Biomedical subjects

R H Ashley

Publications and source records attributed to R H Ashley.

At least 19 recordsLinked to original sources

PubMed2002

Overexpressed chloride intracellular channel protein CLIC4 (p64H1) is an essential component of novel plasma membrane anion channels.

Chloride intracellular channel protein CLIC4 is a putative organellar anion channel or channel regulator with an unusual dual cytoplasmic and integral membrane localisation. To investigate its contribution to cellular anion channel activity, the protein was overexpressed in stably transfected HEK-293 cells. Patch-clamp recording revealed CLIC4-associated indanyloxyacetic acid-sensitive (IC(50) approximately 100 microM) plasma membrane currents showing mild outward rectification, and novel low conductance (approximately 1pS) CLIC4-associated anion channels were resolved at the single-channel level. The CLIC4-associated channels were inhibited by anti-CLIC4 antibodies, including a monoclonal antibody directed against a FLAG epitope fused to the C-terminus of CLIC4, but only when these were applied to the cytoplasmic (not the external) face of the membrane. CLIC4 is thus an essential molecular component of novel cellular anion channels and the C-terminus of the integral membrane form of CLIC4 is cytoplasmic.

Animals↗

PubMed2001

Clostridium perfringens type A enterotoxin forms mepacrine-sensitive pores in pure phospholipid bilayers in the absence of putative receptor proteins.

Clostridium perfringens enterotoxin (CPE) is an important cause of food poisoning with no significant homology to other enterotoxins and its mechanism of action remains uncertain. Although CPE has recently been shown to complex with tight junction proteins, we have previously demonstrated that CPE increases ionic permeability in single Caco-2 cells using the whole-cell patch-clamp technique, thereby excluding any paracellular permeability. In this paper we demonstrate that CPE forms pores in synthetic phospholipid membranes in the absence of receptor proteins. The properties of the pores are consistent with CPE-induced permeability changes in Caco-2 cells suggesting that CPE has innate pore-forming ability.

Bacterial Toxins↗

PubMed2001

Inhibitors can arrest the membrane activity of human islet amyloid polypeptide independently of amyloid formation.

Human islet amyloid polypeptide (hIAPP), co-secreted with insulin from pancreatic beta cells, misfolds to form amyloid deposits in non-insulin-dependent diabetes mellitus (NIDDM). Like many amyloidogenic proteins, hIAPP is membrane-active: this may be significant in the pathogenesis of NIDDM. Non-fibrillar hIAPP induces electrical and physical breakdown in planar lipid bilayers, and IAPP inserts spontaneously into lipid monolayers, markedly increasing their surface area and producing Brewster angle microscopy reflectance changes. Congo red inhibits these activities, and they are completely arrested by rifampicin, despite continued amyloid formation. Our results support the idea that non-fibrillar IAPP is membrane-active, and may have implications for therapy and for structural studies of membrane-active amyloid.

Amyloid↗

PubMed2001

Chloride intracellular channel protein CLIC4 (p64H1) binds directly to brain dynamin I in a complex containing actin, tubulin and 14-3-3 isoforms.

Mammalian chloride intracellular channel (CLIC) (p64-related) proteins are widely expressed, with an unusual dual localization as both soluble and integral membrane proteins. The molecular basis for their cellular localization and ion channel activity remains unclear. To help in addressing these problems, we identified novel rat brain CLIC4 (p64H1) binding partners by affinity chromatography, mass spectrometric analysis and microsequencing. Brain CLIC4 binds dynamin I, alpha-tubulin, beta-actin, creatine kinase and two 14-3-3 isoforms; the interactions are confirmed in vivo by immunoprecipitation. Gel overlay and reverse pull-down assays indicate that the binding of CLIC4 to dynamin I and 14-3-3zeta is direct. In HEK-293 cells, biochemical and immunofluorescence analyses show partial co-localization of recombinant CLIC4 with caveolin and with functional caveolae, which is consistent with a dynamin-associated role for CLIC4 in caveolar endocytosis. We speculate that brain CLIC4 might be involved in the dynamics of neuronal plasma membrane microdomains (micropatches) containing caveolin-like proteins and might also have other cellular roles related to membrane trafficking. Our results provide the basis for new hypotheses concerning novel ways in which CLIC proteins might be associated with cell membrane remodelling, the control of cell shape, and anion channel activity.

