Polarized but Differential Localization and Recruitment of STIM1, Orai1 and TRPC Channels in Secretory Cells

Polarized Ca²⁺ signals in secretory epithelial cells are determined by compartmentalized localization of Ca²⁺ signaling proteins at the apical pole. Recently the ER Ca²⁺ sensor STIM1 (stromal interaction molecule 1) and the Orai channels were shown to play a critical role in store‐dependent Ca²⁺ influx. STIM1 also gates the transient receptor potential‐canonical (TRPC) channels. Here, we asked how cell stimulation affects the localization, recruitment and function of the native proteins in polarized cells. Inhibition of Orai1, STIM1, or deletion of TRPC1 reduces Ca²⁺ influx and frequency of Ca²⁺ oscillations. Orai1 localization is restricted to the apical pole of the lateral membrane. Surprisingly, cell stimulation does not lead to robust clustering of native Orai1, as is observed with expressed Orai1. Unexpectedly, cell stimulation causes polarized recruitment of native STIM1 to both the apical and lateral regions, thus to regions with and without Orai1. Accordingly, STIM1 and Orai1 show only 40% colocalization. Consequently, STIM1 shows higher colocalization with the basolateral membrane marker E‐cadherin than does Orai1, while Orai1 showed higher colocalization with the tight junction protein ZO1. TRPC1 is expressed in both apical and basolateral regions of the plasma membrane. Co‐IP of STIM1/Orai1/IP₃ receptors (IP₃Rs)/TRPCs is enhanced by cell stimulation and disrupted by 2‐aminoethoxydiphenyl borate (2APB). The polarized localization and recruitment of these proteins results in preferred Ca²⁺ entry that is initiated at the apical pole. These findings reveal that in addition to Orai1, STIM1 likely regulates other Ca²⁺ permeable channels, such as the TRPCs. Both channels contribute to the frequency of [Ca²⁺] oscillations and thus impact critical cellular functions.     

Potent functional uncoupling between STIM1 and Orai1 by dimeric 2-aminodiphenyl borinate analogs

The coupling of ER Ca²⁺-sensing STIM proteins and PM Orai Ca²⁺ entry channels generates “store-operated” Ca²⁺ signals crucial in controlling responses in many cell types. The dimeric derivative of 2-aminoethoxydiphenyl borinate (2-APB), DPB162-AE, blocks functional coupling between STIM1 and Orai1 with an IC₅₀ (200 nM) 100-fold lower than 2-APB. Unlike 2-APB, DPB162-AE does not affect L-type or TRPC channels or Ca²⁺ pumps at maximal STIM1–Orai1 blocking levels. DPB162-AE blocks STIM1-induced Orai1 or Orai2, but does not block Orai3 or STIM2-mediated effects. We narrowed the DPB162-AE site of action to the STIM–Orai activating region (SOAR) of STIM1. DPB162-AE does not prevent the SOAR–Orai1 interaction but potently blocks SOAR-mediated Orai1 channel activation, yet its action is not as an Orai1 channel pore blocker. Using the SOAR-F394H mutant which prevents both physical and functional coupling to Orai1, we reveal DPB162-AE rapidly restores SOAR–Orai binding but only slowly restores Orai1 channel-mediated Ca²⁺ entry. With the same SOAR mutant, 2-APB induces rapid physical and functional coupling to Orai1, but channel activation is transient. We infer that the actions of both 2-APB and DPB162-AE are directed toward the STIM1–Orai1 coupling interface. Compared to 2-APB, DPB162-AE is a much more potent and specific STIM1/Orai1 functional uncoupler. DPB162-AE provides an important pharmacological tool and a useful mechanistic probe for the function and coupling between STIM1 and Orai1 channels.     

