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D Clapham

Publications and source records attributed to D Clapham.

16 recordsLinked to original sources
PubMed1997

Calcium release and influx colocalize to the endoplasmic reticulum.

Intracellular Ca2+ is released from intracellular stores in the endoplasmic reticulum (ER) in response to the second messenger inositol (1,4,5) trisphosphate (InsP3) [1,2]. Then, a poorly understood cellular mechanism, termed capacitative Ca2+ entry, is activated [3,4]; this permits Ca2+ to enter cells through Ca(2+)-selective Ca(2+)-release-activated ion channels [5,6] as well as through less selective store-operated channels [7]. The level of stored Ca2+ is sensed by Ca(2+)-permeant channels in the plasma membrane, but the identity of these channels, and the link between them and Ca2+ stores, remain unknown. It has been argued that either a diffusible second messenger (Ca2+ influx factor; CIF) [8] or a physical link [9,10] connects the ER Ca(2+)-release channel and store-operated channels; strong evidence for either mechanism is lacking, however [7,10]. Petersen and Berridge [11] showed that activation of the lysophosphatidic acid receptor in a restricted region of the oocyte membrane results in stimulation of Ca2+ influx only in that region, and concluded that a diffusible messenger was unlikely. To investigate the relationship between ER stores and Ca2+ influx, we used centrifugation to redistribute into specific layers the organelles inside intact Xenopus laevis oocytes, and used laser scanning confocal microscopy with the two-photon technique to 'uncage' InsP3 while recording intracellular Ca2+ concentration. Ca2+ release was localized to the stratified ER layer and Ca2+ entry to regions of the membrane directly adjacent to this layer. We conclude that Ca2+ depletion and entry colocalize to the ER and that the mechanism linking Ca2+ stores to Ca2+ entry is similarly locally constrained.

Animals↗
PubMed1996

A wound-inducible gene from Salix viminalis coding for a trypsin inhibitor.

A gene designated swin1.1 has been isolated by screening a Salix viminalis genomic library with a heterologous probe, win3 from Populus. The region sequenced included the entire coding sequence for a protein with 199 amino acids plus the promoter and terminator. At the 5' end of the coding region is a sequence that encodes a hydrophobic region of 25-30 amino acids, that could form a signal peptide. A putative TATAA box and polyadenylator sequence were identified. Introns were absent. The gene product showed similarities with serine protease inhibitors from the Kunitz family and especially with win3 from wounded leaves of Populus. Southern blot analysis indicated that swin1.1 is a member of a clustered gene family, swin1. An oligonucleotide corresponding to the putative hypervariable region towards the carboxyl end when used as a probe in Southern hybridization showed high specificity for swin1.1. Expression of the swin1.1 gene was enhanced in wounded leaves. The swin1.1 coding region without the signal sequence was highly expressed in Escherichia coli and the protein showed inhibitory activity against trypsin but at most slight activity against the other proteases tested. A systemically induced protein, SVTI, with inhibitor activity against trypsin, was isolated from Salix leaves by affinity chromatography on a column of trypsin-Sepharose 4B and N-terminal sequenced. It corresponded with the translated swin1.1 gene at 16 of the 19 amino acid sites, suggesting that SVTI is encoded by another member of the swin1 gene family.

Amino Acid Sequence↗
PubMed1993

Alpha-helical distorting substitution disrupt coupling between m3 muscarinic receptor and G proteins.

Acetylcholine stimulation of the m3 or m2 muscarinic receptor expressed in Xenopus laevis oocytes induces either a fast transient or slowly oscillating calcium-sensitive chloride current. The speed of these currents reflects the efficiency of receptor coupling to guanine nucleotide-binding proteins and phosphatidylinositol (PI) turnover. Point mutations of the m3 receptor were made in a region of the third cytoplasmic loop to test whether receptor function relied on an alpha-helical structure of the G protein-coupling domain. Proline substitution for glutamate at position 257 disrupted the m3 response. Also, single alanine insertions between residues 259 and 260 disrupted the m3 receptor-stimulated response while double alanine insertions at this site had no effect. Based on these results, we suggest that a region of the third cytoplasmic loop of the m3 receptor possesses an amphipathic alpha-helical conformation.

Acetylcholine↗
PubMed1993

Acceleration of intracellular calcium waves in Xenopus oocytes by calcium influx.

Many cell membrane receptors stimulate the phosphoinositide (PI) cycle, which produces complex intracellular calcium signals that regulate diverse processes such as secretion and transcription. A major messenger of this cycle, inositol 1,4,5-triphosphate (IP3), stimulates its receptor channel on the endoplasmic reticulum to release calcium into the cytosol. Activation of the PI cycle also induces calcium influx, which refills the intracellular calcium stores. Confocal microscopy was used to show that receptor-activated calcium influx, enhanced by hyperpolarization, modulates the frequency and velocity of IP3-dependent calcium waves in Xenopus laevis oocytes. These results demonstrate that transmembrane voltage and calcium influx pathways may regulate spatial and temporal patterns of IP3-dependent calcium release.

Acetylcholine↗
PubMed1993

Calcium wave propagation by calcium-induced calcium release: an unusual excitable system.