14-3-3 Proteins↗

PubMed2001

Mutagenesis and functional reconstitution of chlamydial major outer membrane proteins: VS4 domains are not required for pore formation but modify channel function.

Chlamidial organisms are obligate intracellular pathogens containing highly antigenic porin-like major outer membrane proteins (MOMPs). MOMP epitopes are of substantial medical interest, and they cluster within four relatively short variable (VS) domains. If MOMPs adopt a beta-barrel fold, like bacterial porins, the VS domains may form extramembranous loops and the conserved regions of the protein may correspond to predicted membrane-located beta-strands. However, molecular studies on native MOMPs have been hampered by the need to culture chlamydiae in eukaryotic host cells and purification and reconstitution remain problematic. In addition, the organisms are difficult to manipulate genetically, and it has also been difficult to functionally reconstitute recombinant MOMPs. To help overcome these problems and improve our understanding of MOMP structure and function, we cloned and expressed C. trachomatis and C. psittaci MOMPs and functionally reconstituted them at the single-channel level. We measured significant functional differences between the two proteins, and by removing and exchanging VS4, we tested the hypothesis that the largest variable domain forms an extramembranous loop that contributes to these differences. Proteins in which VS4 was deleted continued to form functional ion channels, consistent with the idea that the domain forms an extramembranous protein loop and incompatible with models in which it contributes to predicted membrane-located beta-strands. Additionally, the properties of the chimeric proteins strongly suggested that the VS4 domain interacts closely with other regions of the protein to form the channel entrance or vestibule. Our approach can be used to probe structure-function relationships in chlamydial MOMPs and may have implications for the generation of effective antichlamydial vaccines.

Amino Acid Sequence↗

PubMed2000

Latrophilin, neurexin, and their signaling-deficient mutants facilitate alpha -latrotoxin insertion into membranes but are not involved in pore formation.

Pure alpha-latrotoxin is very inefficient at forming channels/pores in artificial lipid bilayers or in the plasma membrane of non-secretory cells. However, the toxin induces pores efficiently in COS-7 cells transfected with the heptahelical receptor latrophilin or the monotopic receptor neurexin. Signaling-deficient (truncated) mutants of latrophilin and latrophilin-neurexin hybrids also facilitate pore induction, which correlates with toxin binding irrespective of receptor structure. This rules out the involvement of signaling in pore formation. With any receptor, the alpha-latrotoxin pores are permeable to Ca(2+) and small molecules including fluorescein isothiocyanate and norepinephrine. Bound alpha-latrotoxin remains on the cell surface without penetrating completely into the cytosol. Higher temperatures facilitate insertion of the toxin into the plasma membrane, where it co-localizes with latrophilin (under all conditions) and with neurexin (in the presence of Ca(2+)). Interestingly, on subsequent removal of Ca(2+), alpha-latrotoxin dissociates from neurexin but remains in the membrane and continues to form pores. These receptor-independent pores are inhibited by anti-alpha-latrotoxin antibodies. Our results indicate that (i) alpha-latrotoxin is a pore-forming toxin, (ii) receptors that bind alpha-latrotoxin facilitate its insertion into the membrane, (iii) the receptors are not physically involved in the pore structure, (iv) alpha-latrotoxin pores may be independent of the receptors, and (v) pore formation does not require alpha-latrotoxin interaction with other neuronal proteins.

Animals↗

PubMed2000

Expression of full-length and truncated recombinant human brain type I inositol 1,4,5-trisphosphate receptors in mammalian and insect cells.

