Barbara A. Niemeyer

Competitive regulation of CaT-like-mediated Ca2+ entry by protein kinase C and calmodulin

A finely tuned Ca2+ signaling system is essential for cells to transduce extracellular stimuli, to regulate growth, and to differentiate. We have recently cloned CaT-like (CaT-L), a highly selective Ca2+ channel closely related to the epithelial calcium channels (ECaC) and the calcium transport protein CaT1. CaT-L is expressed in selected exocrine tissues, and its expression also strikingly correlates with the malignancy of prostate cancer. The expression pattern and selective Ca2+ permeation properties suggest an important function in Ca2+ uptake and a role in tumor progression, but not much is known about the regulation of this subfamily of ion channels. We now demonstrate a biochemical and functional mechanism by which cells can control CaT-L activity. CaT-L is regulated by means of a unique calmodulin binding site, which, at the same time, is a target for protein kinase C-dependent phosphorylation. We show that Ca2+-dependent calmodulin binding to CaT-L, which facilitates channel inactivation, can be counteracted by protein kinase C-mediated phosphorylation of the calmodulin binding site.

Differential Redox Regulation of ORAI Ion Channels: A Mechanism to Tune Cellular Calcium Signaling

Redox sensitivity of T cells decreases through ORAI Ca2+ channel subunit switching during T cell differentiation. Adapting to Oxidizing Environments Reactive oxygen species (ROS) were thought for many years to be only detrimental, causing damage to DNA and proteins. However, it has become clear that ROS, particularly H2O2, can act as intracellular signaling molecules that link cellular redox state to such processes as proliferation and differentiation. Bogeski et al. have uncovered a role for ROS in regulating calcium channel activity—and intracellular Ca2+ signals crucial to the immune response—in T lymphocytes. They found that activity of ORAI1 calcium channels was blocked by H2O2, whereas that of the related ORAI3 channels was not. Redox sensitivity decreased as naïve human T helper lymphocytes differentiated into effector T helper lymphocytes, which was associated with an increase in the abundance of mRNA encoding the insensitive ORAI3 protein. The authors suggest that changes in the specific complement of ORAI channels, and thereby sensitivity to ROS, could enable T lymphocytes to fine tune cellular responses in oxidizing environments such as those found during inflammation. Reactive oxygen species (ROS) are involved in many physiological and pathophysiological cellular processes. We used lymphocytes, which are exposed to highly oxidizing environments during inflammation, to study the influence of ROS on cellular function. Calcium ion (Ca2+) influx through Ca2+ release–activated Ca2+ (CRAC) channels composed of proteins of the ORAI family is essential for the activation, proliferation, and differentiation of T lymphocytes, but whether and how ROS affect ORAI channel function have been unclear. Here, we combined Ca2+ imaging, patch-clamp recordings, and measurements of cell proliferation and cytokine secretion to determine the effects of hydrogen peroxide (H2O2) on ORAI channel activity and human T helper lymphocyte (TH cell) function. ORAI1, but not ORAI3, channels were inhibited by oxidation by H2O2. The differential redox sensitivity of ORAI1 and ORAI3 channels depended mainly on an extracellularly located reactive cysteine, which is absent in ORAI3. TH cells became progressively less redox-sensitive after differentiation into effector cells, a shift that would allow them to proliferate, differentiate, and secrete cytokines in oxidizing environments. The decreased redox sensitivity of effector TH cells correlated with increased expression of Orai3 and increased abundance of several cytosolic antioxidants. Knockdown of ORAI3 with small-interfering RNA rendered effector TH cells more redox-sensitive. The differential expression of Orai isoforms between naïve and effector TH cells may tune cellular responses under oxidative stress.

Calcium microdomains at the immunological synapse: how ORAI channels, mitochondria and calcium pumps generate local calcium signals for efficient T-cell activation

Cell polarization enables restriction of signalling into microdomains. Polarization of lymphocytes following formation of a mature immunological synapse (IS) is essential for calcium‐dependent T‐cell activation. Here, we analyse calcium microdomains at the IS with total internal reflection fluorescence microscopy. We find that the subplasmalemmal calcium signal following IS formation is sufficiently low to prevent calcium‐dependent inactivation of ORAI channels. This is achieved by localizing mitochondria close to ORAI channels. Furthermore, we find that plasma membrane calcium ATPases (PMCAs) are re‐distributed into areas beneath mitochondria, which prevented PMCA up‐modulation and decreased calcium export locally. This nano‐scale distribution—only induced following IS formation—maximizes the efficiency of calcium influx through ORAI channels while it decreases calcium clearance by PMCA, resulting in a more sustained NFAT activity and subsequent activation of T cells.

