Bio

Academic Appointments


Administrative Appointments


  • Chair, Stanford University School of Medicine - Molecular & Cellular Physiology (2004 - 2009)

Honors & Awards


  • MERIT Award, National Institute of General Medical Sciences (July 2012 - present)
  • Junior Faculty Scholar, HHMI (1996-1998)

Professional Education


  • B.S., Yale University, Molec Biophysics & Biochem (1978)
  • Ph.D., California Inst. of Technology, Neurobiology (1985)

Research & Scholarship

Current Research and Scholarly Interests


Research in our laboratory is focused on the molecular mechanisms of calcium signaling through store-operated Ca2+ channels (SOCs). This class of ion channels is regulated in a unique way, by the depletion of Ca2+ from the lumen of the endoplasmic reticulum (ER) which normally occurs following stimulation of cell surface receptors that generate IP3. They are expressed in practically all cells, where they contribute to diverse functions including secretion, gene expression, and cell differentiation. A SOC called the Ca2+ release-activated Ca2+ channel, or CRAC channel, is particularly important in T cells, where it generates sustained Ca2+ signals that are essential for triggering T cells to proliferate and carry out immune functions. Loss of function of the CRAC channel by a single mutation in its structural gene leads to a devastating severe combined immunodeficiency (SCID) syndrome in humans.

A major effort in the lab is to understand how the depletion of Ca2+ from the ER triggers the opening of CRAC channels in the plasma membrane. Since the recent discovery of genes encoding the ER Ca2+ sensor (STIM1) and the CRAC channel (Orai1), we have made rapid progress in revealing the choreographic nature of this process. Following store depletion, STIM1 moves from locations throughout the ER to accumulate in ER subregions positioned within 10-25 nm of the plasma membrane (PM). Simultaneously, the CRAC channel gathers at corresponding sites in the PM directly opposite STIM1, where it is opened through an interaction with STIM1. This is an unprecedented mechanism for channel activation, in which a stimulus acts to bring a channel and its activator/sensor together for interaction across apposed membrane compartments. We are currently studying how changes in ER Ca2+ drive the redistribution of STIM1 and the mechanism by which STIM1 and Orai1 interact across the ER-PM gap. For these studies we combine protein engineering with patch-clamp electrophysiology and live-cell imaging using total internal reflection fluorescence (TIRF), confocal, and single-molecule microscopy.

A second major area of interest is how specific information is encoded in Ca2+ signals. Specificity is an acute problem for pluripotent messengers like Ca2+ that are involved in multiple signaling pathways. We have shown that transcriptional specificity in T cells can be achieved through the amplitude and dynamics of Ca2+ signals generated by CRAC channels. We are now studying how these features contribute to cell fate decisions during T cell development. In the thymus, self-reactive thymocytes are deleted through negative selection, while cells with the appropriate avidity for “self” are allowed to mature into T cells and populate the periphery (positive selection). To study the role of Ca2+ in this choice, we have developed a novel thymic slice preparation in which we use two-photon microscopy to track and record Ca2+ signals in single thymocytes as they migrate through tissue engineered to provide defined selection signals. We have found that positive selection is associated with Ca2+ oscillations, which immobilize the cells at locations of self-antigen recognition to promote gene activation. We are currently comparing signaling during positive and negative selection to determine how the Ca2+ signal “signature” helps a T cell decide whether to live and prosper or die.

Teaching

2013-14 Courses


Postdoctoral Advisees


Publications

Journal Articles


  • Store-Operated Calcium Channels: New Perspectives on Mechanism and Function COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY Lewis, R. S. 2011; 3 (12)

    Abstract

    Store-operated calcium channels (SOCs) are a nearly ubiquitous Ca(2+) entry pathway stimulated by numerous cell surface receptors via the reduction of Ca(2+) concentration in the ER. The discovery of STIM proteins as ER Ca(2+) sensors and Orai proteins as structural components of the Ca(2+) release-activated Ca(2+) (CRAC) channel, a prototypic SOC, opened the floodgates for exploring the molecular mechanism of this pathway and its functions. This review focuses on recent advances made possible by the use of STIM and Orai as molecular tools. I will describe our current understanding of the store-operated Ca(2+) entry mechanism and its emerging roles in physiology and disease, areas of uncertainty in which further progress is needed, and recent findings that are opening new directions for research in this rapidly growing field.

    View details for DOI 10.1101/cshperspect.a003970

    View details for Web of Science ID 000298135700003

    View details for PubMedID 21791698

  • Stoichiometric requirements for trapping and gating of Ca2+ release-activated Ca2+ (CRAC) channels by stromal interaction molecule 1 (STIM1) PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hoover, P. J., Lewis, R. S. 2011; 108 (32): 13299-13304

    Abstract

    Store-operated Ca(2+) entry depends critically on physical interactions of the endoplasmic reticulum (ER) Ca(2+) sensor stromal interaction molecule 1 (STIM1) and the Ca(2+) release-activated Ca(2+) (CRAC) channel protein Orai1. Recent studies support a diffusion-trap mechanism in which ER Ca(2+) depletion causes STIM1 to accumulate at ER-plasma membrane (PM) junctions, where it binds to Orai1, trapping and activating mobile CRAC channels in the overlying PM. To determine the stoichiometric requirements for CRAC channel trapping and activation, we expressed mCherry-STIM1 and Orai1-GFP at varying ratios in HEK cells and quantified CRAC current (I(CRAC)) activation and the STIM1:Orai1 ratio at ER-PM junctions after store depletion. By competing for a limited amount of STIM1, high levels of Orai1 reduced the junctional STIM1:Orai1 ratio to a lower limit of 0.3-0.6, indicating that binding of one to two STIM1s is sufficient to immobilize the tetrameric CRAC channel at ER-PM junctions. In cells expressing a constant amount of STIM1, CRAC current was a highly nonlinear bell-shaped function of Orai1 expression and the minimum stoichiometry for channel trapping failed to evoke significant activation. Peak current occurred at a ratio of ?2 STIM1:Orai1, suggesting that maximal CRAC channel activity requires binding of eight STIM1s to each channel. Further increases in Orai1 caused channel activity and fast Ca(2+)-dependent inactivation to decline in parallel. The data are well described by a model in which STIM1 binds to Orai1 with negative cooperativity and channels open with positive cooperativity as a result of stabilization of the open state by STIM1.

