Biomedical subjects

Richard S Lewis

Publications and source records attributed to Richard S Lewis.

13 recordsLinked to original sources

PubMed2006

Ca2+ store depletion causes STIM1 to accumulate in ER regions closely associated with the plasma membrane.

Stromal interacting molecule 1 (STIM1), reported to be an endoplasmic reticulum (ER) Ca(2+) sensor controlling store-operated Ca(2+) entry, redistributes from a diffuse ER localization into puncta at the cell periphery after store depletion. STIM1 redistribution is proposed to be necessary for Ca(2+) release-activated Ca(2+) (CRAC) channel activation, but it is unclear whether redistribution is rapid enough to play a causal role. Furthermore, the location of STIM1 puncta is uncertain, with recent reports supporting retention in the ER as well as insertion into the plasma membrane (PM). Using total internal reflection fluorescence (TIRF) microscopy and patch-clamp recording from single Jurkat cells, we show that STIM1 puncta form several seconds before CRAC channels open, supporting a causal role in channel activation. Fluorescence quenching and electron microscopy analysis reveal that puncta correspond to STIM1 accumulation in discrete subregions of junctional ER located 10-25 nm from the PM, without detectable insertion of STIM1 into the PM. Roughly one third of these ER-PM contacts form in response to store depletion. These studies identify an ER structure underlying store-operated Ca(2+) entry, whose extreme proximity to the PM may enable STIM1 to interact with CRAC channels or associated proteins.

Calcium↗

PubMed2006

The elementary unit of store-operated Ca2+ entry: local activation of CRAC channels by STIM1 at ER-plasma membrane junctions.

The activation of store-operated Ca(2+) entry by Ca(2+) store depletion has long been hypothesized to occur via local interactions of the endoplasmic reticulum (ER) and plasma membrane, but the structure involved has never been identified. Store depletion causes the ER Ca(2+) sensor stromal interacting molecule 1 (STIM1) to form puncta by accumulating in junctional ER located 10-25 nm from the plasma membrane (see Wu et al. on p. 803 of this issue). We have combined total internal reflection fluorescence (TIRF) microscopy and patch-clamp recording to localize STIM1 and sites of Ca(2+) influx through open Ca(2+) release-activated Ca(2+) (CRAC) channels in Jurkat T cells after store depletion. CRAC channels open only in the immediate vicinity of STIM1 puncta, restricting Ca(2+) entry to discrete sites comprising a small fraction of the cell surface. Orai1, an essential component of the CRAC channel, colocalizes with STIM1 after store depletion, providing a physical basis for the local activation of Ca(2+) influx. These studies reveal for the first time that STIM1 and Orai1 move in a coordinated fashion to form closely apposed clusters in the ER and plasma membranes, thereby creating the elementary unit of store-operated Ca(2+) entry.

Calcium↗

PubMed2006

A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function.

Antigen stimulation of immune cells triggers Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels, promoting the immune response to pathogens by activating the transcription factor NFAT. We have previously shown that cells from patients with one form of hereditary severe combined immune deficiency (SCID) syndrome are defective in store-operated Ca2+ entry and CRAC channel function. Here we identify the genetic defect in these patients, using a combination of two unbiased genome-wide approaches: a modified linkage analysis with single-nucleotide polymorphism arrays, and a Drosophila RNA interference screen designed to identify regulators of store-operated Ca2+ entry and NFAT nuclear import. Both approaches converged on a novel protein that we call Orai1, which contains four putative transmembrane segments. The SCID patients are homozygous for a single missense mutation in ORAI1, and expression of wild-type Orai1 in SCID T cells restores store-operated Ca2+ influx and the CRAC current (I(CRAC)). We propose that Orai1 is an essential component or regulator of the CRAC channel complex.

Animals↗

PubMed2006

Regulation of CRAC channel activity by recruitment of silent channels to a high open-probability gating mode.

CRAC (calcium release-activated Ca(2+)) channels attain an extremely high selectivity for Ca(2+) from the blockade of monovalent cation permeation by Ca(2+) within the pore. In this study we have exploited the blockade by Ca(2+) to examine the size of the CRAC channel pore, its unitary conductance for monovalent cations, and channel gating properties. The permeation of a series of methylammonium compounds under divalent cation-free conditions indicates a minimum pore diameter of 3.9 A. Extracellular Ca(2+) blocks monovalent flux in a manner consistent with a single intrapore site having an effective K(i) of 20 microM at -110 mV. Block increases with hyperpolarization, but declines below -100 mV, most likely due to permeation of Ca(2+). Analysis of monovalent current noise induced by increasing levels of block by extracellular Ca(2+) indicates an open probability (P(o)) of approximately 0.8. By extrapolating the variance/mean current ratio to the condition of full blockade (P(o) = 0), we estimate a unitary conductance of approximately 0.7 pS for Na(+), or three to fourfold higher than previous estimates. Removal of extracellular Ca(2+) causes the monovalent current to decline over tens of seconds, a process termed depotentiation. The declining current appears to result from a reduction in the number of active channels without a change in their high open probability. Similarly, low concentrations of 2-APB that enhance I(CRAC) increase the number of active channels while open probability remains constant. We conclude that the slow regulation of whole-cell CRAC current by store depletion, extracellular Ca(2+), and 2-APB involves the stepwise recruitment of silent channels to a high open-probability gating mode.

