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Biomedical subjects

David E Clapham

Publications and source records attributed to David E Clapham.

At least 19 recordsLinked to original sources
PubMed2007

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.

Animals↗
PubMed2006

Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart.

Despite recent advances in delineating the mechanisms involved in cardiogenesis, cellular lineage specification remains incompletely understood. To explore the relationship between developmental fate and potential, we isolated a cardiac-specific Nkx2.5(+) cell population from the developing mouse embryo. The majority of these cells differentiated into cardiomyocytes and conduction system cells. Some, surprisingly, adopted a smooth muscle fate. To address the clonal origin of these lineages, we isolated Nkx2.5(+) cells from in vitro differentiated murine embryonic stem cells and found approximately 28% of these cells expressed c-kit. These c-kit(+) cells possessed the capacity for long-term in vitro expansion and differentiation into both cardiomyocytes and smooth muscle cells from a single cell. We confirmed these findings by isolating c-kit(+)Nkx2.5(+) cells from mouse embryos and demonstrated their capacity for bipotential differentiation in vivo. Taken together, these results support the existence of a common precursor for cardiovascular lineages in the mammalian heart.

Animals↗
PubMed2006

The TRPM7 ion channel functions in cholinergic synaptic vesicles and affects transmitter release.

A longstanding hypothesis is that ion channels are present in the membranes of synaptic vesicles and might affect neurotransmitter release. Here we demonstrate that TRPM7, a member of the transient receptor potential (TRP) ion channel family, resides in the membrane of synaptic vesicles of sympathetic neurons, forms molecular complexes with the synaptic vesicle proteins synapsin I and synaptotagmin I, and directly interacts with synaptic vesicular snapin. In sympathetic neurons, changes in TRPM7 levels and channel activity alter acetylcholine release, as measured by EPSP amplitudes and decay times in postsynaptic neurons. TRPM7 affects EPSP quantal size, an intrinsic property of synaptic vesicle release. Targeted peptide interference of TRPM7's interaction with snapin affects the amplitudes and kinetics of postsynaptic EPSPs. Thus, vesicular TRPM7 channel activity is critical to neurotransmitter release in sympathetic neurons.

Acetylcholine↗
PubMed2006

Bisandrographolide from Andrographis paniculata activates TRPV4 channels.

Many transient receptor potential (TRP) channels are activated or blocked by various compounds found in plants; two prominent examples include the activation of TRPV1 channels by capsaicin and the activation of TRPM8 channels by menthol. We sought to identify additional plant compounds that are active on other types of TRP channels. We screened a library of extracts from 50 Chinese herbal plants using a calcium-imaging assay to find compounds active on TRPV3 and TRPV4 channels. An extract from the plant Andrographis paniculata potently activated TRPV4 channels. The extract was fractionated further, and the active compound was identified as bisandrographolide A (BAA). We used purified compound to characterize the activity of BAA on certain TRPV channel subtypes. Although BAA activated TRPV4 channels with an EC(50) of 790-950 nm, it did not activate or block activation of TRPV1, TRPV2, or TRPV3 channels. BAA activated a large TRPV4-like current in immortalized mouse keratinocytes (308 cells) that have been shown to express TRPV4 protein endogenously. This compound also activated TRPV4 currents in cell-free outside-out patches from HEK293T cells overexpressing TRPV4 cDNA, suggesting that BAA can activate the channel in a membrane-delimited manner. Another related compound, andrographolide, found in abundance in the plant Andrographis was unable to activate or block activation of TRPV4 channels. These experiments show that BAA activates TRPV4 channels, and we discuss the possibility that activation of TRPV4 by BAA could play a role in some of the effects of Andrographis extract described in traditional medicine.

Andrographis↗
PubMed2006

Calbindin-D28K dynamically controls TRPV5-mediated Ca2+ transport.

In Ca(2+)-transporting epithelia, calbindin-D(28K) (CaBP(28K)) facilitates Ca(2+) diffusion from the luminal Ca(2+) entry side of the cell to the basolateral side, where Ca(2+) is extruded into the extracellular compartment. Simultaneously, CaBP(28K) provides protection against toxic high Ca(2+) levels by buffering the cytosolic Ca(2+) concentration ([Ca(2+)](i)) during high Ca(2+) influx. CaBP(28K) consistently colocalizes with the epithelial Ca(2+) channel TRPV5, which constitutes the apical entry step in renal Ca(2+)-transporting epithelial cells. Here, we demonstrate using protein-binding analysis, subcellular fractionation and evanescent-field microscopy that CaBP(28K) translocates towards the plasma membrane and directly associates with TRPV5 at a low [Ca(2+)](i). (45)Ca(2+) uptake measurements, electrophysiological recordings and transcellular Ca(2+) transport assays of lentivirus-infected primary rabbit connecting tubule/distal convolute tubule cells revealed that associated CaBP(28K) tightly buffers the flux of Ca(2+) entering the cell via TRPV5, facilitating high Ca(2+) transport rates by preventing channel inactivation. In summary, CaBP(28K) acts in Ca(2+)-transporting epithelia as a dynamic Ca(2+) buffer, regulating [Ca(2+)] in close vicinity to the TRPV5 pore by direct association with the channel.

