-4), fibrinopeptide B (FP-B), and fibrinopeptide A (FP-A). With the exception of FP-A and FP-B, all peptides were also purified from acid extracts of nonstimulated platelets. The in vitro antimicrobial activities of the seven released peptides were further tested against bacteria (E. coli and Staphylococcus aureus) and fungi (Candida albicans and Cryptococcus neoformans). Each peptide exerted activity against at least two organisms. Generally, the peptides were more potent against bacteria than fungi, activity was greater at acidic pHs, and antimicrobial activities were dose dependent. Exceptions to these observations were observed with PF-4, which displayed a bimodal dose-response relationship in microbicidal assays, and Tbeta-4, which had greater activity at alkaline pHs. At concentrations at which they were individually sublethal, PF-4 and CTAP-3 exerted synergistic microbicidal activity against E. coli. Collectively, these findings suggest a direct antimicrobial role for platelets as they are activated to release peptides in response to trauma or mediators of inflammation.

Homodimeric theta-defensins from rhesus macaque leukocytes: isolation, synthesis, antimicrobial activities, and bacterial binding properties of the cyclic peptides.

Rhesus theta-defensin 1 (RTD-1) is a unique tridisulfide, cyclic antimicrobial peptide formed by the ligation of two 9-residue sequences derived from heterodimeric splicing of similar 76-amino acid, alpha-defensin-related precursors, termed RTD1a and RTD1b (Tang, Y. Q., Yuan, J., Osapay, G., Osapay, K., Tran, D., Miller, C. J., Ouellette, A. J., and Selsted, M. E. (1999) Science 286, 498-502). The structures of RTD-2 and RTD-3 were predicted to exist if homodimeric splicing of the RTD1a and RTD1b occurs in vivo. Western blotting disclosed the presence of putative theta-defensins, distinct from RTD-1, in leukocyte extracts. Two new theta-defensins, RTD-2 and RTD-3, were purified by reverse-phase high performance liquid chromatography and characterized by amino acid analysis, matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, and comparison to the synthetic standards. RTD-2 and RTD-3 are the predicted homodimeric splicing products of RTD1b and RTD1a, respectively. The cellular abundances of RTD-1, -2, and -3 were 29:1:2, indicating that there is a preference for the heterodimeric ligation that generates RTD-1. RTD-1, -2, and -3 had similar antimicrobial activities against Staphylococcus aureus, Candida albicans, and Cryptococcus neoformans, whereas the activity of RTD-2 against Escherichia coli was 2-3-fold less than those of RTD-1 and RTD-3. Equal amounts of each theta-defensin bound to E. coli cells, indicating that the differences in antibacterial activities are the result of post-binding processes.