Boron Compounds↗

PubMed2005

Real-time measurement of signaling and motility during T cell development in the thymus.

Intracellular signals arising from interactions of immature thymocytes with distinct populations of stromal cells in the thymus are central to T cell development. The characteristics of these signals and the mechanisms underlying thymocyte migration between stromal cell compartments have been difficult to identify from static measurements of fixed tissue. Recent advances in two-photon microscopy and the development of three-dimensional models for real-time studies of T cell development have shed light on how single cells navigate the thymus. These studies reveal crosstalk between thymocyte signaling and motility that may integrate the search for potentially rare self-antigens with the requirement for sustained signaling in T cell maturation.

Animals↗

PubMed2005

A severe defect in CRAC Ca2+ channel activation and altered K+ channel gating in T cells from immunodeficient patients.

Engagement of the TCR triggers sustained Ca(2+) entry through Ca(2+) release-activated Ca(2+) (CRAC) channels, which helps drive gene expression underlying the T cell response to pathogens. The identity and activation mechanism of CRAC channels at a molecular level are unknown. We have analyzed ion channel expression and function in T cells from SCID patients which display 1-2% of the normal level of Ca(2+) influx and severely impaired T cell activation. The lack of Ca(2+) influx is not due to deficient regulation of Ca(2+) stores or expression of several genes implicated in controlling Ca(2+) entry in lymphocytes (kcna3/Kv1.3, kcnn4/IKCa1, trpc1, trpc3, trpv6, stim1). Instead, electrophysiologic measurements show that the influx defect is due to a nearly complete absence of functional CRAC channels. The lack of CRAC channel activity is correlated with diminished voltage sensitivity and slowed activation kinetics of the voltage-dependent Kv1.3 channel. These results demonstrate that CRAC channels provide the major, if not sole, pathway for Ca(2+) entry activated by the TCR in human T cells. They also offer evidence for a functional link between CRAC and Kv1.3 channels, and establish a model system for molecular genetic studies of the CRAC channel.

Calcium↗

PubMed2005

Calcium oscillations regulate thymocyte motility during positive selection in the three-dimensional thymic environment.

The three-dimensional thymic microenvironment and calcium signaling pathways are essential for driving positive selection of developing T cells. However, the nature of calcium signals and the diversity of their effects in the thymus are unknown. We describe here a thymic slice preparation for visualizing thymocyte motility and signaling in real time with two-photon microscopy. Naive thymocytes were highly motile at low intracellular calcium concentrations, but during positive selection cells became immobile and showed sustained calcium concentration oscillations. Increased intracellular calcium was necessary and sufficient to arrest thymocyte motility. The calcium dependence of motility acts to prolong thymocyte interactions with antigen-bearing stromal cells, promoting sustained signaling that may enhance the expression of genes underlying positive selection.

Animals↗

PubMed2004

Role of membrane potential and hydrogen bonding in the mechanism of translocation of guanidinium-rich peptides into cells.