    View details for DOI 10.1073/pnas.1101664108

    View details for Web of Science ID 000293691400067

    View details for PubMedID 21788510

  • Essential Role for the CRAC Activation Domain in Store-dependent Oligomerization of STIM1 MOLECULAR BIOLOGY OF THE CELL Covington, E. D., Wu, M. M., Lewis, R. S. 2010; 21 (11): 1897-1907

    Abstract

    Oligomerization of the ER Ca(2+) sensor STIM1 is an essential step in store-operated Ca(2+) entry. The lumenal EF-hand and SAM domains of STIM1 are believed to initiate oligomerization after Ca(2+) store depletion, but the contributions of STIM1 cytosolic domains (coiled-coil 1, CC1; coiled-coil 2, CC2; CRAC activation domain, CAD) to this process are not well understood. By applying coimmunoprecipitation and fluorescence photobleaching and energy transfer techniques to truncated and mutant STIM1 proteins, we find that STIM1 cytosolic domains play distinct roles in forming both "resting" oligomers in cells with replete Ca(2+) stores and higher-order oligomers in store-depleted cells. CC1 supports the formation of resting STIM1 oligomers and appears to interact with cytosolic components to slow STIM1 diffusion. On store depletion, STIM1 lacking all cytosolic domains (STIM1-DeltaC) oligomerizes through EF-SAM interactions alone, but these oligomers are unstable. Addition of CC1 + CAD, but not CC1 alone, enables the formation of stable store-dependent oligomers. Within the CAD, both CC2 and C-terminal residues contribute to oligomer formation. Our results reveal a new function for the CAD: in addition to binding and activating Orai1, it is directly involved in STIM1 oligomerization, the initial event triggering store-operated Ca(2+) entry.

    View details for DOI 10.1091/mbc.E10-02-0145

    View details for Web of Science ID 000278118000011

    View details for PubMedID 20375143

  • Differential Contribution of Chemotaxis and Substrate Restriction to Segregation of Immature and Mature Thymocytes IMMUNITY Ehrlich, L. I., Oh, D. Y., Weissman, I. L., Lewis, R. S. 2009; 31 (6): 986-998

    Abstract

    T cell development requires sequential localization of thymocyte subsets to distinct thymic microenvironments. To address mechanisms governing this segregation, we used two-photon microscopy to visualize migration of purified thymocyte subsets in defined microenvironments within thymic slices. Double-negative (CD4(-)8(-)) and double-positive (CD4(+)8(+)) thymocytes were confined to cortex where they moved slowly without directional bias. DP cells accumulated and migrated more rapidly in a specialized inner-cortical microenvironment, but were unable to migrate on medullary substrates. In contrast, CD4 single positive (SP) thymocytes migrated directionally toward the medulla, where they accumulated and moved very rapidly. Our results revealed a requisite two-step process governing CD4 SP cell medullary localization: the chemokine receptor CCR7 mediated chemotaxis of CD4 SP cells towards medulla, whereas a distinct pertussis-toxin sensitive pathway was required for medullary entry. These findings suggest that developmentally regulated responses to both chemotactic signals and specific migratory substrates guide thymocytes to specific locations in the thymus.

    View details for DOI 10.1016/j.immuni.2009.09.020

    View details for Web of Science ID 000273616400018

    View details for PubMedID 19962328

  • STIM1 and calmodulin interact with Orai1 to induce Ca2+-dependent inactivation of CRAC channels PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mullins, F. M., Park, C. Y., Dolmetsch, R. E., Lewis, R. S. 2009; 106 (36): 15495-15500

    Abstract

    Ca(2+)-dependent inactivation (CDI) is a key regulator and hallmark of the Ca(2+) release-activated Ca(2+) (CRAC) channel, a prototypic store-operated Ca(2+) channel. Although the roles of the endoplasmic reticulum Ca(2+) sensor STIM1 and the channel subunit Orai1 in CRAC channel activation are becoming well understood, the molecular basis of CDI remains unclear. Recently, we defined a minimal CRAC activation domain (CAD; residues 342-448) that binds directly to Orai1 to activate the channel. Surprisingly, CAD-induced CRAC currents lack fast inactivation, revealing a critical role for STIM1 in this gating process. Through truncations of full-length STIM1, we identified a short domain (residues 470-491) C-terminal to CAD that is required for CDI. This domain contains a cluster of 7 acidic amino acids between residues 475 and 483. Neutralization of aspartate or glutamate pairs in this region either reduced or enhanced CDI, whereas the combined neutralization of six acidic residues eliminated inactivation entirely. Based on bioinformatics predictions of a calmodulin (CaM) binding site on Orai1, we also investigated a role for CaM in CDI. We identified a membrane-proximal N-terminal domain of Orai1 (residues 68-91) that binds CaM in a Ca(2+)-dependent manner and mutations that eliminate CaM binding abrogate CDI. These studies identify novel structural elements of STIM1 and Orai1 that are required for CDI and support a model in which CaM acts in concert with STIM1 and the N terminus of Orai1 to evoke rapid CRAC channel inactivation.

    View details for DOI 10.1073/pnas.0906781106

    View details for Web of Science ID 000269632400074

    View details for PubMedID 19706428

  • STIM1 Clusters and Activates CRAC Channels via Direct Binding of a Cytosolic Domain to Orai1 CELL Park, C. Y., Hoover, P. J., Mullins, F. M., Bachhawat, P., Covington, E. D., Raunser, S., Walz, T., Garcia, K. C., Dolmetsch, R. E., Lewis, R. S. 2009; 136 (5): 876-890

    Abstract

    Store-operated Ca(2+) channels activated by the depletion of Ca(2+) from the endoplasmic reticulum (ER) are a major Ca(2+) entry pathway in nonexcitable cells and are essential for T cell activation and adaptive immunity. After store depletion, the ER Ca(2+) sensor STIM1 and the CRAC channel protein Orai1 redistribute to ER-plasma membrane (PM) junctions, but the fundamental issue of how STIM1 activates the CRAC channel at these sites is unresolved. Here, we identify a minimal, highly conserved 107-aa CRAC activation domain (CAD) of STIM1 that binds directly to the N and C termini of Orai1 to open the CRAC channel. Purified CAD forms a tetramer that clusters CRAC channels, but analysis of STIM1 mutants reveals that channel clustering is not sufficient for channel activation. These studies establish a molecular mechanism for store-operated Ca(2+) entry in which the direct binding of STIM1 to Orai1 drives the accumulation and the activation of CRAC channels at ER-PM junctions.