Redox regulation of calcium ion channels: chemical and physiological aspects.

Reactive oxygen species (ROS) are increasingly recognized as second messengers in many cellular processes. While high concentrations of oxidants damage proteins, lipids and DNA, ultimately resulting in cell death, selective and reversible oxidation of key residues in proteins is a physiological mechanism that can transiently alter their activity and function. Defects in ROS producing enzymes cause disturbed immune response and disease. Changes in the intracellular free Ca(2+) concentration are key triggers for diverse cellular functions. Ca(2+) homeostasis thus needs to be precisely tuned by channels, pumps, transporters and cellular buffering systems. Alterations of these key regulatory proteins by reversible or irreversible oxidation alter the physiological outcome following cell stimulation. It is therefore necessary to understand which proteins are regulated and if this regulation is relevant in a physiological- and/or pathophysiological context. Because ROS are inherently difficult to identify and to measure, we first review basic oxygen redox chemistry and methods of ROS detection with special emphasis on electron paramagnetic resonance (EPR) spectroscopy. We then focus on the present knowledge of redox regulation of Ca(2+) permeable ion channels such as voltage-gated (CaV) Ca(2+) channels, transient receptor potential (TRP) channels and Orai channels.

Trafficking and Assembly of the Cold-sensitive TRPM8 Channel

TRPM (transient receptor potential melastatin-like) channels are distinct from many other members of the transient receptor potential family in regard to their overall size (>1000 amino acids), the lack of N-terminal ankyrin-like repeats, and hydrophobicity predictions that may allow for more than six transmembrane regions. Common to each TRPM member is a prominent C-terminal coiled coil region. Here we have shown that TRPM8 channels assemble as multimers using the putative coiled coil region within the intracellular C terminus and that this assembly can be disturbed by a single point mutation within the coiled coil region. This mutant neither gives rise to functional channels nor do its subunits interact or form protein complexes that correspond to a multimer. However, they are still transported to the plasma membrane. Furthermore, wild-type currents can be suppressed by expressing the membrane-attached C-terminal region of TRPM8. To separate assembly from trafficking, we investigated the maturation of TRPM8 protein by identifying and mutating the relevant N-linked glycosylation site and showing that glycosylation is neither essential for multimerization nor for transport to the plasma membrane per se but appears to facilitate efficient multimerization and transport.

Members of the synaptobrevin/vesicle-associated membrane protein (VAMP) family in Drosophila are functionally interchangeable in vivo for neurotransmitter release and cell viability

Synaptobrevins or VAMPs are vesicle-associated membrane proteins, often called v-SNARES, that are important for vesicle transport and fusion at the plasma membrane. Drosophila has two characterized members of this gene family: synaptobrevin (syb) and neuronal synaptobrevin (n-syb). Mutant phenotypes and gene-expression patterns indicate that n-Syb is exclusively neuronal and required only for synaptic vesicle secretion, whereas Syb is ubiquitous and, as shown here, essential for cell viability. When the eye precursor cells were made homozygous for syb−, the eye failed to develop. In contrast, n-syb− eye clones developed appropriately but failed to activate downstream neurons. To determine whether the two proteins are structurally specialized to accomplish these distinct in vivo functions, we have driven the expression of each gene in the absence of the other to look for phenotypic rescue. We find that expression of n-syb during eye development can rescue the cell lethality of the syb mutations, as can rat VAMP2 and cellubrevin. Expression of syb can restore synaptic transmission to n-syb mutants as assayed both by electroretinogram and recordings of excitatory junctional currents at the neuromuscular junction. Therefore, we find that Syb, which usually is not involved in synaptic function, can mediate Ca2+-triggered synaptic activity and that no particular specialization of the v-SNARE is required to differentiate synaptic exocytosis from other forms.