TRPC3 Regulates Agonist-stimulated Ca2+ Mobilization by Mediating the Interaction between Type I Inositol 1,4,5-Trisphosphate Receptor, RACK1, and Orai1

There is a body of evidence suggesting that Ca²⁺ handling proteins assemble into signaling complexes required for a fine regulation of Ca²⁺ signals, events that regulate a variety of critical cellular processes. Canonical transient receptor potential (TRPC) and Orai proteins have both been proposed to form Ca²⁺-permeable channels mediating Ca²⁺ entry upon agonist stimulation. A number of studies have demonstrated that inositol 1,4,5-trisphosphate receptors (IP₃Rs) interact with plasma membrane TRPC channels; however, at present there is no evidence supporting the interaction between Orai proteins and IP₃Rs. Here we report that treatment with thapsigargin or cellular agonists results in association of Orai1 with types I and II IP₃Rs. In addition, we have found that TRPC3, RACK1 (receptor for activated protein kinase C-1), and STIM1 (stromal interaction molecule 1) interact with Orai1 upon stimulation with agonists. TRPC3 expression silencing prevented both the interaction of Orai1 with TRPC3 and, more interestingly, the association of Orai1 with the type I IP₃R, but not with the type II IP₃R, thus suggesting that TRPC3 selectively mediates interaction between Orai1 and type I IP₃R. In addition, TRPC3 expression silencing attenuated ATP- and CCh-stimulated interaction between RACK1 and the type I IP₃R, as well as Ca²⁺ release and entry. In conclusion, our results indicate that agonist stimulation results in the formation of an Orai1-STIM1-TRPC3-RACK1-type I IP₃R complex, where TRPC3 plays a central role. This Ca²⁺ signaling complex might be important for both agonist-induced Ca²⁺ release and entry.     

内质网上的蛋白质RCN2与STIM1-Orai1复合体相互作用

膜蛋白质Orail组成了一类被称为钙释放激活钙通道（CRAC）的离子通道,并且由相互作用的蛋白质STIM1作为其在内质网上的钙感受器．但是这类通道的调节机制还未研究透彻．通过串连亲和纯化STIM1-Orai1复合体,发现与之相互作用的内质网蛋白质RCN2．共聚焦显微术显示RCN2与STIM1在钙库排空前后完全共定位．对RCN2的EFhands结构突变体所作单细胞测钙,结果显示其对钙库操控通道电流特性有微弱影响．全内反射荧光显微术显示,RCN2以花环状围绕包围STIM1聚集堆,这提示RCN2在STIM1聚集中起到一种结构约束作用．     

Evidence for the interaction of peroxiredoxin-4 with the store-operated calcium channel activator STIM1 in liver cells

Ca²⁺ entry through store-operated Ca²⁺ channels (SOCs) in the plasma membrane (PM) of hepatocytes plays a central role in the hormonal regulation of liver metabolism. SOCs are composed of Orai1, the channel pore protein, and STIM1, the activator protein, and are regulated by hormones and reactive oxygen species (ROS). In addition to Orai1, STIM1 also interacts with several other intracellular proteins. Most previous studies of the cellular functions of Orai1 and STIM1 have employed immortalised cells in culture expressing ectopic proteins tagged with a fluorescent polypeptide such as GFP. Little is known about the intracellular distributions of endogenous Orai1 and STIM1. The aims are to determine the intracellular distribution of endogenous Orai1 and STIM1 in hepatocytes and to identify novel STIM1 binding proteins. Subcellular fractions of rat liver were prepared by homogenisation and differential centrifugation. Orai1 and STIM1 were identified and quantified by western blot. Orai1 was found in the PM (0.03%), heavy (44%) and light (27%) microsomal fractions, and STIM1 in the PM (0.09%), and heavy (85%) and light (13%) microsomal fractions. Immunoprecipitation of STIM1 followed by LC/MS or western blot identified peroxiredoxin-4 (Prx-4) as a potential STIM1 binding protein. Prx-4 was found principally in the heavy microsomal fraction. Knockdown of Prx-4 using siRNA, or inhibition of Prx-4 using conoidin A, did not affect Ca²⁺ entry through SOCs but rendered SOCs susceptible to inhibition by H₂O₂. It is concluded that, in hepatocytes, a considerable proportion of endogenous Orai1 and STIM1 is located in the rough ER. In the rough ER, STIM1 interacts with Prx-4, and this interaction may contribute to the regulation by ROS of STIM1 and SOCs.     

