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Clapham, David

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Clapham

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David

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Clapham, David
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    Caspase-11 Controls Interleukin-1β Release through Degradation of TRPC1
    (2014) Py, Bénédicte F.; Jin, Mingzhi; Desai, Bimal N.; Penumaka, Anirudh; Zhu, Hong; Kober, Maike; Dietrich, Alexander; Lipinski, Marta M.; Henry, Thomas; Clapham, David; Yuan, Junying
    SUMMARY Caspase-11 is a highly inducible caspase that controls both inflammatory responses and cell death. Caspase-11 controls interleukin 1β (IL-1β) secretion by potentiating caspase-1 activation and induces caspase-1-independent pyroptosis downstream of noncanonical NLRP3 inflammasome activators such as lipopolysaccharide (LPS) and Gram-negative bacteria. However, we still know very little about the downstream mechanism of caspase-11 in regulating inflammation because the known substrates of caspase-11 are only other caspases. Here, we identify the cationic channel subunit transient receptor potential channel 1 (TRPC1) as a substrate of caspase-11. TRPC1 deficiency increases the secretion of IL-1β without modulating caspase-1 cleavage or cell death in cultured macrophages. Consistently, trpc1−/− mice show higher IL-1β secretion in the sepsis model of intraperitoneal LPS injection. Altogether, our data suggest that caspase-11 modulates the cationic channel composition of the cell and thus regulates the unconventional secretion pathway in a manner independent of caspase-1.
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    CatSperζ regulates the structural continuity of sperm Ca2+ signaling domains and is required for normal fertility
    (eLife Sciences Publications, Ltd, 2017) Chung, Jean-Ju; Miki, Kiyoshi; Kim, Doory; Shim, Sang-Hee; Shi, Huanan F; Hwang, Jae Yeon; Cai, Xinjiang; Iseri, Yusuf; Zhuang, Xiaowei; Clapham, David
    We report that the Gm7068 (CatSpere) and Tex40 (CatSperz) genes encode novel subunits of a 9-subunit CatSper ion channel complex. Targeted disruption of CatSperz reduces CatSper current and sperm rheotactic efficiency in mice, resulting in severe male subfertility. Normally distributed in linear quadrilateral nanodomains along the flagellum, the complex lacking CatSperζ is disrupted at ~0.8 μm intervals along the flagellum. This disruption renders the proximal flagellum inflexible and alters the 3D flagellar envelope, thus preventing sperm from reorienting against fluid flow in vitro and efficiently migrating in vivo. Ejaculated CatSperz-null sperm cells retrieved from the mated female uterus partially rescue in vitro fertilization (IVF) that failed with epididymal spermatozoa alone. Human CatSperε is quadrilaterally arranged along the flagella, similar to the CatSper complex in mouse sperm. We speculate that the newly identified CatSperζ subunit is a late evolutionary adaptation to maximize fertilization inside the mammalian female reproductive tract. DOI: http://dx.doi.org/10.7554/eLife.23082.001
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    Polycystin-2 is an essential ion channel subunit in the primary cilium of the renal collecting duct epithelium
    (eLife Sciences Publications, Ltd, 2018) Liu, Xiaowen; Vien, Thuy; Duan, Jingjing; Sheu, Shu-Hsien; DeCaen, Paul G; Clapham, David
    Mutations in the polycystin genes, PKD1 or PKD2, results in Autosomal Dominant Polycystic Kidney Disease (ADPKD). Although a genetic basis of ADPKD is established, we lack a clear understanding of polycystin proteins’ functions as ion channels. This question remains unsolved largely because polycystins localize to the primary cilium – a tiny, antenna-like organelle. Using a new ADPKD mouse model, we observe primary cilia that are abnormally long in cells associated with cysts after conditional ablation of Pkd1 or Pkd2. Using primary cultures of collecting duct cells, we show that polycystin-2, but not polycystin-1, is a required subunit for the ion channel in the primary cilium. The polycystin-2 channel preferentially conducts K+ and Na+; intraciliary Ca2+, enhances its open probability. We introduce a novel method for measuring heterologous polycystin-2 channels in cilia, which will have utility in characterizing PKD2 variants that cause ADPKD.
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    Role of the C-terminal domain in the structure and function of tetrameric sodium channels
    (Nature Pub. Group, 2013) Bagnéris, Claire; DeCaen, Paul; Hall, Benjamin A.; Naylor, Claire E.; Clapham, David; Kay, Christopher W. M.; Wallace, B. A.
