The store-operated Ca2+ channel Orai1α is required for agonist-evoked NF-κB activation by a mechanism dependent on PKCß2.

Store-operated Ca2+ entry is a ubiquitous mechanism for Ca2+ influx in mammalian cells that regulates a variety of physiological processes. The identification of two forms of Orai1, the predominant store-operated channel, Orai1α and Orai1ß, raises the question whether they differentially regulate cell function. Orai1α is the full-length Orai1, containing 301 amino acids, whereas Orai1ß lacks the N-terminal 63 amino acids. Here, using a combination of biochemistry and imaging combined with the use of human embryonic kidney 293 KO cells, missing the native Orai1, transfected with plasmids encoding for either Orai1α or Orai1ß, we show that Orai1α plays a relevant role in agonist-induced NF-κB transcriptional activity. In contrast, functional Orai1ß is not required for the activation of these transcription factors. The role of Orai1α in the activation of NF-κB is entirely dependent on Ca2+ influx and involves PKCß activation. Our results indicate that Orai1α interacts with PKCß2 by a mechanism involving the Orai1α exclusive AKAP79 association region, which strongly suggests a role for AKAP79 in this process. These findings provide evidence of the role of Orai1α in agonist-induced NF-κB transcriptional activity and reveal functional differences between Orai1 variants.

Combining Sound and Deep Neural Networks for the Measurement of Jump Height in Sports Science.

Jump height tests are employed to measure lower-limb muscle power of athletic and non-athletic populations. The most popular instruments for this purpose are jump mats and, in recent years, smartphone apps, which compute jump height through the manual annotation of video recordings and recently automatically using the sound produced during the jump to extract the flight time. In a previous work, the afore-mentioned sound systems were presented by the authors in which the take-off and landing events from the audio recordings of jump executions were obtained using classical signal processing. In this work, a more precise, noise-immune, and robust system, capable of working in the most unfavorable environments, is presented. The system uses a deep neural network trained specifically for this purpose. More than 300 jumps were recorded to train and validate the network performance. The ground truth was a jump mat, providing a slightly better accuracy in quiet and medium quiet environments but excellent accuracy in noisy and complicated ones. The developed audio-based system is a trustworthy instrument for measuring jump height accurately in any kind of environment, providing a perfect measurement tool that can be accessed through a mobile phone in the form of an app.

Functional differences in agonist-induced plasma membrane expression of Orai1α and Orai1ß.

Orai1 is the pore-forming subunit of the store-operated Ca2+ release-activated Ca2+ (CRAC) channels involved in a variety of cellular functions. Two Orai1 variants have been identified, the long form, Orai1α, containing 301 amino acids, and the short form, Orai1ß, which arises from alternative translation initiation from methionines 64 or 71, in Orai1α. Orai1 is mostly expressed in the plasma membrane, but a subset of Orai1 is located in intracellular compartments. Here we show that Ca2+ store depletion leads to trafficking and insertion of compartmentalized Orai1α in the plasma membrane via a mechanism that is independent on changes in cytosolic free-Ca2+ concentration, as demonstrated by cell loading with the fast intracellular Ca2+ chelator dimethyl BAPTA in the absence of extracellular Ca2+ . Interestingly, thapsigargin (TG) was found to be unable to induce translocation of Orai1ß to the plasma membrane when expressed individually; by contrast, when Orai1ß is co-expressed with Orai1α, cell treatment with TG induced rapid trafficking and insertion of compartmentalized Orai1ß in the plasma membrane. Translocation of Orai1 forms to the plasma membrane was found to require the integrity of the actin cytoskeleton. Finally, expression of a dominant negative mutant of the small GTPase ARF6, and ARF6-T27N, abolished the translocation of compartmentalized Orai1 variants to the plasma membrane upon store depletion. These findings provide new insights into the mechanism that regulate the plasma membrane abundance of Orai1 variants after Ca2+ store depletion.

Role of Orai-family channels in the activation and regulation of transcriptional activity.