CRAC channels in secretory epithelial cell function and disease

The receptor-evoked Ca²⁺ signal in secretory epithelia mediate many cellular functions essential for cell survival and their most fundamental functions of secretory granules exocytosis and fluid and electrolyte secretion. Ca²⁺ influx is a key component of the receptor-evoked Ca²⁺ signal in secretory cell and is mediated by both TRPC and the STIM1-activated Orai1 channels that mediates the Ca²⁺ release-activated current (CRAC) I_crac. The core components of the receptor-evoked Ca²⁺ signal are assembled at the ER/PM junctions where exchange of materials between the plasma membrane and internal organelles take place, including transfer of lipids and Ca²⁺. The Ca²⁺ signal generated at the confined space of the ER/PM junctions is necessary for activation of the Ca²⁺-regulated proteins and ion channels that mediate exocytosis with high fidelity and tight control. In this review we discuss the general properties of Ca²⁺ signaling, PI(4,5)P₂ and other lipids at the ER/PM junctions with regard to secretory cells function and disease caused by uncontrolled Ca²⁺ influx.     

The distinct role of STIM1 and STIM2 in the regulation of store-operated Ca2+ entry and cellular function

Stromal interaction molecules STIM1 and STIM2 are endoplasmic reticulum (ER) Ca²⁺ sensors that initiate store-operated Ca ²⁺ entry (SOCE). The roles of STIM1-mediated SOCE in cancer biology have been highlighted in different types of cancer, but that of STIM2 is unknown. By the model of cervical cancer, here we focus on the cooperative regulation of SOCE by STIM proteins and their distinct roles in cellular function. Immunofluorescent stainings of surgical specimens of cervical cancer show that STIM1 and STIM2 are abundant in tumor tissues, but STIM1 is the major ER Ca ²⁺ sensor identified in the invasive front of cancer tissues. STIM1 or STIM2 overexpression in cervical cancer SiHa cells induces an upregulated SOCE. Regarding cellular function, STIM1 and STIM2 are necessary for cell proliferation, whereas STIM1 is the dominant ER Ca ²⁺ sensor involved in cell migration. During SOCE, STIM1 is aggregated and translocated towards the Orai1-containing plasma membrane in association with the microtubule plus-end binding protein EB1. In contrast, STIM2 is constitutively aggregated without significant trafficking or association with microtubules. These results show the distinct role of STIM1 and STIM2 in SOCE and cellular function of cervical cancer cells.     

TRPC1, Orai1, and STIM1 in SOCE: Friends in tight spaces

Store-operated calcium entry (SOCE) is a ubiquitous Ca²⁺ entry pathway that is activated in response to depletion of ER-Ca²⁺ stores and critically controls the regulation of physiological functions in miscellaneous cell types. The transient receptor potential canonical 1 (TRPC1) is the first member of the TRPC channel subfamily to be identified as a molecular component of SOCE. While TRPC1 has been shown to contribute to SOCE and regulate various functions in many cells, none of the reported TRPC1-mediated currents resembled I_CRAC, the highly Ca²⁺-selective store-dependent current first identified in lymphocytes and mast cells. Almost a decade after the cloning of TRPC1 two proteins were identified as the primary components of the CRAC channel. The first, STIM1, is an ER-Ca²⁺ sensor protein involved in activating SOCE. The second, Orai1 is the pore-forming component of the CRAC channel. Co-expression of STIM1 and Orai1 generated robust I_CRAC. Importantly, STIM1 was shown to also activate TRPC1 via its C-terminal polybasic domain, which is distinct from its Orai1-activating domain, SOAR. In addition, TRPC1 function critically depends on Orai1-mediated Ca²⁺ entry which triggers recruitment of TRPC1 into the plasma membrane where it is then activated by STIM1. TRPC1 and Orai1 form discrete STIM1-gated channels that generate distinct Ca²⁺ signals and regulate specific cellular functions. Surface expression of TRPC1 can be modulated by trafficking of the channel to and from the plasma membrane, resulting in changes to the phenotype of TRPC1-mediated current and [Ca²⁺]_i signals. Thus, TRPC1 is activated downstream of Orai1 and modifies the initial [Ca²⁺]_i signal generated by Orai1 following store depletion. This review will summarize the important findings that underlie the current concepts for activation and regulation of TRPC1, as well as its impact on cell function.     