    View details for DOI 10.1016/j.cell.2009.02.014

    View details for Web of Science ID 000263930900011

    View details for PubMedID 19249086

  • Oligomerization of STIM1 couples ER calcium depletion to CRAC channel activation NATURE Luik, R. M., Wang, B., Prakriya, M., Wu, M. M., Lewis, R. S. 2008; 454 (7203): 538-U11

    Abstract

    Ca(2+)-release-activated Ca(2+) (CRAC) channels generate sustained Ca(2+) signals that are essential for a range of cell functions, including antigen-stimulated T lymphocyte activation and proliferation. Recent studies have revealed that the depletion of Ca(2+) from the endoplasmic reticulum (ER) triggers the oligomerization of stromal interaction molecule 1 (STIM1), the ER Ca(2+) sensor, and its redistribution to ER-plasma membrane (ER-PM) junctions where the CRAC channel subunit ORAI1 accumulates in the plasma membrane and CRAC channels open. However, how the loss of ER Ca(2+) sets into motion these coordinated molecular rearrangements remains unclear. Here we define the relationships among [Ca(2+)](ER), STIM1 redistribution and CRAC channel activation and identify STIM1 oligomerization as the critical [Ca(2+)](ER)-dependent event that drives store-operated Ca(2+) entry. In human Jurkat leukaemic T cells expressing an ER-targeted Ca(2+) indicator, CRAC channel activation and STIM1 redistribution follow the same function of [Ca(2+)](ER), reaching half-maximum at approximately 200 microM with a Hill coefficient of approximately 4. Because STIM1 binds only a single Ca(2+) ion, the high apparent cooperativity suggests that STIM1 must first oligomerize to enable its accumulation at ER-PM junctions. To assess directly the causal role of STIM1 oligomerization in store-operated Ca(2+) entry, we replaced the luminal Ca(2+)-sensing domain of STIM1 with the 12-kDa FK506- and rapamycin-binding protein (FKBP12, also known as FKBP1A) or the FKBP-rapamycin binding (FRB) domain of the mammalian target of rapamycin (mTOR, also known as FRAP1). A rapamycin analogue oligomerizes the fusion proteins and causes them to accumulate at ER-PM junctions and activate CRAC channels without depleting Ca(2+) from the ER. Thus, STIM1 oligomerization is the critical transduction event through which Ca(2+) store depletion controls store-operated Ca(2+) entry, acting as a switch that triggers the self-organization and activation of STIM1-ORAI1 clusters at ER-PM junctions.

    View details for DOI 10.1038/nature07065

    View details for Web of Science ID 000257860300056

    View details for PubMedID 18596693

  • The molecular choreography of a store-operated calcium channel NATURE Lewis, R. S. 2007; 446 (7133): 284-287

    Abstract

    Store-operated calcium channels (SOCs) serve essential functions from secretion and motility to gene expression and cell growth. A fundamental mystery is how the depletion of Ca2+ from the endoplasmic reticulum (ER) activates Ca2+ entry through SOCs in the plasma membrane. Recent studies using genetic approaches have identified genes encoding the ER Ca2+ sensor and a prototypic SOC, the Ca2+-release-activated Ca2+ (CRAC) channel. New findings reveal a unique mechanism for channel activation, in which the CRAC channel and its sensor migrate independently to closely apposed sites of interaction in the ER and the plasma membrane.

    View details for DOI 10.1038/nature05637

    View details for Web of Science ID 000244892900036

    View details for PubMedID 17361175

  • Ca2+ store depletion causes STIM1 to accumulate in ER regions closely associated with the plasma membrane JOURNAL OF CELL BIOLOGY Wu, M. M., Buchanan, J., Luik, R. M., Lewis, R. S. 2006; 174 (6): 803-813

    Abstract

    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.

    View details for Web of Science ID 000240697100010

    View details for PubMedID 16966422

  • The elementary unit of store-operated Ca2+ entry: local activation of CRAC channels by STIM1 at ER-plasma membrane junctions JOURNAL OF CELL BIOLOGY Luik, R. M., Wu, M. M., Buchanan, J., Lewis, R. S. 2006; 174 (6): 815-825

    Abstract

    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.

    View details for Web of Science ID 000240697100011

    View details for PubMedID 16966423

  • Regulation of CRAC channel activity by recruitment of silent channels to a high open-probability gating mode JOURNAL OF GENERAL PHYSIOLOGY Prakriya, M., Lewis, R. S. 2006; 128 (3): 373-386

    Abstract

    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.

    View details for DOI 10.1085/jgp.200609588

    View details for Web of Science ID 000240622100011

    View details for PubMedID 16940559

  • Calcium oscillations regulate thymocyte motility during positive selection in the three-dimensional thymic environment NATURE IMMUNOLOGY Bhakta, N. R., Oh, D. Y., Lewis, R. S. 2005; 6 (2): 143-151

    Abstract

    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.

    View details for DOI 10.1038/ni1161

    View details for Web of Science ID 000226468100017

    View details for PubMedID 15654342

  • Calcium oscillations increase the efficiency and specificity of gene expression NATURE Dolmetsch, R. E., XU, K. L., Lewis, R. S. 1998; 392 (6679): 933-936

    Abstract

    Cytosolic calcium ([Ca2+]i) oscillations are a nearly universal mode of signalling in excitable and non-excitable cells. Although Ca2+ is known to mediate a diverse array of cell functions, it is not known whether oscillations contribute to the efficiency or specificity of signalling or are merely an inevitable consequence of the feedback control of [Ca2+]i. We have developed a Ca2+ clamp technique to investigate the roles of oscillation amplitude and frequency in regulating gene expression driven by the proinflammatory transcription factors NF-AT, Oct/OAP and NF-kappaB. Here we report that oscillations reduce the effective Ca2+ threshold for activating transcription factors, thereby increasing signal detection at low levels of stimulation. In addition, specificity is encoded by the oscillation frequency: rapid oscillations stimulate all three transcription factors, whereas infrequent oscillations activate only NF-kappaB. The genes encoding the cytokines interleukin (IL)-2 and IL-8 are also frequency-sensitive in a way that reflects their degree of dependence on NF-AT versus NF-kappaB. Our results provide direct evidence that [Ca2+]i oscillations increase both the efficacy and the information content of Ca2+ signals that lead to gene expression and cell differentiation.