    Voltage-gated sodium channels have essential roles in electrical signalling. Prokaryotic sodium channels are tetramers consisting of transmembrane (TM) voltage-sensing and pore domains, and a cytoplasmic carboxy-terminal domain. Previous crystal structures of bacterial sodium channels revealed the nature of their TM domains but not their C-terminal domains (CTDs). Here, using electron paramagnetic resonance (EPR) spectroscopy combined with molecular dynamics, we show that the CTD of the NavMs channel from Magnetococcus marinus includes a flexible region linking the TM domains to a four-helix coiled-coil bundle. A 2.9 Å resolution crystal structure of the NavMs pore indicates the position of the CTD, which is consistent with the EPR-derived structure. Functional analyses demonstrate that the coiled-coil domain couples inactivation with channel opening, and is enabled by negatively charged residues in the linker region. A mechanism for gating is proposed based on the structure, whereby splaying of the bottom of the pore is possible without requiring unravelling of the coiled-coil.
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    Sperm BerserKers
    (eLife Sciences Publications, Ltd, 2013) Clapham, David
    Human sperm cells rely on an unusual type of potassium ion channel.
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    Simultaneous knockout of Slo3 and CatSper1 abolishes all alkalization- and voltage-activated current in mouse spermatozoa
    (The Rockefeller University Press, 2013) Zeng, Xu-Hui; Navarro, Betsy; Xia, Xiao-Ming; Clapham, David; Lingle, Christopher J.
    During passage through the female reproductive tract, mammalian sperm undergo a maturation process termed capacitation that renders sperm competent to produce fertilization. Capacitation involves a sequence of changes in biochemical and electrical properties, the onset of a hyperactivated swimming behavior, and development of the ability to undergo successful fusion and penetration with an egg. In mouse sperm, the development of hyperactivated motility is dependent on cytosolic alkalization that then results in an increase in cytosolic Ca2+. The elevation of Ca2+ is thought to be primarily driven by the concerted interplay of two alkalization-activated currents, a K+ current (KSPER) composed of pore-forming subunits encoded by the Kcnu1 gene (also termed Slo3) and a Ca2+ current arising from a family of CATSPER subunits. After deletion of any of four CATSPER subunit genes (CATSPER1–4), the major remaining current in mouse sperm is alkalization-activated KSPER current. After genetic deletion of the Slo3 gene, KSPER current is abolished, but there remains a small voltage-activated K+ current hypothesized to reflect monovalent flux through CATSPER. Here, we address two questions. First, does the residual outward K+ current present in the Slo3 −/− sperm arise from CATSPER? Second, can any additional membrane K+ currents be detected in mouse sperm by patch-clamp methods other than CATSPER and KSPER? Here, using mice bred to lack both SLO3 and CATSPER1 subunits, we show conclusively that the voltage-activated outward current present in Slo3 −/− sperm is abolished when CATSPER is also deleted. Any leak currents that may play a role in setting the resting membrane potential in noncapacitated sperm are likely smaller than the pipette leak current and thus cannot be resolved within the limitation of the patch-clamp technique. Together, KSPER and CATSPER appear to be the sole ion channels present in mouse sperm that regulate membrane potential and Ca2+ influx in response to alkalization.
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    The G-protein–gated K+ channel, IKACh, is required for regulation of pacemaker activity and recovery of resting heart rate after sympathetic stimulation
    (The Rockefeller University Press, 2013) Mesirca, Pietro; Marger, Laurine; Toyoda, Futoshi; Rizzetto, Riccardo; Audoubert, Matthieu; Dubel, Stefan; Torrente, Angelo G.; DiFrancesco, Mattia L.; Muller, Jana Christina; Leoni, Anne-Laure; Couette, Brigitte; Nargeot, Joël; Clapham, David; Wickman, Kevin; Mangoni, Matteo E.
    Parasympathetic regulation of sinoatrial node (SAN) pacemaker activity modulates multiple ion channels to temper heart rate. The functional role of the G-protein–activated K+ current (IKACh) in the control of SAN pacemaking and heart rate is not completely understood. We have investigated the functional consequences of loss of IKACh in cholinergic regulation of pacemaker activity of SAN cells and in heart rate control under physiological situations mimicking the fight or flight response. We used knockout mice with loss of function of the Girk4 (Kir3.4) gene (Girk4−/− mice), which codes for an integral subunit of the cardiac IKACh channel. SAN pacemaker cells from Girk4−/− mice completely lacked IKACh. Loss of IKACh strongly reduced cholinergic regulation of pacemaker activity of SAN cells and isolated intact hearts. Telemetric recordings of electrocardiograms of freely moving mice showed that heart rate measured over a 24-h recording period was moderately increased (10%) in Girk4−/− animals. Although the relative extent of heart rate regulation of Girk4−/− mice was similar to that of wild-type animals, recovery of resting heart rate after stress, physical exercise, or pharmacological β-adrenergic stimulation of SAN pacemaking was significantly delayed in Girk4−/− animals. We conclude that IKACh plays a critical role in the kinetics of heart rate recovery to resting levels after sympathetic stimulation or after direct β-adrenergic stimulation of pacemaker activity. Our study thus uncovers a novel role for IKACh in SAN physiology and heart rate regulation.