Regulation of endogenous and heterologous Ca release-activated Ca currents by pH

Deviations from physiological pH (∼pH 7.2) as well as altered Ca²⁺ signaling play important roles in immune disease and cancer. One of the most ubiquitous pathways for cellular Ca²⁺ influx is the store-operated Ca²⁺ entry (SOCE) or Ca²⁺ release-activated Ca²⁺ current (I_CRAC), which is activated upon depletion of intracellular Ca²⁺ stores. We here show that extracellular and intracellular changes in pH regulate both endogenous I_CRAC in Jurkat T lymphocytes and RBL2H3 cells, and heterologous I_CRAC in HEK293 cells expressing the molecular components STIM1/2 and Orai1/2/3 (CRACM1/2/3). We find that external acidification suppresses, and alkalization facilitates IP₃-induced I_CRAC. In the absence of IP₃, external alkalization did not elicit endogenous I_CRAC but was able to activate heterologous I_CRAC in HEK293 cells expressing Orai1/2/3 and STIM1 or STIM2. Similarly, internal acidification reduced IP₃-induced activation of endogenous and heterologous I_CRAC, while alkalization accelerated its activation kinetics without affecting overall current amplitudes. Mutation of two aspartate residues to uncharged alanine amino acids (D110/112A) in the first extracellular loop of Orai1 significantly attenuated both the inhibition of I_CRAC by external acidic pH as well as its facilitation by alkaline conditions. We conclude that intra- and extracellular pH differentially regulates I_CRAC. While intracellular pH might affect aggregation and/or binding of STIM to Orai, external pH seems to modulate I_CRAC through its channel pore, which in Orai1 is partially mediated by residues D110 and D112.     

A Novel Native Store-operated Calcium Channel Encoded by Orai3: SELECTIVE REQUIREMENT OF Orai3 VERSUS Orai1 IN ESTROGEN RECEPTOR-POSITIVE VERSUS ESTROGEN RECEPTOR-NEGATIVE BREAST CANCER CELLS*

Store-operated calcium (Ca²⁺) entry (SOCE) mediated by STIM/Orai proteins is a ubiquitous pathway that controls many important cell functions including proliferation and migration. STIM proteins are Ca²⁺ sensors in the endoplasmic reticulum and Orai proteins are channels expressed at the plasma membrane. The fall in endoplasmic reticulum Ca²⁺ causes translocation of STIM1 to subplasmalemmal puncta where they activate Orai1 channels that mediate the highly Ca²⁺-selective Ca²⁺ release-activated Ca²⁺ current (I_CRAC). Whereas Orai1 has been clearly shown to encode SOCE channels in many cell types, the role of Orai2 and Orai3 in native SOCE pathways remains elusive. Here we analyzed SOCE in ten breast cell lines picked in an unbiased way. We used a combination of Ca²⁺ imaging, pharmacology, patch clamp electrophysiology, and molecular knockdown to show that native SOCE and I_CRAC in estrogen receptor-positive (ER⁺) breast cancer cell lines are mediated by STIM1/2 and Orai3 while estrogen receptor-negative (ER⁻) breast cancer cells use the canonical STIM1/Orai1 pathway. The ER⁺ breast cancer cells represent the first example where the native SOCE pathway and I_CRAC are mediated by Orai3. Future studies implicating Orai3 in ER⁺ breast cancer progression might establish Orai3 as a selective target in therapy of ER⁺ breast tumors.     

Regulation of CRAC channels by protein interactions and post-translational modification

Store-operated Ca²⁺ entry (SOCE) is a widespread mechanism to elevate the intracellular Ca²⁺ concentrations and stimulate downstream signaling pathways affecting proliferation, secretion, differentiation and death in different cell types. In immune cells, immune receptor stimulation induces intracellular Ca²⁺ store depletion that subsequently activates Ca²⁺-release-activated-Ca²⁺ (CRAC) channels, a prototype of store-operated Ca²⁺ (SOC) channels. CRAC channel opening leads to activation of diverse downstream signaling pathways affecting proliferation, differentiation, cytokine production and cell death. Recent identification of STIM1 as the endoplasmic reticulum Ca²⁺ sensor and Orai1 as the pore subunit of CRAC channels has provided the much-needed molecular tools to dissect the mechanism of activation and regulation of CRAC channels. In this review, we discuss the recent advances in understanding the associating partners and posttranslational modifications of Orai1 and STIM1 proteins that regulate diverse aspects of CRAC channel function.     