The results described herein support a mechanistic hypothesis for how guanidine-rich transporters attached to small cargos (MW ca. <3000) can migrate across the lipid membrane of a cell and directly enter the cytosol. Arginine oligomers are found to partition almost completely into the aqueous layer of a water-octanol bilayer. However, when the same partitioning experiment is conducted in the presence of sodium laurate, a representative negatively charged membrane constituent, the arginine oligomer partitions almost completely (>95%) into the octanol layer. In contrast, ornithine oligomers partition almost exclusively into the water layer with and without added sodium laurate. The different partitioning between guanidinium-rich and ammonium-rich oligomers in the presence of sodium laurate is consistent with the ability of the former to form a bidentate hydrogen bonded ion pair. Mono- and dimethylated arginine oligomers, which like ornithine can only efficiently form monodentate hydrogen bonds, were prepared and found to exhibit poor cellular uptake. Ion pair formation converts a once water-soluble agent to a lipid-soluble agent, thereby reducing the energetic penalty for passage of guanidine-rich transporters through the lipid bilayer. Uptake of guanidine-rich transporters is known to be an energy-dependent process, and this requirement for cellular ATP is now rationalized by the inhibition of guanidine-rich transporter uptake in the presence of agents that reduce the membrane potential. Specifically, incubation of cells in buffers with high potassium ion concentrations or pretreatment of cells with gramicidin A reduces the cellular uptake of Fl-aca-arg8-CONH2 by >90%. Furthermore, the reciprocal experiment of hyperpolarizing the cell with valinomycin increased uptake by >1.5 times. In summary, we propose that the water-soluble, positively charged guanidinium headgroups of the transporter form bidentate hydrogen bonds with H-bond acceptor functionality on the cell surface. The resultant ion pair complexes partition into the lipid bilayer and migrate across at a rate related to the membrane potential. The complex dissociates on the inner leaf of the membrane, and the transporter enters the cytosol. This hypothesis does not preclude uptake by other mechanisms, including endocytosis, which is likely to dominate with large cargos.

Guanidine↗

PubMed2004

Modulation of plasma membrane calcium-ATPase activity by local calcium microdomains near CRAC channels in human T cells.

The spatial distribution of Ca(2+) signalling molecules is critical for establishing specific interactions that control Ca(2+) signal generation and transduction. In many cells, close physical coupling of Ca(2+) channels and their targets enables precise and robust activation of effector molecules through local [Ca(2+)](i) elevation in microdomains. In T cells, the plasma membrane Ca(2+)-ATPase (PMCA) is a major target of Ca(2+) influx through Ca(2+) release-activated Ca(2+) (CRAC) channels. Elevation of [Ca(2+)](i) slowly modulates pump activity to ensure the stability and enhance the dynamic nature of Ca(2+) signals. In this study we probed the functional organization of PMCA and CRAC channels in T cells by manipulating Ca(2+) microdomains near CRAC channels and measuring the resultant modulation of PMCAs. The amplitude and spatial extent of microdomains was increased by elevating the rate of Ca(2+) entry, either by raising extracellular [Ca(2+)], by increasing the activity of CRAC channels with 2-aminoethoxyborane (2-APB), or by hyperpolarizing the plasma membrane. Surprisingly, doubling the rate of Ca(2+) influx does not further increase global [Ca(2+)](i) in a substantial fraction of cells, due to a compensatory increase in PMCA activity. The enhancement of PMCA activity without changes in global [Ca(2+)](i) suggests that local [Ca(2+)](i) microdomains near CRAC channels effectively promote PMCA modulation. These results reveal an intimate functional association between CRAC channels and Ca(2+) pumps in the plasma membrane which may play an important role in governing the time course and magnitude of Ca(2+) signals in T cells.

Algorithms↗

PubMed2002

Enhancement of calcium signalling dynamics and stability by delayed modulation of the plasma-membrane calcium-ATPase in human T cells.

In addition to its homeostatic role of maintaining low resting levels of intracellular calcium ([Ca2+](i)), the plasma-membrane calcium-ATPase (PMCA) may actively contribute to the generation of complex Ca2+ signals. We have investigated the role of the PMCA in shaping Ca2+ signals in Jurkat human leukaemic T cells using single-cell voltage-clamp and calcium-imaging techniques. Crosslinking the T-cell receptor with the monoclonal antibody OKT3 induces a biphasic elevation in [Ca2+](i) consisting of a rapid overshoot to a level > 1 microM, followed by a slow decay to a plateau of approximately 0.5 microM. A similar overshoot was triggered by a constant level of Ca2+ influx through calcium-release-activated Ca2+ (CRAC) channels in thapsigargin-treated cells, due to a delayed increase in the rate of Ca2+ clearance by the PMCA. Following a rise in [Ca2+](i), PMCA activity increased in two phases: a rapid increase followed by a further calcium-dependent increase of up to approximately fivefold over 10-60 s, termed modulation. After the return of [Ca2+](i) to baseline levels, the PMCA recovered slowly from modulation (tau approximately 4 min), effectively retaining a 'memory' of the previous [Ca2+](i) elevation. Using a Michaelis-Menten model with appropriate corrections for cytoplasmic Ca2+ buffering, we found that modulation extended the dynamic range of PMCA activity by increasing both the maximal pump rate and Ca2+ sensitivity (reduction of K(M)). A simple flux model shows how pump modulation and its reversal produce the initial overshoot of the biphasic [Ca2+](i) response. The modulation of PMCA activity enhanced the stability of Ca2+ signalling by adjusting the efflux rate to match influx through CRAC channels, even at high [Ca2+](i) levels that saturate the transport sites and would otherwise render the cell defenceless against additional Ca2+ influx. At the same time, the delay in modulation enables small Ca2+ fluxes to transiently elevate [Ca2+](i), thus enhancing Ca2+ signalling dynamics.