    View details for Web of Science ID 000073359900052

    View details for PubMedID 9582075

  • Abnormal Calcium Handling Properties Underlie Familial Hypertrophic Cardiomyopathy Pathology in Patient-Specific Induced Pluripotent Stem Cells CELL STEM CELL Lan, F., Lee, A. S., Liang, P., Sanchez-Freire, V., Nguyen, P. K., Wang, L., Han, L., Yen, M., Wang, Y., Sun, N., Abilez, O. J., Hu, S., Ebert, A. D., Navarrete, E. G., Simmons, C. S., Wheeler, M., Pruitt, B., Lewis, R., Yamaguchi, Y., Ashley, E. A., Bers, D. M., Robbins, R. C., Longaker, M. T., Wu, J. C. 2013; 12 (1): 101-113

    Abstract

    Familial hypertrophic cardiomyopathy (HCM) is a prevalent hereditary cardiac disorder linked to arrhythmia and sudden cardiac death. While the causes of HCM have been identified as genetic mutations in the cardiac sarcomere, the pathways by which sarcomeric mutations engender myocyte hypertrophy and electrophysiological abnormalities are not understood. To elucidate the mechanisms underlying HCM development, we generated patient-specific induced pluripotent stem cell cardiomyocytes (iPSC-CMs) from a ten-member family cohort carrying a hereditary HCM missense mutation (Arg663His) in the MYH7 gene. Diseased iPSC-CMs recapitulated numerous aspects of the HCM phenotype including cellular enlargement and contractile arrhythmia at the single-cell level. Calcium (Ca(2+)) imaging indicated dysregulation of Ca(2+) cycling and elevation in intracellular Ca(2+) ([Ca(2+)](i)) are central mechanisms for disease pathogenesis. Pharmacological restoration of Ca(2+) homeostasis prevented development of hypertrophy and electrophysiological irregularities. We anticipate that these findings will help elucidate the mechanisms underlying HCM development and identify novel therapies for the disease.

    View details for DOI 10.1016/j.stem.2012.10.010

    View details for Web of Science ID 000313839500014

    View details for PubMedID 23290139

  • Molecular Basis of Calcium Signaling in Lymphocytes: STIM and ORAI ANNUAL REVIEW OF IMMUNOLOGY, VOL 28 Hogan, P. G., Lewis, R. S., Rao, A. 2010; 28: 491-533

    Abstract

    Ca(2+) entry into cells of the peripheral immune system occurs through highly Ca(2+)-selective channels known as CRAC (calcium release-activated calcium) channels. CRAC channels are a very well-characterized example of store-operated Ca(2+) channels, so designated because they open when the endoplasmic reticulum (ER) Ca(2+) store becomes depleted. Physiologically, Ca(2+) is released from the ER lumen into the cytoplasm when activated receptors couple to phospholipase C and trigger production of the second messenger inositol 1,4,5-trisphosphate (IP(3)). IP(3) binds to IP(3) receptors in the ER membrane and activates Ca(2+) release. The proteins STIM and ORAI were discovered through limited and genome-wide RNAi screens, respectively, performed in Drosophila cells and focused on identifying modulators of store-operated Ca(2+) entry. STIM1 and STIM2 sense the depletion of ER Ca(2+) stores, whereas ORAI1 is a pore subunit of the CRAC channel. In this review, we discuss selected aspects of Ca(2+) signaling in cells of the immune system, focusing on the roles of STIM and ORAI proteins in store-operated Ca(2+) entry.

    View details for DOI 10.1146/annurev.immunol.021908.132550

    View details for Web of Science ID 000277363600019

    View details for PubMedID 20307213

  • Some assembly required: Constructing the elementary units of store-operated Ca2+ entry CELL CALCIUM Wu, M. M., Luik, R. M., Lewis, R. S. 2007; 42 (2): 163-172

    Abstract

    The means by which Ca(2+) store depletion evokes the opening of store-operated Ca(2+) channels (SOCs) in the plasma membrane of excitable and non-excitable cells has been a longstanding mystery. Indirect evidence has supported local interactions between the ER and SOCs as well as long-range interactions mediated through a diffusible activator. The recent molecular identification of the ER Ca(2+) sensor (STIM1) and a subunit of the CRAC channel (Orai1), a prototypic SOC, has now made it possible to visualize directly the sequence of events that links store depletion to CRAC channel opening. Following store depletion, STIM1 moves from locations throughout the ER to accumulate in ER subregions positioned within 10-25nm of the plasma membrane. Simultaneously, Orai1 gathers at discrete sites in the plasma membrane directly opposite STIM1, resulting in local CRAC channel activation. These new studies define the elementary units of store-operated Ca(2+) entry, and reveal an unprecedented mechanism for channel activation in which the stimulus brings a channel and its activator/sensor together for interaction across apposed membrane compartments. We discuss the implications of this choreographic mechanism with regard to Ca(2+) dynamics, specificity of Ca(2+) signaling, and the existence of a specialized ER subset dedicated to the control of the CRAC channel.