Ca signaling and regulation of fluid secretion in salivary gland acinar cells

Neurotransmitter stimulation of plasma membrane receptors stimulates salivary gland fluid secretion via a complex process that is determined by coordinated temporal and spatial regulation of several Ca²⁺ signaling processes as well as ion flux systems. Studies over the past four decades have demonstrated that Ca²⁺ is a critical factor in the control of salivary gland function. Importantly, critical components of this process have now been identified, including plasma membrane receptors, calcium channels, and regulatory proteins. The key event in activation of fluid secretion is an increase in intracellular [Ca²⁺] ([Ca²⁺]_i) triggered by IP₃-induced release of Ca²⁺ from ER via the IP₃R. This increase regulates the ion fluxes required to drive vectorial fluid secretion. IP₃Rs determine the site of initiation and the pattern of [Ca²⁺]_i signal in the cell. However, Ca²⁺ entry into the cell is required to sustain the elevation of [Ca²⁺]_i and fluid secretion. This Ca²⁺ influx pathway, store-operated calcium influx pathway (SOCE), has been studied in great detail and the regulatory mechanisms as well as key molecular components have now been identified. Orai1, TRPC1, and STIM1 are critical components of SOCE and among these, Ca²⁺ entry via TRPC1 is a major determinant of fluid secretion. The receptor-evoked Ca²⁺ signal in salivary gland acinar cells is unique in that it starts at the apical pole and then rapidly increases across the cell. The basis for the polarized Ca²⁺ signal can be ascribed to the polarized arrangement of the Ca²⁺ channels, transporters, and signaling proteins. Distinct localization of these proteins in the cell suggests compartmentalization of Ca²⁺ signals during regulation of fluid secretion. This chapter will discuss new concepts and findings regarding the polarization and control of Ca²⁺ signals in the regulation of fluid secretion.     

Evidence that Orai1 does not contribute to store-operated TRPC1 channels in vascular smooth muscle cells

Ca²⁺-permeable store-operated channels (SOCs) mediate Ca²⁺ entry pathways which are involved in many cellular functions such as contraction, growth, and proliferation. Prototypical SOCs are formed of Orai1 proteins and are activated by the endo/sarcoplasmic reticulum Ca²⁺ sensor stromal interaction molecule 1 (STIM1). There is considerable debate about whether canonical transient receptor potential 1 (TRPC1) proteins also form store-operated channels (SOCs), and if they do, is Orai1 involved. We recently showed that stimulation of TRPC1-based SOCs involves store depletion inducing STIM1-evoked Gαq/PLCβ1 activity in contractile vascular smooth muscle cells (VSMCs). Therefore the present work investigates the role of Orai1 in activation of TRPC1-based SOCs in freshly isolated mesenteric artery VSMCs from wild-type (WT) and Orai1^?/? mice. Store-operated whole-cell and single channel currents recorded from WT and Orai1^?/? VSMCs had similar properties, with relatively linear current-voltage relationships, reversal potentials of about +20mV, unitary conductances of about 2pS, and inhibition by anti-TRPC1 and anti-STIM1 antibodies. In Orai1^?/? VSMCs, store depletion induced PLCβ1 activity measured with the fluorescent phosphatidylinositol 4,5-bisphosphate/inositol 1,4,5-trisphosphate biosensor GFP-PLCδ1-PH, which was prevented by knockdown of STIM1. In addition, in Orai1^?/? VSMCs, store depletion induced translocation of STIM1 from within the cell to the plasma membrane where it formed STIM1-TRPC1 interactions at discrete puncta-like sites. These findings indicate that activation of TRPC1-based SOCs through a STIM1-activated PLCβ1 pathway are likely to occur independently of Orai1 proteins, providing evidence that TRPC1 channels form genuine SOCs in VSMCs with a contractile phenotype.     

Store-independent Orai1-mediated Ca2+ entry and cancer

Ca²⁺ channels play an important role in the development of different types of cancer, and considerable progress has been made to understand the pathophysiological mechanisms underlying the role of Ca²⁺ influx in the development of different cancer hallmarks. Orai1 is among the most ubiquitous and multifunctional Ca²⁺ channels. Orai1 mediates the highly Ca²⁺-selective Ca²⁺ release-activated current (I_CRAC) and participates in the less Ca²⁺-selective store-operated current (I_SOC), along with STIM1 or STIM1 and TRPC1, respectively. Furthermore, Orai1 contributes to a variety of store-independent Ca²⁺ influx mechanisms, including the arachidonate-regulated Ca²⁺ current, together with Orai3 and the plasma membrane resident pool of STIM1, as well as the constitutive Ca²⁺ influx processes activated by the secretory pathway Ca²⁺-ATPase-2 (SPCA2) or supported by physical and functional interaction with the small conductance Ca²⁺-activated K⁺ channel 3 (SK3) or the voltage-dependent K_v10.1 channel. This review summarizes the current knowledge concerning the store-independent mechanisms of Ca²⁺ influx activation through Orai1 channels and their role in the development of different cancer features.     