Intracellular inositol 1,4,5-trisphosphate receptors (IP(3)Rs) form tetrameric Ca2+-release channels that are crucial for Ca2+ signalling in many eukaryotic cells. IP(3)R subunits contain an N-terminal, cytoplasmic, ligand binding domain linked by a modulatory domain to a channel-forming, hydrophobic C-terminal domain. We assembled and sequenced cDNAs encoding the SI-/SII+/SIII+ splice variant of the human brain type I IP(3)R, and functionally expressed the full-length receptor, and a C-terminally truncated receptor lacking the final 20% of the protein, in mammalian and insect cells. Both proteins were insoluble, consistent with in vivo immunofluorescence and ligand binding studies. This contrasted with the behaviour of recombinant FIKBP12 (a soluble control protein). The truncated receptor also fractionated with the "membrane" pellet after alkaline carbonate treatment. We conclude that the human type I IP(3)R forms high MW aggregates or complexes in cells when expressed without the C-terminal hydrophobic domain. This behaviour should be considered when expressing and refolding "soluble" human type I IP(3)R domains for structural studies.

Alternative Splicing↗

PubMed1999

Changes in the expression of myometrial ryanodine receptor mRNAs during human pregnancy.

Uterine contraction is triggered by a rise in intracellular free Ca(2+) concentration ([Ca2+]i), and although ryanodine-sensitive Ca(2+) release channels (RyRs) play a key role in the regulation of [Ca(2+)](i) in skeletal and cardiac muscle, much less is known about their role in smooth muscle. In this study, we investigated the expression of RyR mRNAs (ryr1-3) during human pregnancy by examining myometrial samples (n=18) taken, with informed consent and ethical approval, from non-pregnant patients undergoing hysterectomy, and patients undergoing elective caesarean section (at term, prior to or following the onset of labour). Ca(2+) release channel expression was determined both qualitatively and quantitatively, using reverse transcription-polymerase chain reaction (RT-PCR) analysis, RNase protection assays, and in situ mRNA hybridisation. RT-PCR analysis demonstrated that all three ryr genes, as well as the gene encoding the type I inositol 1,4,5-trisphosphate receptor (InsP(3)RI), are expressed in human myometrium. Quantitation by RNase protection assays showed that ryr3 and InsP(3)RI mRNAs are the most abundant, while ryr2 mRNA is barely detectable. In situ mRNA hybridisation confirmed that ryr3 and InsP(3)RI mRNAs are both localised to myometrial smooth muscle cells. The expression of ryr2 and ryr3 mRNA is down-regulated at the end of pregnancy compared to non-pregnant myometrium, indicating that ryanodine-sensitive Ca(2+) release channels are differentially expressed. The relative conservation of ryr1 expression is consistent with a role for Ca(2+) release from ryanodine-sensitive stores in the mechanism of uterine contractility during labour.

Adolescent↗

PubMed1999

Characterization of sheep brain ryanodine receptor ATP binding site by photoaffinity labeling.

Two high Mr protein bands (440 and 420 kDa) in sheep brain microsomal membranes were labeled with the photoaffinity ATP analog, O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (Bz2ATP). The 420 kDa band is labeled by [alpha-32P]-Bz2ATP with about 1000-fold higher affinity than the 440 kDa band. The heavily labeled 420 kDa band is identified as dynein heavy chain based on its partial amino acid sequence, and cross-reactivity with anti-dynein antibodies. The 440 kDa protein is immunologically identified as the type-2 RyR. Bz2ATP binding is obtained in the absence of divalent cations. Bz2ATP and ATP increased the binding of ryanodine to its receptor up to 3-fold, and increased the binding affinity up to 6-fold. Other nucleotides stimulate ryanodine binding with decreasing effectiveness: Bz2ATP > ATP > ADP > AMP > AMP-PNP > GTP > cAMP. With respect to nucleotide specificity, this binding site is similar to the skeletal muscle RyR (type 1). However, the brain RyR may have additional one or more sites with lower affinity with inhibitory effect on ryanodine binding. These results suggest that the major RyR isoform in sheep brain corresponds to the type-2 isoform, and that modulation of ryanodine binding by ATP involves its binding to the RyR protein. The association of dynein with brain microsomal membranes may reflect a linkage of RyR to the cytoskeleton.

Adenosine Triphosphate↗

PubMed1999

Single channel analysis of recombinant major outer membrane protein porins from Chlamydia psittaci and Chlamydia pneumoniae.