Store operated Ca2+ entry (SOCE) is a cornerstone for the maintenance of intracellular Ca2+ homeostasis and the regulation of a variety of cellular functions. SOCE is mediated by STIM and Orai proteins following the activation of inositol 1,4,5-trisphosphate receptors. Then, a reduction of the endoplasmic reticulum intraluminal Ca2+ concentration is sensed by STIM proteins, which undergo a conformational change and activate plasma membrane Ca2+ channels comprised by Orai proteins. STIM1/Orai-mediated Ca2+ signals are finely regulated and modulate the activity of different transcription factors, including certain isoforms of the nuclear factor of activated T-cells, the cAMP-response element binding protein, the nuclear factor κ-light chain-enhancer of activated B cells, c-fos, and c-myc. These transcription factors associate SOCE with a plethora of signaling events and cellular functions. Here we provide an overview of the current knowledge about the role of Orai channels in the regulation of transcription factors through Ca2+ -dependent signaling pathways.

Cross-Talk Between the Adenylyl Cyclase/cAMP Pathway and Ca2+ Homeostasis.

Cyclic AMP and Ca2+ are the first second or intracellular messengers identified, unveiling the cellular mechanisms activated by a plethora of extracellular signals, including hormones. Cyclic AMP generation is catalyzed by adenylyl cyclases (ACs), which convert ATP into cAMP and pyrophosphate. By the way, Ca2+, as energy, can neither be created nor be destroyed; Ca2+ can only be transported, from one compartment to another, or chelated by a variety of Ca2+-binding molecules. The fine regulation of cytosolic concentrations of cAMP and free Ca2+ is crucial in cell function and there is an intimate cross-talk between both messengers to fine-tune the cellular responses. Cancer is a multifactorial disease resulting from a combination of genetic and environmental factors. Frequent cases of cAMP and/or Ca2+ homeostasis remodeling have been described in cancer cells. In those tumoral cells, cAMP and Ca2+ signaling plays a crucial role in the development of hallmarks of cancer, including enhanced proliferation and migration, invasion, apoptosis resistance, or angiogenesis. This review summarizes the cross-talk between the ACs/cAMP and Ca2+ intracellular pathways with special attention to the functional and reciprocal regulation between Orai1 and AC8 in normal and cancer cells.

Orai1α, but not Orai1ß, co-localizes with TRPC1 and is required for its plasma membrane location and activation in HeLa cells.

The identification of two variants of the canonical pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel Orai1, Orai1α and Orai1ß, in mammalian cells arises the question whether they exhibit different functional characteristics. Orai1α and Orai1ß differ in the N-terminal 63 amino acids, exclusive of Orai1α, and show different sensitivities to Ca2+-dependent inactivation, as well as distinct ability to form arachidonate-regulated channels. We have evaluated the role of both Orai1 variants in the activation of TRPC1 in HeLa cells. We found that Orai1α and Orai1ß are required for the maintenance of regenerative Ca2+ oscillations, while TRPC1 plays a role in agonist-induced Ca2+ influx but is not essential for Ca2+ oscillations. Using APEX2 proximity labeling, co-immunoprecipitation and the fluorescence of G-GECO1.2 fused to Orai1α our results indicate that agonist stimulation and Ca2+ store depletion enhance Orai1α-TRPC1 interaction. Orai1α is essential for TRPC1 plasma membrane location and activation. Thus, TRPC1 function in HeLa cells depends on Ca2+ influx through Orai1α exclusively.

Similarities and Differences between the Orai1 Variants: Orai1α and Orai1ß.

Orai1, the first identified member of the Orai protein family, is ubiquitously expressed in the animal kingdom. Orai1 was initially characterized as the channel responsible for the store-operated calcium entry (SOCE), a major mechanism that allows cytosolic calcium concentration increments upon receptor-mediated IP3 generation, which results in intracellular Ca2+ store depletion. Furthermore, current evidence supports that abnormal Orai1 expression or function underlies several disorders. Orai1 is, together with STIM1, the key element of SOCE, conducting the Ca2+ release-activated Ca2+ (CRAC) current and, in association with TRPC1, the store-operated Ca2+ (SOC) current. Additionally, Orai1 is involved in non-capacitative pathways, as the arachidonate-regulated or LTC4-regulated Ca2+ channel (ARC/LRC), store-independent Ca2+ influx activated by the secretory pathway Ca2+-ATPase (SPCA2) and the small conductance Ca2+-activated K+ channel 3 (SK3). Furthermore, Orai1 possesses two variants, Orai1α and Orai1ß, the latter lacking 63 amino acids in the N-terminus as compared to the full-length Orai1α form, which confers distinct features to each variant. Here, we review the current knowledge about the differences between Orai1α and Orai1ß, the implications of the Ca2+ signals triggered by each variant, and their downstream modulatory effect within the cell.