We discuss in detail the behaviour of a model, proposed by Goldbeter et al. (1990. Proc. natn. Acad. Sci. 87, 1461-1465), for intracellular calcium wave propagation by calcium-induced calcium release, focusing our attention on excitability and the propagation of waves in one spatial dimension. The model with no diffusion behaves like a generic excitable system, and threshold behaviour, excitability and oscillations can be understood within this general framework. However, when diffusion is included, the model no longer behaves like a generic excitable system; the fast and slow variables are not distinct and previous results on excitable systems do not necessarily apply. We consider a piecewise linear simplification of the model, and construct travelling pulse and periodic plane wave solutions to the simplified model. The analogous behaviour in the full model is studied numerically. Goldbeter's model for calcium-induced calcium release is an excitable system of a type not previously studied in detail.

Animals↗
PubMed1993

Calcium waves.

Intracellular Ca2+ oscillations and waves are commonly observed both in excitable cells, including neurons, and in non-excitable cells. Current attempts to describe and explain these complex intracellular signals suggest that the oscillations are the result of a highly regulated mechanism, the details of which vary among different cells. Recently, the Xenopus oocyte has become an important model system in which a single pool of IP3 receptors release Ca2+ to initiate waves. The intrinsic bell-shaped dependence of the IP3 receptor on Ca2+ is sufficient to explain the regenerative wave phenomenon.

Animals↗
PubMed1993

A single-pool model for intracellular calcium oscillations and waves in the Xenopus laevis oocyte.

We construct a minimal model of cytosolic free Ca2+ oscillations based on Ca2+ release via the inositol 1,4,5-trisphosphate (IP3) receptor/Ca2+ channel (IP3R) of a single intracellular Ca2+ pool. The model relies on experimental evidence that the cytosolic free calcium concentration ([Ca2+]c) modulates the IP3R in a biphasic manner, with Ca2+ release inhibited by low and high [Ca2+]c and facilitated by intermediate [Ca2+]c, and that channel inactivation occurs on a slower time scale than activation. The model produces [Ca2+]c oscillations at constant [IP3] and reproduces a number of crucial experiments. The two-dimensional spatial model with IP3 dynamics, cytosolic diffusion of IP3 (Dp = 300 microns 2 s-1), and cytosolic diffusion of Ca2+ (Dc = 20 microns 2 s-1) produces circular, planar, and spiral waves of Ca2+ with speeds of 7-15 microns.s-1, which annihilate upon collision. Increasing extracellular [Ca2+] influx increases wave speed and baseline [Ca2+]c. A [Ca2+]c-dependent Ca2+ diffusion coefficient does not alter the qualitative behavior of the model. An important model prediction is that channel inactivation must occur on a slower time scale than activation in order for waves to propagate. The model serves to capture the essential macroscopic mechanisms that are involved in the production of intracellular Ca2+ oscillations and traveling waves in the Xenopus laevis oocyte.

Animals↗
PubMed1992

New mammalian chloride channel identified by expression cloning.

Ion channels selectively permeable to chloride ions regulate cell functions as diverse as excitability and control of cell volume. Using expression cloning techniques, a complementary DNA from an epithelial cell line has been isolated, sequenced and its putative structure examined by site-directed mutagenesis. This cDNA, encoding a 235-amino-acid protein, gave rise to a chloride-selective outward current when expressed in Xenopus oocytes. The expressed, outwardly rectifying chloride current was calcium-insensitive and was blocked by nucleotides applied to the cell surface. Mutation of a putative nucleotide-binding site resulted in loss of nucleotide block but incurred dependence on extracellular calcium concentration. The unusual sequence of this putative channel protein suggests a new class of ion channels not related to other previously cloned chloride channels.

Amino Acid Sequence↗
PubMed1992

Two-dimensional model of calcium waves reproduces the patterns observed in Xenopus oocytes.

Biological excitability enables the rapid transmission of physiological signals over distance. Using confocal fluorescence microscopy, we previously reported circular, planar, and spiral waves of Ca2+ in Xenopus laevis oocytes that annihilated one another upon collision. We present experimental evidence that the excitable process underlying wave propagation depends on Ca2+ diffusion and does not require oscillations in inositol (1,4,5)trisphosphate (IP3) concentration. Extending an existing ordinary differential equation (ODE) model of Ca2+ oscillations to two spatial dimensions, we develop a partial differential equation (PDE) model of Ca2+ excitability. The model assumes that cytosolic Ca2+ couples neighboring Ca2+ release sites. This simple PDE model qualitatively reproduces our experimental observations.

Animals↗
PubMed1991

Subcellular patterns of calcium release determined by G protein-specific residues of muscarinic receptors.