We recently demonstrated that the major outer membrane protein of Chlamydia psittaci, the primary vaccine candidate for combating chlamydial infections, functions as a porin-like ion channel. In this study, we have cloned, expressed and functionally reconstituted recombinant major outer membrane proteins from C. psittaci and Chlamydia pneumoniae and analysed them at the single channel level. Both form porin-like ion channels that are functionally similar to those formed by native C. psittaci major outer membrane protein. Also, like the native channels, recombinant C. psittaci channels are modified by a native major outer membrane protein-specific monoclonal antibody. This is the first time that native function has been demonstrated for recombinant chlamydial major outer membrane proteins. Future bilayer reconstitution will provide a strategy for detailed structure/function studies of this new subclass of bacterial porins and the work also has important implications for successful protein refolding and the development of improved subunit vaccines.

Antibodies, Monoclonal↗

PubMed1999

Pregnant rat myometrial cells show heterogeneous ryanodine- and caffeine-sensitive calcium stores.

Intracellular Ca(2+) release channels such as ryanodine receptors play crucial roles in the Ca(2+)-mediated signaling that triggers excitation-contraction coupling in muscles. Although the existence and the role of these channels are well characterized in skeletal and cardiac muscles, their existence in smooth muscles, and more particularly in the myometrium, is very controversial. We have now clearly demonstrated the expression of ryanodine receptor Ca(2+) release channels in rat myometrial smooth muscle, and for the first time, intracellular Ca(2+) concentration experiments with indo 1 on single myometrial cells have revealed the existence of a functional ryanodine- and caffeine-sensitive Ca(2+) release mechanism in 30% of rat myometrial cells. RT-PCR and RNase protection assay on whole myometrial smooth muscle demonstrate the existence of all three ryr mRNAs in the myometrium: ryr3 mRNA is the predominant subtype, with much lower levels of expression for ryr1 and ryr2 mRNAs, suggesting that the ryanodine Ca(2+) release mechanism in rat myometrium is largely encoded by ryr3. Moreover, using intracellular Ca(2+) concentration measurements and RNase protection assays, we have demonstrated that the expression, the percentage of cells responding to ryanodine, and the function of these channels are not modified during pregnancy.

Animals↗

PubMed1999

The mechanism of islet amyloid polypeptide toxicity is membrane disruption by intermediate-sized toxic amyloid particles.

NIDDM is characterized by islet amyloid deposits and decreased beta-cell mass. Islet amyloid is derived from the locally expressed protein islet amyloid polypeptide (IAPP). While it is now widely accepted that abnormal aggregation of IAPP has a role in beta-cell death in NIDDM, the mechanism remains unknown. We hypothesized that small IAPP aggregates, rather than mature large amyloid deposits, are cytotoxic. Consistent with this hypothesis, freshly dissolved human (h)-IAPP was cytotoxic when added to dispersed mouse and human islet cells, provoking the formation of abnormal vesicle-like membrane structures in association with vacuolization and cell death. Human islet cell death occurred by both apoptosis and necrosis, predominantly between 24 and 48 h after exposure to h-IAPP. In contrast, the addition to dispersed islet cells of matured h-IAPP containing large amyloid deposits of organized fibrils was seldom associated with vesicle-like structures or features of cell death, even though the cells were often encased in the larger amyloid deposits. Based on these observations, we hypothesized that h-IAPP cytotoxicity is mediated by membrane damage induced by early h-IAPP aggregates. Consistent with this hypothesis, application of freshly dissolved h-IAPP to voltage-clamped planar bilayer membranes (a cell-free in vitro system) also caused membrane instability manifested as a marked increase in conductance, increased membrane electrical noise, and accelerated membrane breakage, effects that were absent using matured h-IAPP or rat IAPP solutions. Light-scattering techniques showed that membrane toxicity corresponded to h-IAPP aggregates containing approximately 25-6,000 IAPP molecules, an intermediate-sized amyloid particle that we term intermediate-sized toxic amyloid particles (ISTAPs). We conclude that freshly dissolved h-IAPP is cytotoxic and that this cytotoxicity is mediated through an interaction of ISTAPs with cellular membranes. Once ISTAPs mature into amyloid deposits comprising >10(6) molecules, the capacity of h-IAPP to cause membrane instability and islet cell death is significantly reduced or abolished. These data may have implications for the mechanism of cell death in other diseases characterized by local amyloid formation (such as Alzheimer's disease).