Functional role of TRPC6 and STIM2 in cytosolic and endoplasmic reticulum Ca2+ content in resting estrogen receptor-positive breast cancer cells.

TRPC6 forms non-selective cation channels activated by a variety of stimuli that are involved in a wide number of cellular functions. In estrogen receptor-positive (ER+) breast cancer cells, the store-operated Ca2+ entry has been reported to be dependent on STIM1, STIM2 and Orai3, with TRPC6 playing a key role in the activation of store-operated Ca2+ entry as well as in proliferation, migration and viability of breast cancer cells. We have used a combination of biotinylation, Ca2+ imaging as well as protein knockdown and overexpression of a dominant-negative TRPC6 mutant (TRPC6dn) to show that TRPC6 and STIM2 are required for the maintenance of cytosolic and endoplasmic reticulum Ca2+ content under resting conditions in ER+ breast cancer MCF7 cells. These cells exhibit a greater plasma membrane expression of TRPC6 under resting conditions than non-tumoral breast epithelial cells. Attenuation of STIM2, TRPC6 and Orai3, alone or in combination, results in impairment of resting cytosolic and endoplasmic reticulum Ca2+ homeostasis. Similar results were observed when cells were transfected with expression plasmid for TRPC6dn. TRPC6 co-immunoprecipitates with STIM2 in resting MCF7 cells, a process that is impaired by rises in cytosolic Ca2+ concentration. Impairment of TRPC6 function leads to abnormal Ca2+ homeostasis and endoplasmic reticulum stress, thus, suggesting that TRPC6 might be a potential target for the development of anti-tumoral therapies.

Video-Based System for Automatic Measurement of Barbell Velocity in Back Squat.

Velocity-based training is a contemporary method used by sports coaches to prescribe the optimal loading based on the velocity of movement of a load lifted. The most employed and accurate instruments to monitor velocity are linear position transducers. Alternatively, smartphone apps compute mean velocity after each execution by manual on-screen digitizing, introducing human error. In this paper, a video-based instrument delivering unattended, real-time measures of barbell velocity with a smartphone high-speed camera has been developed. A custom image-processing algorithm allows for the detection of reference points of a multipower machine to autocalibrate and automatically track barbell markers to give real-time kinematic-derived parameters. Validity and reliability were studied by comparing the simultaneous measurement of 160 repetitions of back squat lifts executed by 20 athletes with the proposed instrument and a validated linear position transducer, used as a criterion. The video system produced practically identical range, velocity, force, and power outcomes to the criterion with low and proportional systematic bias and random errors. Our results suggest that the developed video system is a valid, reliable, and trustworthy instrument for measuring velocity and derived variables accurately with practical implications for use by coaches and practitioners.

Role of Orai3 in the Pathophysiology of Cancer.

The mammalian exclusive Orai3 channel participates in the generation and/or modulation of two independent Ca2+ currents, the store-operated current, Icrac, involving functional interactions between the stromal interaction molecules (STIM), STIM1/STIM2, and Orai1/Orai2/Orai3, as well as the store-independent arachidonic acid (AA) (or leukotriene C4)-regulated current Iarc, which involves Orai1, Orai3 and STIM1. Overexpression of functional Orai3 has been described in different neoplastic cells and cancer tissue samples as compared to non-tumor cells or normal adjacent tissue. In these cells, Orai3 exhibits a cell-specific relevance in Ca2+ influx. In estrogen receptor-positive breast cancer cells and non-small cell lung cancer (NSCLC) cells store-operated Ca2+ entry (SOCE) is strongly dependent on Orai3 expression while in colorectal cancer and pancreatic adenocarcinoma cells Orai3 predominantly modulates SOCE. On the other hand, in prostate cancer cells Orai3 expression has been associated with the formation of Orai1/Orai3 heteromeric channels regulated by AA and reduction in SOCE, thus leading to enhanced proliferation. Orai3 overexpression is associated with supporting several cancer hallmarks, including cell cycle progression, proliferation, migration, and apoptosis resistance. This review summarizes the current knowledge concerning the functional role of Orai3 in the pathogenesis of cancer.