Adenosine Triphosphate↗

PubMed2002

Dynamics of thymocyte-stromal cell interactions visualized by two-photon microscopy.

Thymocytes are selected to mature according to their ability to interact with self major histocompatibility complex (MHC)-peptide complexes displayed on the thymic stroma. Using two-photon microscopy, we performed real-time analysis of the cellular contacts made by developing thymocytes undergoing positive selection in a three-dimensional thymic organ culture. A large fraction of thymocytes within these cultures were highly motile. MHC recognition was found to increase the duration of thymocyte-stromal cell interactions and occurred as both long-lived cellular associations displaying stable cell-cell contacts and as shorter, highly dynamic contacts. Our results identify the diversity and dynamics of thymocyte interactions during positive selection.

Animals↗

PubMed2002

Separation and characterization of currents through store-operated CRAC channels and Mg2+-inhibited cation (MIC) channels.

Although store-operated calcium release-activated Ca(2+) (CRAC) channels are highly Ca(2+)-selective under physiological ionic conditions, removal of extracellular divalent cations makes them freely permeable to monovalent cations. Several past studies have concluded that under these conditions CRAC channels conduct Na(+) and Cs(+) with a unitary conductance of approximately 40 pS, and that intracellular Mg(2+) modulates their activity and selectivity. These results have important implications for understanding ion permeation through CRAC channels and for screening potential CRAC channel genes. We find that the observed 40-pS channels are not CRAC channels, but are instead Mg(2+)-inhibited cation (MIC) channels that open as Mg(2+) is washed out of the cytosol. MIC channels differ from CRAC channels in several critical respects. Store depletion does not activate MIC channels, nor does store refilling deactivate them. Unlike CRAC channels, MIC channels are not blocked by SKF 96365, are not potentiated by low doses of 2-APB, and are less sensitive to block by high doses of the drug. By applying 8-10 mM intracellular Mg(2+) to inhibit MIC channels, we examined monovalent permeation through CRAC channels in isolation. A rapid switch from 20 mM Ca(2+) to divalent-free extracellular solution evokes Na(+) current through open CRAC channels (Na(+)-I(CRAC)) that is initially eightfold larger than the preceding Ca(2+) current and declines by approximately 80% over 20 s. Unlike MIC channels, CRAC channels are largely impermeable to Cs(+) (P(Cs)/P(Na) = 0.13 vs. 1.2 for MIC). Neither the decline in Na(+)-I(CRAC) nor its low Cs(+) permeability are affected by intracellular Mg(2+) (90 microM to 10 mM). Single openings of monovalent CRAC channels were not detectable in whole-cell recordings, but a unitary conductance of 0.2 pS was estimated from noise analysis. This new information about the selectivity, conductance, and regulation of CRAC channels forces a revision of the biophysical fingerprint of CRAC channels, and reveals intriguing similarities and differences in permeation mechanisms of voltage-gated and store-operated Ca(2+) channels.

Calcium↗

PubMedDate not supplied

CRAC channels: activation, permeation, and the search for a molecular identity.

The Ca2+ release-activated Ca2+ (CRAC) channel is a highly Ca2+-selective store-operated channel that is expressed in T lymphocytes, mast cells, and other hematopoietic cells. In T cells, CRAC channels are essential for generating the prolonged intracellular Ca2+ ([Ca2+](i)) elevation required for the expression of T-cell activation genes. Here we review recent work addressing CRAC channel regulation, pore properties, and the search for CRAC channel genes. Of the current models for CRAC current (I(CRAC)) activation, several new studies argue against a conformational coupling mechanism in which IP(3) receptors communicate store depletion to CRAC channels through direct physical interaction. The study of CRAC channels has been complicated by the fact that they lose activity in the absence of extracellular Ca2+. Attempts to maintain current size by removing intracellular Mg2+ have been found to unmask Mg2+-inhibited cation (MIC/MagNuM/TRPM7) channels, which have been mistaken in several studies for the CRAC channel. Recent studies under conditions that prevent MIC activation reveal that CRAC channels use high-affinity binding of Ca2+ in the pore to achieve high Ca2+ selectivity but have a surprisingly low conductance for both Ca2+ (approximately 10fS) and Na+ (approximately 0.2pS). Pore properties provide a unique fingerprint that provides a stringent test for potential CRAC channel genes and suggest models for the ion selectivity mechanism.

Animals↗