Amyloid↗

PubMed1998

The structure, function, and cellular regulation of ryanodine-sensitive Ca2+ release channels.

The fundamental biological process of Ca2+ signaling is known to be important in most eukaryotic cells, and inositol 1,2,5-trisphosphate and ryanodine receptors, intracellular Ca2+ release channels encoded by two distantly related gene families, are central to this phenomenon. Ryanodine receptors in the sarcoplasmic reticulum of skeletal and cardiac muscle have a predominant role in excitation-contraction coupling, but the channels are also present in the endoplasmic reticulum of noncontractile tissues including the central nervous system and the immune system. In all, three highly homologous ryanodine receptor isoforms have been identified, all very large proteins which assemble as (homo)tetramers of approximately 2 MDa. They contain large cytoplasmically disposed regulatory domains and are always associated with other structural or regulatory proteins, including calmodulin and immunophilins, which can have marked effects on channel function. The type 1 isoform in skeletal muscle is electromechanically coupled to surface membrane voltage sensors, whereas the remaining isoforms appear to be activated solely by endogenous cytoplasmic second messengers or other ligands, including Ca2+ itself ("Ca(2+)-induced Ca2+ release"). This review concentrates on ryanodine receptor structure-function relationships as probed by a variety of methods and on the molecular mechanisms of channel modulation at the cellular level (including evidence for the regulation of gene expression and transcription). It also touches on the relevance of ryanodine receptors to complex cellular functions and disease.

Animals↗

PubMed1998

Partial cloning and differential expression of ryanodine receptor/calcium-release channel genes in human tissues including the hippocampus and cerebellum.

Cellular Ca2+ signalling is an important factor in the control of neuronal metabolism and electrical activity. Although the roles of Ca2+-release channels are well established for skeletal and cardiac muscle, less is known about their expression and roles in the central nervous system, especially in the human brain. We have isolated partial complementary DNAs derived from the human ryanodine receptor Ca2+-release channel genes (ryr1, ryr2 and ryr3), and examined their expression in the human hippocampus and cerebellum. For comparison, we have included in our analysis an inositol trisphosphate Ca2+-release channel type I complementary RNA probe. All four messenger RNAs show widespread distribution in the human hippocampus, where ryr2 is the most abundant isoform, and all four are expressed in the human cerebellum. However, striking differences were seen between ryr and inositol trisphosphate Ca2+-release channel type I complementary RNA expression in the cerebellum, with inositol trisphosphate Ca2+-release channel type I messenger RNA being largely restricted to, and very highly expressed, in Purkinje cells, whereas ryr1, ryr2 and ryr3 were all expressed predominantly in the granular layer. The widespread expression of ryr isoforms in the human hippocampus and cerebellum suggests that ryanodine receptor proteins may have a central role in Ca2+ signalling and Ca2+ homeostasis in the human central nervous system. These may include roles in fundamental processes like synaptic plasticity. Furthermore, these Ca2+-release channels may be involved in pathogenic processes such as excitotoxicity, where excessive rises in intracellular Ca2+ concentration mediate neuronal cell death.

Aged↗

PubMed1998

The major outer membrane protein of Chlamydia psittaci functions as a porin-like ion channel.

The major outer membrane protein (MOMP) of Chlamydia species shares several biochemical properties with classical porin proteins. Secondary structure analysis by circular dichroism now reveals that MOMP purified from Chlamydia psittaci has a predominantly beta-sheet content (62%), which is also typical of bacterial porins. Can MOMP form functional ion channels? To directly test the "porin channel" hypothesis at the molecular level, the MOMP was reconstituted into planar lipid bilayers, where it gave rise to multibarreled channels, probably trimers, which were modified by an anti-MOMP monoclonal antibody. These observations are consistent with the well-characterized homo-oligomeric nature of MOMP previously revealed by biochemical analysis and with the triple-barreled behavior of other porins. MOMP channels were weakly anion selective (PCl/PK approximately 2) and permeable to ATP. They may therefore be a route by which Chlamydia can take advantage of host nucleoside triphosphates and explain why some anti-MOMP antibodies neutralize infection. These findings have broad implications on the search for an effective chlamydial vaccine to control the significant human and animal diseases caused by these organisms.