    View details for DOI 10.1016/j.ceca.2007.03.003

    View details for Web of Science ID 000248833400007

    View details for PubMedID 17499354

  • New insights into the molecular mechanisms of store-operated Ca2+ signaling in T cells TRENDS IN MOLECULAR MEDICINE Luik, R. M., Lewis, R. S. 2007; 13 (3): 103-107

    Abstract

    The activation of Ca(2+) entry through store-operated channels by agonists that deplete Ca(2+) from the endoplasmic reticulum (ER) is an ubiquitous signaling mechanism, the molecular basis of which has remained elusive for the past 20 years. In T lymphocytes, store-operated Ca(2+)-release-activated Ca(2+) (CRAC) channels constitute the sole pathway for Ca(2+) entry following antigen-receptor engagement, and their function is essential for driving the program of gene expression that underlies T-cell activation by antigen. The first molecular components of this pathway have recently been identified: stromal interaction molecule 1 (STIM1), the ER Ca(2+) sensor, and Orai1, a pore-forming subunit of the CRAC channel. Recent work shows that CRAC channels are activated in a complex fashion that involves the co-clustering of STIM1 in junctional ER directly opposite Orai1 in the plasma membrane. These studies reveal an abundance of sites where Ca(2+) signaling might be controlled to modulate the activity of T cells during the immune response.

    View details for DOI 10.1016/j.molmed.2007.01.004

    View details for Web of Science ID 000245393200003

    View details for PubMedID 17267286

  • Real-time measurement of signaling and motility during T cell development in the thymus SEMINARS IN IMMUNOLOGY Bhakta, N. R., Lewis, R. S. 2005; 17 (6): 411-420

    Abstract

    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.

    View details for DOI 10.1016/j.smim.2005.09.004

    View details for Web of Science ID 000233992400004

    View details for PubMedID 16256363

  • Modulation of plasma membrane calcium-ATPase activity by local calcium microdomains near CRAC channels in human T cells JOURNAL OF PHYSIOLOGY-LONDON Bautista, D. M., Lewis, R. S. 2004; 556 (3): 805-817

    Abstract

    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.

    View details for DOI 10.1113/jphysiol.2003.060004

    View details for Web of Science ID 000221266600011

    View details for PubMedID 14966303

  • CRAC channels: activation, permeation, and the search for a molecular identity CELL CALCIUM Prakriya, M., Lewis, R. S. 2003; 33 (5-6): 311-321

    Abstract

    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.

    View details for DOI 10.1016/S0143-4160(03)00045-9

    View details for Web of Science ID 000183673700004

    View details for PubMedID 12765678

  • Enhancement of calcium signalling dynamics and stability by delayed modulation of the plasma-membrane calcium-ATPase in human T cells JOURNAL OF PHYSIOLOGY-LONDON Bautista, D. M., Hoth, M., Lewis, R. S. 2002; 541 (3): 877-894

    Abstract

    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.

    View details for DOI 10.1113/jphysiol.2001.016154

    View details for Web of Science ID 000176746400017

    View details for PubMedID 12068047

  • Separation and characterization of currents through store-operated CRAC channels and Mg2+-inhibited cation (MIC) channels JOURNAL OF GENERAL PHYSIOLOGY Prakriya, M., Lewis, R. S. 2002; 119 (5): 487-507

    Abstract

    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.

    View details for Web of Science ID 000176142500006

    View details for PubMedID 11981025

  • Potentiation and inhibition of Ca2+ release-activated Ca2+ channels by 2-aminoethyldiphenyl borate (2-APB) occurs independently of IP3 receptors JOURNAL OF PHYSIOLOGY-LONDON Prakriya, M., Lewis, R. S. 2001; 536 (1): 3-19

    Abstract

    1. The effects of the IP(3)-receptor antagonist 2-aminoethyldiphenyl borate (2-APB) on the Ca(2+) release-activated Ca(2+) current (I(CRAC)) in Jurkat human T cells, DT40 chicken B cells and rat basophilic leukaemia (RBL) cells were examined. 2. 2-APB elicited both stimulatory and inhibitory effects on Ca(2+) influx through CRAC channels. At concentrations of 1-5 microM, 2-APB enhanced Ca(2+) entry in intact cells and increased I(CRAC) amplitude by up to fivefold. At levels > or = 10 microM, 2-APB caused a transient enhancement of I(CRAC) followed by inhibition. 3. 2-APB altered the kinetics of fast Ca(2+)-dependent inactivation of I(CRAC). At concentrations of 1-5 microM, 2-APB increased the rate of fast inactivation. In contrast, 2-APB at higher concentrations (> or = 10 microM) reduced or completely blocked inactivation. 4. 2-APB inhibited Ca(2+) efflux from mitochondria. 5. 2-APB inhibited I(CRAC) more potently when applied extracellularly than intracellularly. Furthermore, increased protonation of 2-APB at low pH did not affect potentiation or inhibition. Thus, 2-APB may have an extracellular site of action. 6. Neither I(CRAC) activation by passive store depletion nor the effects of 2-APB were altered by intracellular dialysis with 500 microg ml(-1) heparin. 7. I(CRAC) is present in wild-type as well as mutant DT40 B cells lacking all three IP(3) receptor isoforms. 2-APB also potentiates and inhibits I(CRAC) in both cell types, indicating that 2-APB exerts its effects independently of IP(3) receptors. 8. Our results show that CRAC channel activation does not require physical interaction with IP(3) receptors as proposed in the conformational coupling model. Potentiation of I(CRAC) by 2-APB may be a useful diagnostic feature for positive identification of putative CRAC channel genes, and provides a novel tool for exploring the physiological functions of store-operated channels.

    View details for Web of Science ID 000171427200003

    View details for PubMedID 11579153

  • Calcium signaling mechanisms in T lymphocytes ANNUAL REVIEW OF IMMUNOLOGY Lewis, R. S. 2001; 19: 497-521

    Abstract

    Elevation of intracellular free Ca(2+) is one of the key triggering signals for T-cell activation by antigen. A remarkable variety of Ca(2+) signals in T cells, ranging from infrequent spikes to sustained oscillations and plateaus, derives from the interactions of multiple Ca(2+) sources and sinks in the cell. Following engagement of the T cell receptor, intracellular channels (IP3 and ryanodine receptors) release Ca(2+) from intracellular stores, and by depleting the stores trigger prolonged Ca(2+) influx through store-operated Ca(2+) (CRAC) channels in the plasma membrane. The amplitude and dynamics of the Ca(2+) signal are shaped by several mechanisms, including K(+) channels and membrane potential, slow modulation of the plasma membrane Ca(2+)-ATPase, and mitochondria that buffer Ca(2+) and prevent the inactivation of CRAC channels. Ca(2+) signals have a number of downstream targets occurring on multiple time scales. At short times, Ca(2+) signals help to stabilize contacts between T cells and antigen-presenting cells through changes in motility and cytoskeletal reorganization. Over periods of minutes to hours, the amplitude, duration, and kinetic signature of Ca(2+) signals increase the efficiency and specificity of gene activation events. The complexity of Ca(2+) signals contains a wealth of information that may help to instruct lymphocytes to choose between alternate fates in response to antigenic stimulation.