Orai1-NFAT signalling pathway triggered by T cell receptor stimulation

T cell receptor (TCR) stimulation plays a crucial role in development, homeostasis, proliferation, cell death, cytokine production, and differentiation of T cells. Thus, in depth understanding of TCR signalling is crucial for development of therapy targeting inflammatory diseases, improvement of vaccination efficiency, and cancer therapy utilizing T cell-based strategies. TCR activation turns on various signalling pathways, one of the important one being the Ca²⁺-calcineurin-nuclear factor of activated T cells (NFAT) signalling pathway. Stimulation of TCRs triggers depletion of intracellular Ca²⁺ store and in turn, initiates store-operated Ca²⁺ entry (SOCE), one of the major mechanisms to raise the intracellular Ca²⁺ concentrations in T cells. Ca²⁺-release-activated-Ca²⁺ (CRAC) channels are a prototype of store-operated Ca²⁺ (SOC) channels in immune cells that are very well characterized. Recent identification of STIM1 as the endoplasmic reticulum (ER) Ca²⁺ sensor and Orai1 as the pore subunit has dramatically advanced the understanding of CRAC channels and provides a molecular tool to investigate the physiological outcomes of Ca²⁺ signalling during immune responses. In this review, we focus on our current understanding of CRAC channel activation, regulation, and downstream calcineurin-NFAT signaling pathway.     

A novel STIM1-Orai1 gating interface essential for CRAC channel activation

Calcium signalling through store-operated calcium (SOC) entry is of crucial importance for T-cell activation and the adaptive immune response. This entry occurs via the prototypic Ca²⁺ release-activated Ca²⁺ (CRAC) channel. STIM1, a key molecular component of this process, is located in the membrane of the endoplasmic reticulum (ER) and is initially activated upon Ca²⁺ store depletion. This activation signal is transmitted to the plasma membrane via a direct physical interaction that takes place between STIM1 and the highly Ca²⁺-selective ion channel Orai1. The activation of STIM1 induces an extended cytosolic conformation. This, in turn, exposes the CAD/SOAR domain and leads to the formation of STIM1 oligomers. In this study, we focused on a small helical segment (STIM1 α3, aa 400–403), which is located within the CAD/SOAR domain. We determined this segment’s specific functional role in terms of STIM1 activation and Orai1 gating. The STIM1 α3 domain appears not essential for STIM1 to interact with Orai1. Instead, it represents a key domain that conveys STIM1 interaction into Orai1 channel gating. The results of cysteine crosslinking experiments revealed the close proximity of STIM1 α3 to a region within Orai1, which was located at the cytosolic extension of transmembrane helix 3, forming a STIM1-Orai1 gating interface (SOGI). We suggest that the interplay between STIM1 α3 and Orai1 TM3 allows STIM1 coupling to be transmitted into physiological CRAC channel activation.     

Calcium signaling in lacrimal glands

Lacrimal glands provide the important function of lubricating and protecting the ocular surface. Failure of proper lacrimal gland function results in a number of debilitating dry eye diseases. Lacrimal glands secrete lipids, mucins, proteins, salts and water and these secretions are at least partially regulated by neurotransmitter-mediated cell signaling. The predominant signaling mechanism for lacrimal secretion involves activation of phospholipase C, generation of the Ca²⁺-mobilizing messenger, IP₃, and release of Ca²⁺ stored in the endoplasmic reticulum. The loss of Ca²⁺ from the endoplasmic reticulum then triggers a process known as store-operated Ca²⁺ entry, involving a Ca²⁺ sensor in the endoplasmic reticulum, STIM1, which activates plasma membrane store-operated channels comprised of Orai subunits. Recent studies with deletions of the channel subunit, Orai1, confirm the important role of SOCE in both fluid and protein secretion in lacrimal glands, both in vivo and in vitro.