A Fibrin Coating Method of Polypropylene Meshes Enables the Adhesion of Menstrual Blood-Derived Mesenchymal Stromal Cells: A New Delivery Strategy for Stem Cell-Based Therapies.

Polypropylene (PP) mesh is well-known as a gold standard of all prosthetic materials of choice for the reinforcement of soft tissues in case of hernia, organ prolapse, and urinary incontinence. The adverse effects that follow surgical mesh implantation remain an unmet medical challenge. Herein, it is outlined a new approach to allow viability and adhesion of human menstrual blood-derived mesenchymal stromal cells (MenSCs) on PP surgical meshes. A multilayered fibrin coating, based on fibrinogen and thrombin from a commercial fibrin sealant, was optimized to guarantee a homogeneous and stratified film on PP mesh. MenSCs were seeded on the optimized fibrin-coated meshes and their adhesion, viability, phenotype, gene expression, and immunomodulatory capacity were fully evaluated. This coating guaranteed MenSC viability, adhesion and did not trigger any change in their stemness and inflammatory profile. Additionally, MenSCs seeded on fibrin-coated meshes significantly decreased CD4+ and CD8+ T cell proliferation, compared to in vitro stimulated lymphocytes (p < 0.0001). Hence, the proposed fibrin coating for PP surgical meshes may allow the local administration of stromal cells and the reduction of the exacerbated inflammatory response following mesh implantation surgery. Reproducible and easy to adapt to other cell types, this method undoubtedly requires a multidisciplinary and translational approach to be improved for future clinical uses.

Molecular Basis and Regulation of Store-Operated Calcium Entry.

Store-operated Ca2+ entry (SOCE) is a ubiquitous mechanism for Ca2+ influx in mammalian cells with important physiological implications. Since the discovery of SOCE more than three decades ago, the mechanism that communicates the information about the amount of Ca2+ accumulated in the intracellular Ca2+ stores to the plasma membrane channels and the nature of these channels have been matters of intense investigation and debate. The stromal interaction molecule-1 (STIM1) has been identified as the Ca2+ sensor of the intracellular Ca2+ compartments that activates the store-operated channels. STIM1 regulates two types of store-dependent channels: the Ca2+ release-activated Ca2+ (CRAC) channels, formed by Orai1 subunits, that conduct the highly Ca2+ selective current I CRAC and the cation permeable store-operated Ca2+ (SOC) channels, which consist of Orai1 and TRPC1 proteins and conduct the non-selective current I SOC. While the crystal structure of Drosophila CRAC channel has already been solved, the architecture of the SOC channels still remains unclear. The dynamic interaction of STIM1 with the store-operated channels is modulated by a number of proteins that either support the formation of the functional STIM1-channel complex or protect the cell against Ca2+ overload.

Arachidonic Acid Attenuates Cell Proliferation, Migration and Viability by a Mechanism Independent on Calcium Entry.

Arachidonic acid (AA) is a phospholipase A2 metabolite that has been reported to mediate a plethora of cellular mechanisms involved in healthy and pathological states such as platelet aggregation, lymphocyte activation, and tissue inflammation. AA has been described to activate Ca2+ entry through the arachidonate-regulated Ca2+-selective channels (ARC channels). Here, the analysis of the changes in the intracellular Ca2+ homeostasis revealed that, despite MDA-MB-231 cells expressing the ARC channel components Orai1, Orai3, and STIM1, AA does not evoke Ca2+ entry in these cells. We observed that AA evokes Ca2+ entry in MDA-MB-231 cells transiently expressing ARC channels. Nevertheless, MDA-MB-231 cell treatment with AA reduces cell proliferation and migration while inducing cell death through apoptosis. The latter mostly likely occurs via mitochondria membrane depolarization and the activation of caspases-3, -8, and -9. Altogether, our results indicate that AA exerts anti-tumoral effects on MDA-MB-231 cells, without having any effect on non-tumoral breast epithelial cells, by a mechanism that is independent on the activation of Ca2+ influx via ARC channels.

Fine-tuning of store-operated calcium entry by fast and slow Ca2+-dependent inactivation: Involvement of SARAF.