    View details for Web of Science ID 000168234600018

    View details for PubMedID 11244045

  • Mitochondrial control of calcium-channel gating: A mechanism for sustained signaling and transcriptional activation in T lymphocytes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hoth, M., Button, D. C., Lewis, R. S. 2000; 97 (19): 10607-10612

    Abstract

    In addition to their well-known functions in cellular energy transduction, mitochondria play an important role in modulating the amplitude and time course of intracellular Ca(2+) signals. In many cells, mitochondria act as Ca(2+) buffers by taking up and releasing Ca(2+), but this simple buffering action by itself often cannot explain the organelle's effects on Ca(2+) signaling dynamics. Here we describe the functional interaction of mitochondria with store-operated Ca(2+) channels in T lymphocytes as a mechanism of mitochondrial Ca(2+) signaling. In Jurkat T cells with functional mitochondria, prolonged depletion of Ca(2+) stores causes sustained activation of the store-operated Ca(2+) current, I(CRAC) (CRAC, Ca(2+) release-activated Ca(2+)). Inhibition of mitochondrial Ca(2+) uptake by compounds that dissipate the intramitochondrial potential unmasks Ca(2+)-dependent inactivation of I(CRAC). Thus, functional mitochondria are required to maintain CRAC-channel activity, most likely by preventing local Ca(2+) accumulation near sites that govern channel inactivation. In cells stimulated through the T-cell antigen receptor, acute blockade of mitochondrial Ca(2+) uptake inhibits the nuclear translocation of the transcription factor NFAT in parallel with CRAC channel activity and [Ca(2+)](i) elevation, indicating a functional link between mitochondrial regulation of I(CRAC) and T-cell activation. These results demonstrate a role for mitochondria in controlling Ca(2+) channel activity and signal transmission from the plasma membrane to the nucleus.

    View details for Web of Science ID 000089341400058

    View details for PubMedID 10973476

  • Store-operated calcium channels ION CHANNEL REGULATION Lewis, R. S. 1999; 33: 279-307

    View details for Web of Science ID 000080418200013

    View details for PubMedID 10218123

  • Mitochondrial regulation of store-operated calcium signaling in T lymphocytes JOURNAL OF CELL BIOLOGY Hoth, M., Fanger, C. M., Lewis, R. S. 1997; 137 (3): 633-648

    Abstract

    Mitochondria act as potent buffers of intracellular Ca2+ in many cells, but a more active role in modulating the generation of Ca2+ signals is not well established. We have investigated the ability of mitochondria to modulate store-operated or "capacitative" Ca2+ entry in Jurkat leukemic T cells and human T lymphocytes using fluorescence imaging techniques. Depletion of the ER Ca2+ store with thapsigargin (TG) activates Ca2+ release-activated Ca2+ (CRAC) channels in T cells, and the ensuing influx of Ca2+ loads a TG-insensitive intracellular store that by several criteria appears to be mitochondria. Loading of this store is prevented by carbonyl cyanide m-chlorophenylhydrazone or by antimycin A1 + oligomycin, agents that are known to inhibit mitochondrial Ca2+ import by dissipating the mitochondrial membrane potential. Conversely, intracellular Na+ depletion, which inhibits Na+-dependent Ca2+ export from mitochondria, enhances store loading. In addition, we find that rhod-2 labels mitochondria in T cells, and it reports changes in Ca2+ levels that are consistent with its localization in the TG-insensitive store. Ca2+ uptake by the mitochondrial store is sensitive (threshold is <400 nM cytosolic Ca2+), rapid (detectable within 8 s), and does not readily saturate. The rate of mitochondrial Ca2+ uptake is sensitive to extracellular [Ca2+], indicating that mitochondria sense Ca2+ gradients near CRAC channels. Remarkably, mitochondrial uncouplers or Na+ depletion prevent the ability of T cells to maintain a high rate of capacitative Ca2+ entry over prolonged periods of >10 min. Under these conditions, the rate of Ca2+ influx in single cells undergoes abrupt transitions from a high influx to a low influx state. These results demonstrate that mitochondria not only buffer the Ca2+ that enters T cells via store-operated Ca2+ channels, but also play an active role in modulating the rate of capacitative Ca2+ entry.

    View details for Web of Science ID A1997WY01900009

    View details for PubMedID 9151670

  • Differential activation of transcription factors induced by Ca2+ response amplitude and duration NATURE Dolmetsch, R. E., Lewis, R. S., Goodnow, C. C., Healy, J. I. 1997; 386 (6627): 855-858

    Abstract

    An increase in the intracellular calcium ion concentration ([Ca2+]i) controls a diverse range of cell functions, including adhesion, motility, gene expression and proliferation. Calcium signalling patterns can occur as single transients, repetitive oscillations or sustained plateaux, but it is not known whether these patterns are responsible for encoding the specificity of cellular responses. We report here that the amplitude and duration of calcium signals in B lymphocytes controls differential activation of the pro-inflammatory transcriptional regulators NF-kappaB, c-Jun N-terminal kinase (JNK) and NFAT. NF-kappaB and JNK are selectively activated by a large transient [Ca2+]i rise, whereas NFAT is activated by a low, sustained Ca2+ plateau. Differential activation results from differences in the Ca2+ sensitivities and kinetic behaviour of the three pathways. Our results show how downstream effectors can decode information contained in the amplitude and duration of Ca2+ signals, revealing a mechanism by which a multifunctional second messenger such as Ca2+ can achieve specificity in signalling to the nucleus.