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    A Novel Gene Required for Male Fertility and Functional CATSPER Channel Formation in Spermatozoa
    (2014) Chung, Jean-Ju; Navarro, Betsy; Krapivinsky, Grigory; Krapivinsky, Luba; Clapham, David
    Summary Calcium signaling is critical for successful fertilization. In spermatozoa, capacitation, hyperactivation of motility, and the acrosome reaction are all mediated by increases in intracellular Ca2+. Cation channels of sperm proteins (CATSPERS1-4) form an alkalinization-activated Ca2+-selective channel required for the hyperactivated motility of spermatozoa and male fertility. Each of the CatSper1-4 genes encodes a subunit of a tetramer surrounding a Ca2+-selective pore, in analogy with other six-transmembrane ion channel α subunits. In addition to the pore-forming proteins, the sperm Ca2+ channel contains auxiliary subunits, CATSPERβ and CATSPERγ. Here, we identify the Tmem146 gene product as a novel subunit, CATSPERδ, required for CATSPER channel function. We find that mice lacking the sperm tail-specific CATSPERδ are infertile and their spermatozoa lack both Ca2+ current and hyperactivated motility. We show that CATSPERδ is an essential element of the CATSPER channel complex and propose that CATSPERδ is required for proper CATSPER channel assembly and/or transport.
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    MCU encodes the pore conducting mitochondrial calcium currents
    (eLife Sciences Publications, Ltd, 2013) Chaudhuri, Dipayan; Sancak, Yasemin; Mootha, Vamsi; Clapham, David
    Mitochondrial calcium (Ca2+) import is a well-described phenomenon regulating cell survival and ATP production. Of multiple pathways allowing such entry, the mitochondrial Ca2+ uniporter is a highly Ca2+-selective channel complex encoded by several recently-discovered genes. However, the identity of the pore-forming subunit remains to be established, since knockdown of all the candidate uniporter genes inhibit Ca2+ uptake in imaging assays, and reconstitution experiments have been equivocal. To definitively identify the channel, we use whole-mitoplast voltage-clamping, the technique that originally established the uniporter as a Ca2+ channel. We show that RNAi-mediated knockdown of the mitochondrial calcium uniporter (MCU) gene reduces mitochondrial Ca2+ current (IMiCa), whereas overexpression increases it. Additionally, a classic feature of IMiCa, its sensitivity to ruthenium red inhibition, can be abolished by a point mutation in the putative pore domain without altering current magnitude. These analyses establish that MCU encodes the pore-forming subunit of the uniporter channel. DOI: http://dx.doi.org/10.7554/eLife.00704.001
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    Functional reconstitution of the mitochondrial Ca2+/H+ antiporter Letm1
    (The Rockefeller University Press, 2014) Tsai, Ming-Feng; Jiang, Dawei; Zhao, Linlin; Clapham, David; Miller, Christopher
    The leucine zipper, EF hand–containing transmembrane protein 1 (Letm1) gene encodes a mitochondrial inner membrane protein, whose depletion severely perturbs mitochondrial Ca2+ and K+ homeostasis. Here we expressed, purified, and reconstituted human Letm1 protein in liposomes. Using Ca2+ fluorophore and 45Ca2+-based assays, we demonstrate directly that Letm1 is a Ca2+ transporter, with apparent affinities of cations in the sequence of Ca2+ ≈ Mn2+ > Gd3+ ≈ La3+ > Sr2+ >> Ba2+, Mg2+, K+, Na+. Kinetic analysis yields a Letm1 turnover rate of 2 Ca2+/s and a Km of ∼25 µM. Further experiments show that Letm1 mediates electroneutral 1 Ca2+/2 H+ antiport. Letm1 is insensitive to ruthenium red, an inhibitor of the mitochondrial calcium uniporter, and CGP-37157, an inhibitor of the mitochondrial Na+/Ca2+ exchanger. Functional properties of Letm1 described here are remarkably similar to those of the H+-dependent Ca2+ transport mechanism identified in intact mitochondria.