Store-operated Ca2+ entry (SOCE) is a functionally relevant mechanism for Ca2+ influx present in electrically excitable and non-excitable cells. Regulation of Ca2+ entry through store-operated channels is essential to maintain an appropriate intracellular Ca2+ homeostasis and prevent cell damage. Calcium-release activated channels exhibit Ca2+-dependent inactivation mediated by two temporally separated mechanisms: fast Ca2+-dependent inactivation takes effect in the order of milliseconds and involves the interaction of Ca2+ with residues in the channel pore while slow Ca2+-dependent inactivation (SCDI) develops over tens of seconds, requires a global rise in [Ca2+]cyt and is a mechanism regulated by mitochondria. Recent studies have provided evidence that the protein SARAF (SOCE-associated regulatory factor) is involved in the mechanism underlying SCDI of Orai1. SARAF is an endoplasmic reticulum (ER) membrane protein that associates with STIM1 and translocate to plasma membrane-ER junctions in a STIM1-dependent manner upon store depletion to modulate SOCE. SCDI mediated by SARAF depends on the location of the STIM1-Orai1 complex within a PI(4,5)P2-rich microdomain. SARAF also interacts with Orai1 and TRPC1 in cells endogenously expressing STIM1 and cells with a low STIM1 expression and modulates channel function. This review focuses on the modulation by SARAF of SOCE and other forms of Ca2+ influx mediated by Orai1 and TRPC1 in order to provide spatio-temporally regulated Ca2+ signals.

Filamin A Modulates Store-Operated Ca2+ Entry by Regulating STIM1 (Stromal Interaction Molecule 1)-Orai1 Association in Human Platelets.

OBJECTIVE: Here, we provide evidence for the role of FLNA (filamin A) in the modulation of store-operated calcium entry (SOCE). APPROACH AND RESULTS: SOCE is a major mechanism for calcium influx controlled by the intracellular Ca2+ stores. On store depletion, the endoplasmic reticulum calcium sensor STIM1 (stromal interaction molecule 1) redistributes into puncta at endoplasmic reticulum/plasma membrane junctions, a process supported by the cytoskeleton, where it interacts with the calcium channels; however, the mechanism for fine-tuning SOCE is not completely understood. Our results demonstrate that STIM1 interacts with FLNA on calcium store depletion in human platelets. The interaction is dependent on the phosphorylation of FLNA at Ser2152 by the cAMP-dependent protein kinase. Impairment of FLNA phosphorylation and knockdown of FLNA expression using siRNA increased SOCE in platelets. Similarly, SOCE was significantly greater in FLNA-deficient melanoma M2 cells than in the FLNA-expressing M2 subclone A7. Expression of FLNA in M2 cells attenuated SOCE, an effect prevented when the cells were transfected with the nonphosphorylatable FLNA S2152A mutant. Transfection of M2 cells with the STIM1(K684,685E) mutant reduced the STIM1-FLNA interaction. In platelets, attenuation of FLNA expression using siRNA resulted in enhanced association of STIM1 with the cytoskeleton, greater STIM1-Orai1 interaction, and SOCE. Introduction of an anti-FLNA (2597-2647) antibody attenuated the STIM1-FLNA interaction and enhanced thrombin-induced platelet aggregation. CONCLUSIONS: Our results indicate that FLNA modulates SOCE and then the correct platelet function, by fine-tuning the distribution of STIM1 in the cytoskeleton and the interaction with Orai1 channels.

Audio-Based System for Automatic Measurement of Jump Height in Sports Science.

Jump height tests are employed to measure the lower-limb muscle power of athletic and non-athletic populations. The most popular instruments for this purpose are jump mats and, more recently, smartphone apps, which compute jump height through manual annotation of video recordings to extract flight time. This study developed a non-invasive instrument that automatically extracts take-off and landing events from audio recordings of jump executions. An audio signal processing algorithm, specifically developed for this purpose, accurately detects and discriminates the landing and take-off events in real time and computes jump height accordingly. Its temporal resolution theoretically outperforms that of flight-time-based mats (typically 1000 Hz) and high-speed video rates from smartphones (typically 240 fps). A validation study was carried out by comparing 215 jump heights from 43 active athletes, measured simultaneously with the audio-based system and with of a validated, commercial jump mat. The audio-based system produced nearly identical jump heights than the criterion with low and proportional systematic bias and random errors. The developed audio-based system is a trustworthy instrument for accurately measuring jump height that can be readily automated as an app to facilitate its use both in laboratories and in the field.