    View details for Web of Science ID A1997WV70600060

    View details for PubMedID 9126747

  • Function follows form: The role of store-operated calcium channels in T-cell activation CELLULAR PHYSIOLOGY AND BIOCHEMISTRY Fanger, C. M., Zweifach, A., Dolmetsch, R. E., Hoth, M., Lewis, R. S. 1997; 7 (3-4): 203-218
  • Calcium-dependent potentiation of store-operated calcium channels in T lymphocytes JOURNAL OF GENERAL PHYSIOLOGY Zweifach, A., Lewis, R. S. 1996; 107 (5): 597-610

    Abstract

    The depletion of intracellular Ca2+ stores triggers the opening of Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane of T lymphocytes. We have investigated the additional role of extracellular Ca2+ (Ca02+) in promoting CRAC channel activation in Jurkat leukemic T cells. Ca2+ stores were depleted with 1 microM thapsigargin in the nominal absence of Ca02+ with 12 mM EGTA or BAPTA in the recording pipette. Subsequent application of Ca02+ caused ICRAC to appear in two phases. The initial phase was complete within 1 s and reflects channels that were open in the absence of Ca02+. The second phase consisted of a severalfold exponential increase in current amplitude with a time constant of 5-10 s; we call this increase Ca(2+)-dependent potentiation, or CDP. The shape of the current-voltage relation and the inferred single-channel current amplitude are unchanged during CDP, indicating that CDP reflects an alteration in channel gating rather than permeation. The extent of CDP is modulated by voltage, increasing from approximately 50% at +50 mV to approximately 350% at -75 mV in the presence of 2 mM Ca02+. The voltage dependence of CDP also causes ICRAC to increase slowly during prolonged hyperpolarizations in the constant presence of Ca02+. CDP is not affected by exogenous intracellular Ca2+ buffers, and Ni2+, a CRAC channel blocker, can cause potentiation. Thus, the underlying Ca2+ binding site is not intracellular. Ba2+ has little or no ability to potentiate CRAC channels. These results demonstrate that the store-depletion signal by itself triggers only a small fraction of capacitative Ca2+ entry and establish Ca2+ as a potent cofactor in this process. CDP confers a previously unrecognized voltage dependence and slow time dependence on CRAC channel activation that may contribute to the dynamic behavior of ICRAC.

    View details for Web of Science ID A1996UJ38600004

    View details for PubMedID 8740373

  • CHARACTERIZATION OF T-CELL MUTANTS WITH DEFECTS IN CAPACITATIVE CALCIUM-ENTRY - GENETIC-EVIDENCE FOR THE PHYSIOLOGICAL ROLES OF CRAC CHANNELS JOURNAL OF CELL BIOLOGY Fanger, C. M., Hoth, M., Crabtree, G. R., Lewis, R. S. 1995; 131 (3): 655-667

    Abstract

    Prolonged Ca2+ influx is an essential signal for the activation of T lymphocytes by antigen. This influx is thought to occur through highly selective Ca2+ release-activated Ca2+ (CRAC) channels that are activated by the depletion of intracellular Ca2+ stores. We have isolated mutants of the Jurkat human T cell line NZdipA to explore the molecular mechanisms that underlie capacitative Ca2+ entry and to allow a genetic test of the functions of CRAC channels in T cells. Five mutant cell lines (CJ-1 through CJ-5) were selected based on their failure to express a lethal diphtheria toxin A chain gene and a lacZ reporter gene driven by NF-AT, a Ca(2+)- and protein kinase C-dependent transcription factor. The rate of Ca2+ influx evoked by thapsigargin was reduced to varying degrees in the mutant cells whereas the dependence of NF-AT/lacZ gene transcription on [Ca2+]i was unaltered, suggesting that the transcriptional defect in these cells is caused by a reduced level of capacitative Ca2+ entry. We examined several factors that determine the rate of Ca2+ entry, including CRAC channel activity, K(+)-channel activity, and Ca2+ clearance mechanisms. The only parameter found to be dramatically altered in most of the mutant lines was the amplitude of the Ca2+ current (ICRAC), which ranged from 1 to 41% of that seen in parental control cells. In each case, the severity of the ICRAC defect was closely correlated with deficits in Ca2+ influx rate and Ca(2-)-dependent gene transcription. Behavior of the mutant cells provides genetic evidence for several roles of ICRAC in T cells. First, mitogenic doses of ionomycin appear to elevate [Ca2+]i primarily by activating CRAC channels. Second, ICRAC promotes the refilling of empty Ca2+ stores. Finally, CRAC channels are solely responsible for the Ca2+ influx that underlies antigen-mediated T cell activation. These mutant cell lines may provide a useful system for isolating, expressing, and exploring the functions of genes involved in capacitative Ca2+ entry.

    View details for Web of Science ID A1995TC10100009

    View details for PubMedID 7593187

  • SLOW CALCIUM-DEPENDENT INACTIVATION OF DEPLETION-ACTIVATED CALCIUM CURRENT - STORE-DEPENDENT AND STORE-INDEPENDENT MECHANISMS JOURNAL OF BIOLOGICAL CHEMISTRY Zweifach, A., Lewis, R. S. 1995; 270 (24): 14445-14451

    Abstract

    Feedback regulation of Ca2+ release-activated Ca2+ (CRAC) channels was studied in Jurkat leukemic T lymphocytes using whole cell recording and [Ca2+]i measurement techniques. CRAC channels were activated by passively depleting intracellular Ca2+ stores in the absence of extracellular Ca2+. Under conditions of moderate intracellular Ca2+ buffering, elevating [Ca2+]o to 22 mM initiated an inward current through CRAC channels that declined slowly with a half-time of approximately 30 s. This slow inactivation was evoked by a rise in [Ca2+]i, as it was effectively suppressed by an elevated level of EFTA in the recording pipette that prevented increases in [Ca2+]i. Blockade of Ca2+ uptake into stores by thapsigargin with or without intracellular inositol 1,4,5-trisphosphate reduced the extent of slow inactivation by approximately 50%, indicating that store refilling normally contributes significantly to this process. The store-independent (thapsigargin-insensitive) portion of slow inactivation was largely prevented by the protein phosphatase inhibitor, okadaic acid, and by a structurally related compound, 1-norokadaone, but not by calyculin A nor by cyclosporin A and FK506 at concentrations that fully inhibit calcineurin (protein phosphatase 2B) in T cells. These results argue against the involvement of protein phosphatases 1, 2A, 2B, or 3 in store-independent inactivation. We conclude that calcium acts through at least two slow negative feedback pathways to inhibit CRAC channels. Slow feedback inhibition of CRAC current is likely to play important roles in controlling the duration and dynamic behavior of receptor-generated Ca2+ signals.