Calcium release from intracellular stores is a point of convergence for a variety of receptors involved in cell signaling. Consequently, the mechanism(s) by which cells differentiate between individual receptor signals is central to transmembrane communication. There are significant differences in timing and magnitude of Ca2+ release stimulated by the m2 and m3 muscarinic acetylcholine receptors. The m2 receptors couple to a pertussis toxin-sensitive G protein to activate phosphatidyl inositol hydrolysis weakly and to stimulate small, delayed and oscillatory chloride currents. In contrast, m3 receptors potently activate phosphatidyl inositol hydrolysis and stimulate large, rapid and transient chloride currents by a pertussis toxin-insensitive G protein pathway. Using confocal microscopy, we now show that the m2- and m3-coupled Ca2+ release pathways can also be spatially distinguished. At submaximal acetylcholine concentrations, both receptors stimulated pulses of Ca2+ release from discrete foci in random, periodic and frequently bursting patterns of activity. But maximal stimulation of m2 receptors increased the number of focal release sites, whereas m3 receptors invariably evoked a Ca2+ wave propagating rapidly just beneath the plasma membrane surface. Analysis of pertussis toxin sensitivity and hybrid m2-m3 muscarinic acetylcholine receptors confirmed that these Ca2+ release patterns represent distinct cell signalling pathways.

Acetylcholine↗
PubMed1991

Spiral calcium wave propagation and annihilation in Xenopus laevis oocytes.

Intracellular calcium (Ca2+) is a ubiquitous second messenger. Information is encoded in the magnitude, frequency, and spatial organization of changes in the concentration of cytosolic free Ca2+. Regenerative spiral waves of release of free Ca2+ were observed by confocal microscopy in Xenopus laevis oocytes expressing muscarinic acetylcholine receptor subtypes. This pattern of Ca2+ activity is characteristic of an intracellular milieu that behaves as a regenerative excitable medium. The minimal critical radius for propagation of focal Ca2+ waves (10.4 micrometers) and the effective diffusion constant for the excitation signal (2.3 x 10(-6) square centimeters per second) were estimated from measurements of velocity and curvature of circular wavefronts expanding from foci. By modeling Ca2+ release with cellular automata, the absolute refractory period for Ca2+ stores (4.7 seconds) was determined. Other phenomena expected of an excitable medium, such as wave propagation of undiminished amplitude and annihilation of colliding wavefronts, were observed.

Animals↗
PubMed1990

Distinct sequence elements control the specificity of G protein activation by muscarinic acetylcholine receptor subtypes.

Relatively little is understood concerning the mechanisms by which subtypes of receptors, G proteins and effector enzymes interact to transduce specific signals. Through expression of normal, hybrid and deletion mutant receptors in Xenopus oocytes, we determined the G protein coupling characteristics of the functionally distinct m2 and m3 muscarinic acetylcholine receptor (mAChR) subtypes and identified the critical receptor sequences responsible for G protein specificity. Activation of a pertussis toxin insensitive G protein pathway, leading to a rapid and transient release of intracellular Ca2+ characteristic of the m3 receptor, could be specified by the transfer of as few as nine amino acids from the m3 to the m2 receptor. In a reciprocal manner, transfer of no more than 21 residues from the m2 to the m3 receptor was sufficient to specify activation of a pertussis toxin sensitive G protein coupled to a slow and oscillatory Ca2+ release pathway typical of the m2 subtype. Notably, these critical residues occur within the same region of the third cytoplasmic domain of functionally distinct mAChR subtypes.

Amino Acid Sequence↗
PubMed1989

Identification and subcellular localization of a putative cell-to-cell transport protein from red clover mottle virus.

To investigate the mode of gene expression of red clover mottle virus (RCMV) middle component (M) RNA, we have synthesized an oligopeptide corresponding to the predicted carboxy-terminus of the RCMV counterparts of the cowpea mosaic virus (CPMV) 48K and 58K proteins. Using an antiserum raised against this synthetic oligopeptide, we have detected a 43-kDa protein in the 30,000 g pellet from extracts of RCMV-infected cowpea protoplasts. Immunogold cytochemistry further localized this protein to the plasmodesmata of RCMV-infected pea tissue. This subcellular location, taken together with other evidence, suggests that this 43-kDa protein has a role in the cell-to-cell spread of RCMV.

Antibody Specificity↗
PubMed1989

Diverse functions of muscarinic acetylcholine receptor subtypes.

Muscarinic acetylcholine receptor subtypes m1, m3 and m5 couple strongly to phosphatidylinositol turnover and hence to intracellular Ca2+ concentration via pertussis toxin (PTX) sensitive and insensitive G proteins. The m2 and m4 muscarinic receptor subtypes strongly inhibit adenylyl cyclase production via PTX sensitive G proteins. Additionally, the cardiac M2 receptor is closely coupled to a K+ current (IK.ACh). To characterize this functional diversity more completely, we measured the ACh-induced Ca2+ responses of cells transfected with the muscarinic receptor subtypes m1, m2, m3 and m4. As expected, cells transfected with m1 or m3 receptors exhibited large dose-dependent increases in Ca2+ in response to ACh application. Unexpectedly, cells transfected with m2 or m4 receptors also exhibited increases in Ca2+ in response to agonist application. The m2- or m4-coupled responses were smaller in amplitude, required higher concentrations of agonist and were much more sensitive to PTX treatment when compared to m1- or m3-coupled responses. We discuss this remarkable diversity of function in terms of the receptor subtype's coupling to G proteins.

Animals↗