Animals↗
PubMed2006

CACNA1H mutations in autism spectrum disorders.

Autism spectrum disorders (ASD) are neurodevelopmental conditions characterized by impaired social interaction, communication skills, and restricted and repetitive behavior. The genetic causes for autism are largely unknown. Previous studies implicate CACNA1C (L-type Ca(V)1.2) calcium channel mutations in a disorder associated with autism (Timothy syndrome). Here, we identify missense mutations in the calcium channel gene CACNA1H (T-type Ca(V)3.2) in 6 of 461 individuals with ASD. These mutations are located in conserved and functionally relevant domains and are absent in 480 ethnically matched controls (p = 0.014, Fisher's exact test). Non-segregation within the pedigrees between the mutations and the ASD phenotype clearly suggest that the mutations alone are not responsible for the condition. However, functional analysis shows that all these mutations significantly reduce Ca(V)3.2 channel activity and thus could affect neuronal function and potentially brain development. We conclude that the identified mutations could contribute to the development of the ASD phenotype.

Autistic Disorder↗
PubMed2006

Oregano, thyme and clove-derived flavors and skin sensitizers activate specific TRP channels.

Carvacrol, eugenol and thymol are major components of plants such as oregano, savory, clove and thyme. When applied to the tongue, these flavors elicit a warm sensation. They are also known to be skin sensitizers and allergens. The transient receptor potential channel (TRPV3) is a warm-sensitive Ca2+-permeable cation channel highly expressed in the skin, tongue and nose. Here we show that TRPV3 is strongly activated and sensitized by carvacrol, thymol and eugenol. Tongue and skin epithelial cells respond to carvacrol and eugenol with an increase in intracellular Ca2+ levels. We also show that this TRPV3 activity is strongly potentiated by phospholipase C-linked, G protein-coupled receptor stimulation. In addition, carvacrol activates and rapidly desensitizes TRPA1, which may explain the pungency of oregano. Our results support a role for temperature-sensitive TRP channels in chemesthesis in oral and nasal epithelium and suggest that TRPV3 may be a molecular target of plant-derived skin sensitizers.

Animals↗
PubMed2006

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.

Cell Line↗
PubMed2006

Whole-cell patch-clamp measurements of spermatozoa reveal an alkaline-activated Ca2+ channel.

In mammals, sperm cells become motile during ejaculation and swim up the female reproductive tract. Before fertilization and to overcome various barriers, their motility must be hyperactivated, a motion that is characterized by vigorous asymmetric tail beating. Hyperactivation requires an increase in calcium in the flagella, a process that probably involves plasmalemmal ion channels. Numerous attempts in the past two decades to understand sperm cell channels have been frustrated by the difficulty of measuring spermatozoan transmembrane ion currents. Here, by using a simple approach to patch-clamp spermatozoa and to characterize whole-spermatozoan currents, we describe a constitutively active flagellar calcium channel that is strongly potentiated by intracellular alkalinization. This current is not present in spermatozoa lacking the sperm-specific putative ion channel protein, CatSper1. This plasma membrane protein of the six transmembrane-spanning ion channel superfamily is specifically localized to the principal piece of the sperm tail and is required for sperm cell hyperactivation and male fertility. Our results identify CatSper1 as a component of the key flagellar calcium channel, and suggest that intracellular alkalinization potentiates CatSper current to increase intraflagellar calcium and induce sperm hyperactivation.

Alkalies↗
PubMed2006

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.

Amino Acid Sequence↗
PubMed2005

Functional TRPM7 channels accumulate at the plasma membrane in response to fluid flow.

Many cells are constantly exposed to fluid mechanical forces generated by flowing blood, and wall shear stresses modulate aspects of their structure and function. However, the mechanisms for mechanotransduction of flow are not well understood. Here we report that TRPM7, which is both an ion channel and a functional kinase, is translocated within cells in response to laminar flow. After exposure of cells to physiological values of laminar fluid flow, the number of TRPM7 molecules localized at or near the plasma membrane increased up to 2-fold, in less than 100 seconds. This increase in membrane-localized GFP-TRPM7, as seen by total internal reflection fluorescence microscopy, closely correlated with increases in TRPM7 current. Both endogenous and heterologously expressed TRPM7 was found in tubulovesicular structures that were translocated to the region of the plasma membrane on induction of shear stress. In vascular smooth muscle cells, but not in several types of endothelial cells, fluid flow increased endogenous native TRPM7 current amplitude. We hypothesize that TRPM7 plays a role in pathological response to vessel wall injury.