    View details for Web of Science ID A1995RD45500034

    View details for PubMedID 7540169

  • RAPID INACTIVATION OF DEPLETION-ACTIVATED CALCIUM CURRENT (I-CRAC) DUE TO LOCAL CALCIUM FEEDBACK JOURNAL OF GENERAL PHYSIOLOGY Zweifach, A., Lewis, R. S. 1995; 105 (2): 209-226

    Abstract

    Rapid inactivation of Ca2+ release-activated Ca2+ (CRAC) channels was studied in Jurkat leukemic T lymphocytes using whole-cell patch clamp recording and [Ca2+]i measurement techniques. In the presence of 22 mM extracellular Ca2+, the Ca2+ current declined with a biexponential time course (time constants of 8-30 ms and 50-150 ms) during hyperpolarizing pulses to potentials more negative than -40 mV. Several lines of evidence suggest that the fast inactivation process is Ca2+ but not voltage dependent. First, the speed and extent of inactivation are enhanced by conditions that increase the rate of Ca2+ entry through open channels. Second, inactivation is substantially reduced when Ba2+ is present as the charge carrier. Third, inactivation is slowed by intracellular dialysis with BAPTA (12 mM), a rapid Ca2+ buffer, but not by raising the cytoplasmic concentration of EGTA, a slower chelator, from 1.2 to 12 mM. Recovery from fast inactivation is complete within 200 ms after repolarization to -12 mV. Rapid inactivation is unaffected by changes in the number of open CRAC channels or global [Ca2+]i. These results demonstrate that rapid inactivation of ICRAC results from the action of Ca2+ in close proximity to the intracellular mouths of individual channels, and that Ca2+ entry through one CRAC channel does not affect neighboring channels. A simple model for Ca2+ diffusion in the presence of a mobile buffer predicts multiple Ca2+ inactivation sites situated 3-4 nm from the intracellular mouth of the pore, consistent with a location on the CRAC channel itself.

    View details for Web of Science ID A1995QK81100002

    View details for PubMedID 7760017

  • SIGNALING BETWEEN INTRACELLULAR CA2+ STORES AND DEPLETION-ACTIVATED CA2+ CHANNELS GENERATES [CA2+](I) OSCILLATIONS IN T-LYMPHOCYTES JOURNAL OF GENERAL PHYSIOLOGY Dolmetsch, R. E., Lewis, R. S. 1994; 103 (3): 365-388

    Abstract

    Stimulation through the antigen receptor (TCR) of T lymphocytes triggers cytosolic calcium ([Ca2+]i) oscillations that are critically dependent on Ca2+ entry across the plasma membrane. We have investigated the roles of Ca2+ influx and depletion of intracellular Ca2+ stores in the oscillation mechanism, using single-cell Ca2+ imaging techniques and agents that deplete the stores. Thapsigargin (TG; 5-25 nM), cyclopiazonic acid (CPA; 5-20 microM), and tert-butylhydroquinone (tBHQ; 80-200 microM), inhibitors of endoplasmic reticulum Ca(2+)-ATPases, as well as the Ca2+ ionophore ionomycin (5-40 nM), elicit [Ca2+]i oscillations in human T cells. The oscillation frequency is approximately 5 mHz (for ATPase inhibitors) to approximately 10 mHz (for ionomycin) at 22-24 degrees C. The [Ca2+]i oscillations resemble those evoked by TCR ligation in terms of their shape, amplitude, and an absolute dependence on Ca2+ influx. Ca(2+)-ATPase inhibitors and ionomycin induce oscillations only within a narrow range of drug concentrations that are expected to cause partial depletion of intracellular stores. Ca(2+)-induced Ca2+ release does not appear to be significantly involved, as rapid removal of extracellular Ca2+ elicits the same rate of [Ca2+]i decline during the rising and falling phases of the oscillation cycle. Both transmembrane Ca2+ influx and the content of ionomycin-releasable Ca2+ pools fluctuate in oscillating cells. From these data, we propose a model in which [Ca2+]i oscillations in T cells result from the interaction between intracellular Ca2+ stores and depletion-activated Ca2+ channels in the plasma membrane.

    View details for Web of Science ID A1994NC00500001

    View details for PubMedID 8195779

  • MITOGEN-REGULATED CA2+ CURRENT OF T-LYMPHOCYTES IS ACTIVATED BY DEPLETION OF INTRACELLULAR CA2+ STORES PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zweifach, A., Lewis, R. S. 1993; 90 (13): 6295-6299

    Abstract

    Stimulated influx of Ca2+ across the plasma membrane of T lymphocytes is an essential triggering signal for T-cell activation by antigen. Regulation of the T-cell Ca2+ conductance is not understood; conflicting evidence supports direct activation by inositol 1,4,5-trisphosphate (IP3) or by a signal generated by the depletion of intracellular Ca2+ stores. We have used the perforated-patch recording technique to compare the biophysical properties of Ca2+ currents activated by T-cell receptor stimulation and by thapsigargin, a Ca(2+)-ATPase inhibitor that depletes intracellular stores without generating IP3. Both currents are blocked by Ni2+, are inwardly rectifying, are highly Ca(2+)-selective, and exhibit voltage-independent gating with a unitary chord conductance of approximately 24 fS in isotonic Ca2+. Fluctuation analysis suggests that the underlying Ca2+ transporter is a channel rather than an iron carrier. Thus, in terms of ion permeation, gating, and unitary conductance, the Ca2+ current activated by thapsigargin is indistinguishable from the elicited by crosslinking of T-cell receptors. Moreover, the unitary Ca2+ conductance is > 100-fold smaller than that of previously described IP3-gated, Ca(2+)-permeable channels in T cells [Kuno, M. & Gardner, P. (1987) Nature (London) 326, 301-304]. These results demonstrate that mitogen-activated Ca2+ influx is controlled by the state of intracellular Ca2+ stores rather than by the direct action of IP3 on Ca2+ channels in the plasma membrane.

    View details for Web of Science ID A1993LM03500087

    View details for PubMedID 8392195

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