Mutations of the Ca2+-sensing Stromal Interaction Molecule STIM1 regulate Ca2+ influx by altered oligomerization of STIM1 and by destabilization of the Ca2+ channel Orai1

Background: Calcium influx (ICRAC) is important for proper cell function. Results: A novel STIM1 mutant increases ICRAC, Ca2+-dependently destabilizes Orai1, and alters clustering. A new mathematical model explains the phenotype. Conclusion: The molecular kinetics of STIM1 and Orai1 are major determinants of ICRAC. Significance: The diffusion trap model and alteration of Orai1 stability provide a tool for understanding ICRAC regulation. A drop of endoplasmic reticulum Ca2+ concentration triggers its Ca2+ ssensor protein stromal interaction molecule 1 (STIM1) to oligomerize and accumulate within endoplasmic reticulum-plasma membrane junctions where it activates Orai1 channels, providing store-operated Ca2+ entry. To elucidate the functional significance of N-glycosylation sites of STIM1, we created different mutations of asparagine-131 and asparagine-171. STIM1 NN/DQ resulted in a strong gain of function. Patch clamp, Total Internal Reflection Fluorescent (TIRF) microscopy, and fluorescence recovery after photobleaching (FRAP) analyses revealed that expression of STIM1 DQ mutants increases the number of active Orai1 channels and the rate of STIM1 translocation to endoplasmic reticulum-plasma membrane junctions with a decrease in current latency. Surprisingly, co-expression of STIM1 DQ decreased Orai1 protein, altering the STIM1:Orai1 stoichiometry. We describe a novel mathematical tool to delineate the effects of altered STIM1 or Orai1 diffusion parameters from stoichiometrical changes. The mutant uncovers a novel mechanism whereby “superactive” STIM1 DQ leads to altered oligomerization rate constants and to degradation of Orai1 with a change in stoichiometry of activator (STIM1) to effector (Orai1) ratio leading to altered Ca2+ homeostasis.

ORMDL3 modulates store-operated calcium entry and lymphocyte activation

T lymphocytes rely on a Ca(2+) signal known as store-operated calcium entry (SOCE) for their activation. This Ca(2+) signal is generated by activation of a T-cell receptor, depletion of endoplasmic reticulum (ER) Ca(2+) stores and activation of Ca(2+) release-activated Ca(2+) currents (I(CRAC)). Here, we report that the ER protein orosomucoid like 3 (ORMDL3), the product of the ORMDL3 gene associated with several autoimmune and/or inflammatory diseases, negatively modulates I(CRAC), SOCE, nuclear factor of activated T cells nuclear translocation and interleukin-2 production. ORMDL3 inhibits the Ca(2+) influx mechanism at the outer mitochondrial membrane, resulting in a Ca(2+)-dependent inhibition of I(CRAC) and reduced SOCE. The effect of ORMDL3 could be mimicked by interventions that decreased mitochondrial Ca(2+) influx and reverted by buffering of cytosolic Ca(2+) or activation of mitochondrial Ca(2+) influx. In conclusion, ORMDL3 modifies key steps in the process of T-lymphocyte activation, providing a functional link between the genetic associations of the ORMDL3 gene with autoimmune and/or inflammatory diseases.

Inverse regulation of melanoma growth and migration by Orai1/STIM2‐dependent calcium entry

Spontaneous melanoma phenotype switching is controlled by unknown environmental factors and may determine melanoma outcome and responsiveness to anticancer therapy. We show that Orai1 and STIM2 are highly expressed and control store‐operated Ca2+ entry in human melanoma. Lower extracellular Ca2+ or silencing of Orai1/STIM2 caused a decrease in intracellular Ca2+, which correlated with enhanced proliferation and increased expression of microphthalmia‐associated transcription factor, a marker for proliferative melanoma phenotype. In contrast, the invasive and migratory potential of melanoma cells was reduced upon silencing of Orai1 and/or STIM2. Accordingly, markers for a non‐proliferative, tumor‐maintaining phenotype such as JARID1B and Brn2 decreased. Immunohistochemical staining of primary melanomas and lymph node metastases revealed a heterogeneous distribution of Orai1 and STIM2 with elevated expression in the invasive rim of the tumor. In summary, our results support a dynamic model in which Orai1 and STIM2 inversely control melanoma growth and invasion. Pharmacological tuning of Orai1 and particularly STIM2 might thus prevent metastatic spread and render melanomas more susceptible to conventional therapy.

Structure-function analysis of TRPV channels

In recent years many new members of the family of TRP ion channels have been identified. These channels are classified into several subgroups and participate in many sensory and physiological functions. TRPV channels are important for the perception of pain, temperature sensing, osmotic regulation, and maintenance of calcium homeostasis, and much recent research concerns the identification of protein domains involved in mediating specific channel functions. Recent literature on TRPV channel subunit composition, protein domains required for subunit assembly, trafficking, and regulation will be reviewed and discussed.