Bacterial Outer Membrane Proteins↗

PubMed1997

Rat brain p64H1, expression of a new member of the p64 chloride channel protein family in endoplasmic reticulum.

Many plasma membrane Cl- channels have been cloned, including the cystic fibrosis transmembrane conductance regulator and several members of the voltage-gated ClC family. In contrast, very little is known about the molecular identity of intracellular Cl- channels. We used a polymerase chain reaction-based approach to identify candidate genes in mammalian brain and cloned the cDNA corresponding to rat brain p64H1. This encoded a microsomal membrane protein of predicted Mr 28,635 homologous to the putative intracellular bovine kidney Cl- channel p64. In situ mRNA hybridization histochemistry showed marked expression in hippocampus and cerebellum, and in vitro expression revealed a large cytoplasmic domain, one membrane-spanning segment, and a small nonglycosylated N-terminal luminal domain. The predicted protein contained consensus phosphorylation sites for protein kinase C and protein kinase A, and protein kinase C-mediated phosphorylation increased the Mr of p64H1 to approximately 43,000, characteristic of the native protein in Western blots. Recombinant p64H1 was immunolocalized to the endoplasmic reticulum of human embryonic kidney 293 and HT-4 cells, and incorporation of human embryonic kidney 293 endoplasmic reticulum vesicles into planar lipid bilayers gave rise to intermediate conductance, outwardly rectifying anion channels. Although p64H1 is the first intracellular Cl- channel component or regulator to be identified in brain, Northern blotting revealed transcripts in many other rat tissues. This suggests that p64H1 may contribute widely to intracellular Cl- transport.

Amino Acid Sequence↗

PubMed1997

Single-channel properties of a rat brain endoplasmic reticulum anion channel.

Many intracellular membranes contain ion channels, although their physiological roles are often poorly understood. In this study we incorporated single anion channels colocalized with rat brain endoplasmic reticulum (ER) ryanodine-sensitive Ca(2+)-release channels into planar lipid bilayers. The channels opened in bursts, with more activity at negative (cytoplasm-ER lumen) membrane potentials, and they occupied four open conductance levels with frequencies well described by the binomial equation. The probability of a protomer being open decreased from approximately 0.7 at -40 mV to approximately 0.2 at +40 mV, and the channels selected between different anions in the order PSCN > PNO3 > PBr > PCl > PF. They were also permeant to cations, including the large cation Tris+ (PTris/PCl = 0.16). Their conductance saturated at 170 pS in choline Cl. The channels were inactivated by 15 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and blocked with low affinity (KD of 1-100 microM) by anthracene-9-carboxylic acid, ethacrynic acid, frusemide (furosemide), HEPES, the indanyloxyacetic acid derivative IAA-94, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), and Zn2+. Unlike protein translocation pores, the channels were unaffected by high salt concentrations or puromycin. They may regulate ER Ca2+ release, or be channel components en route to their final cellular destinations. Alternatively, they may contribute to the fusion machinery involved in intracellular membrane trafficking.

Animals↗

PubMed1996

Identification and characterisation of a homologue of p64 in rat tissues.

Previous work has suggested that the gene encoding p64, a component of a bovine kidney intracellular chloride channel, may be a member of a gene family. We have raised a polyclonal antibody to an E. coli fusion protein which has sequence similarity to p64. Immunoblotting detected a protein in rat brain, kidney, liver and lung. In rat brain, the protein was enriched in cerebellar microsomal membranes. Western blot analyses of denaturing and blue native polyacrylamide gels indicated that the protein is a single non-disulphide-linked polypeptide chain with an apparent M(r) of 43 kDa that contributes to a native protein complex with an apparent M(r) of 130 kDa.

Amino Acid Sequence↗