Blood Circulation↗
PubMed2005

Camphor activates and strongly desensitizes the transient receptor potential vanilloid subtype 1 channel in a vanilloid-independent mechanism.

Camphor is a naturally occurring compound that is used as a major active ingredient of balms and liniments supplied as topical analgesics. Despite its long history of common medical use, the underlying molecular mechanism of camphor action is not understood. Capsaicin and menthol, two other topically applied agents widely used for similar purposes, are known to excite and desensitize sensory nerves by acting on two members of transient receptor potential (TRP) channel superfamily: heat-sensitive TRP vanilloid subtype 1 (TRPV1) and cold-sensitive TRP channel M8, respectively. Camphor has recently been shown to activate TRPV3, and here we show that camphor also activates heterologously expressed TRPV1, requiring higher concentrations than capsaicin. Activation was enhanced by phospholipase C-coupled receptor stimulation mimicking inflamed conditions. Similar camphor-activated TRPV1-like currents were observed in isolated rat DRG neurons and were strongly potentiated after activation of protein kinase C with phorbol-12-myristate-13-acetate. Camphor activation of rat TRPV1 was mediated by distinct channel regions from capsaicin, as indicated by camphor activation in the presence of the competitive inhibitor capsazepine and in a capsaicin-insensitive point mutant. Camphor did not activate the capsaicin-insensitive chicken TRPV1. TRPV1 desensitization is believed to contribute to the analgesic actions of capsaicin. We found that, although camphor activates TRPV1 less effectively, camphor application desensitized TRPV1 more rapidly and completely than capsaicin. Conversely, TRPV3 current sensitized after repeated camphor applications, which is inconsistent with the analgesic role of camphor. We also found that camphor inhibited several other related TRP channels, including ankyrin-repeat TRP 1 (TRPA1). The camphor-induced desensitization of TRPV1 and block of TRPA1 may underlie the analgesic effects of camphor.

Animals↗
PubMed2005

TRP channels and mice deficient in TRP channels.

Transient receptor potential (TRP) channels are a superfamily of functionally versatile cation-permeant ion channels present in almost all mammalian cell types. Although they were initially proposed as store-operated calcium channels, recent progress shows that they exhibit a variety of regulatory and functional themes. Here, we summarize the most salient features of TRP channels, the approaches that are providing meaningful discoveries, and the challenges ahead. We primarily emphasize the understanding gleaned from mouse models engineered to be deficient in various members of TRP superfamily and from the human patients that suffer clinically due to defects in TRP channels.

Animals↗
PubMed2005

TRPC6 is a glomerular slit diaphragm-associated channel required for normal renal function.

Progressive kidney failure is a genetically and clinically heterogeneous group of disorders. Podocyte foot processes and the interposed glomerular slit diaphragm are essential components of the permeability barrier in the kidney. Mutations in genes encoding structural proteins of the podocyte lead to the development of proteinuria, resulting in progressive kidney failure and focal segmental glomerulosclerosis. Here, we show that the canonical transient receptor potential 6 (TRPC6) ion channel is expressed in podocytes and is a component of the glomerular slit diaphragm. We identified five families with autosomal dominant focal segmental glomerulosclerosis in which disease segregated with mutations in the gene TRPC6 on chromosome 11q. Two of the TRPC6 mutants had increased current amplitudes. These data show that TRPC6 channel activity at the slit diaphragm is essential for proper regulation of podocyte structure and function.

Adolescent↗
PubMed2005

TATA-binding protein (TBP)-like factor (TLF) is a functional regulator of transcription: reciprocal regulation of the neurofibromatosis type 1 and c-fos genes by TLF/TRF2 and TBP.

The lack of direct targets for TATA-binding protein (TBP)-like factors (TLFs) confounds the understanding of their role in gene expression. Here we report that human TLF (also called TBP-related factor 2 [TRF2]) activates a number of different genes, including the neurofibromatosis type 1 (NF1) gene. The overexpression of TLF increases the amount of NF1 mRNA in cells. In vivo, TLF binds to and upregulates transcription from a fragment of the NF1 promoter. In vitro, purified TLF-TFIIA binds directly to the same NF1 promoter fragment that is required for TLF responsiveness in cells. Furthermore, targeted deletion of TLF in mice reduces NF1 levels. In contrast, TLF inhibits transcription driven by a fragment from the TATA-containing c-fos promoter by sequestering TFIIA. TBP affects the NF1 and c-fos promoters in a manner reciprocal to that of TLF, stimulating the c-fos promoter and inhibiting NF1 transcription. We conclude that TLF is a functional regulator of transcription with targets distinct from those of TBP.

Amino Acid Sequence↗