EFHB is a Novel Cytosolic Ca2+ Sensor That Modulates STIM1-SARAF Interaction.

BACKGROUND/AIMS: STIM1 and Orai1 are the key components of store-operated Ca2+ entry (SOCE). Among the proteins involved in the regulation of SOCE, SARAF prevents spontaneous activation of SOCE and modulates STIM1 function. METHODS: Cytosolic Ca2+ mobilization was estimated in fura-2-loaded cells using an epifluorescence inverted microscope. STIM1 interaction with Orai1, EFHB (EF-hand domain family member B, also known as CFAP21) and SARAF was detected by immunoprecipitation followed by Western blotting using specific antibodies. The involvement of EFHB in the translocation of NFAT to the nucleus was detected by confocal microscopy. RESULTS: Here, we report the identification of EFHB as a new SOCE regulator. EFHB interacts with STIM1 upon store depletion and dissociates through a Ca2+-dependent mechanism. RNAi-mediated silencing as well as overexpression studies revealed that EFHB plays a relevant role in the interaction of STIM1 and Orai1 upon store depletion, the activation of SOCE and NFAT translocation from the cytosol to the nucleus. Silencing EFHB expression abolished the dissociation of SARAF from STIM1, which indicates that EFHB might play an important role in the dynamic interaction between both proteins, which is relevant for the activation of Orai1 channels upon Ca2+ store depletion and their subsequent modulation via slow Ca2+-dependent inactivation. CONCLUSION: Our results indicate that EFHB is a new SOCE regulator that modulates STIM1-SARAF interaction.

Perception of nonlinear distortion on emulation of frequency responses of headphones.

The equalization of headphones can force transducers to work in a non-linear condition, producing non-linear distortion. Depending on the headphone model and the reproduction level, that distortion can be audible. In this study, headphones of diverse quality and price were compelled to emulate the same target frequency response and the non-linear distortion was measured. A Diagonal Volterra model was used to simulate the different headphones with and without distortion. A perceptual test was carried out to determine the level of reproduction above which non-linear distortion is perceived for each headphone model. High correlation has been found between the level of detected distortion and retail prices of headphones.

Store-Operated Ca2+ Entry in Breast Cancer Cells: Remodeling and Functional Role.

Breast cancer is the most common type of cancer in women. It is a heterogeneous disease that ranges from the less undifferentiated luminal A to the more aggressive basal or triple negative breast cancer molecular subtype. Ca2+ influx from the extracellular medium, but more specifically store-operated Ca2+ entry (SOCE), has been reported to play an important role in tumorigenesis and the maintenance of a variety of cancer hallmarks, including cell migration, proliferation, invasion or epithelial to mesenchymal transition. Breast cancer cells remodel the expression and functional role of the molecular components of SOCE. This review focuses on the functional role and remodeling of SOCE in breast cancer cells. The current studies suggest the need to deepen our understanding of SOCE in the biology of the different breast cancer subtypes in order to develop new and specific therapeutic strategies.

Store-operated Ca2+ Entry-associated Regulatory factor (SARAF) Plays an Important Role in the Regulation of Arachidonate-regulated Ca2+ (ARC) Channels.

The store-operated Ca(2+)entry-associated regulatory factor (SARAF) has recently been identified as a STIM1 regulatory protein that facilitates slow Ca(2+)-dependent inactivation of store-operated Ca(2+)entry (SOCE). Both the store-operated channels and the store-independent arachidonate-regulated Ca(2+)(ARC) channels are regulated by STIM1. In the present study, we show that, in addition to its location in the endoplasmic reticulum, SARAF is constitutively expressed in the plasma membrane, where it can interact with plasma membrane (PM)-resident ARC forming subunits in the neuroblastoma cell line SH-SY5Y. Using siRNA-based and overexpression approaches we report that SARAF negatively regulates store-independent Ca(2+)entry via the ARC channels. Arachidonic acid (AA) increases the association of PM-resident SARAF with Orai1. Finally, our results indicate that SARAF modulates the ability of AA to promote cell survival in neuroblastoma cells. In addition to revealing new insight into the biology of ARC channels in neuroblastoma cells, these findings provide evidence for an unprecedented location of SARAF in the plasma membrane.