Elena Oancea

Protein Kinase C as a Molecular Machine for Decoding Calcium and Diacylglycerol Signals

The specificity of many signal transduction pathways relies on the temporal coordination of different second messenger signals. Here we found a molecular mechanism which guarantees that conventional protein kinase C (PKC) isoforms are sequentially activated by calcium and diacylglycerol signals. Receptor stimuli that triggered repetitive calcium spikes induced a parallel repetitive translocation of GFP-tagged PKCgamma to the plasma membrane. While calcium acted rapidly, diacylglycerol binding to PKCgamma was initially prevented by a pseudosubstrate clamp, which kept the diacylglycerol-binding site inaccessible and delayed calcium- and diacylglycerol-mediated kinase activation. After termination of calcium signals, bound diacylglycerol prolonged kinase activity. The properties of this molecular decoding machine make PKCgamma responsive to persistent diacylglycerol increases combined with high- but not low-frequency calcium spikes.

TRPC5 is a regulator of hippocampal neurite length and growth cone morphology

Growth cone motility is regulated by both fast voltage-dependent Ca2+ channels and by unknown receptor-operated Ca2+ entry mechanisms. Transient receptor potential (TRP) homomeric TRPC5 ion channels are receptor-operated, Ca2+-permeable channels predominantly expressed in the brain. Here we show that TRPC5 is expressed in growth cones of young rat hippocampal neurons. Our results indicate that TRPC5 channel subunits interact with the growth cone–enriched protein stathmin 2, are packaged into vesicles and are carried to newly forming growth cones and synapses. Once in the growth cone, TRPC5 channels regulate neurite extension and growth-cone morphology. Dominant-negative TRPC5 expression allowed significantly longer neurites and filopodia to form. We conclude that TRPC5 channels are important components of the mechanism controlling neurite extension and growth cone morphology.

Functional TRPM7 Channels Accumulate at the Plasma Membrane in Response to Fluid Flow

Many cells are constantly exposed to fluid mechanical forces generated by flowing blood, and wall shear stresses modulate aspects of their structure and function. However, the mechanisms for mechanotransduction of flow are not well understood. Here we report that TRPM7, which is both an ion channel and a functional kinase, is translocated within cells in response to laminar flow. After exposure of cells to physiological values of laminar fluid flow, the number of TRPM7 molecules localized at or near the plasma membrane increased up to 2-fold, in less than 100 seconds. This increase in membrane-localized GFP-TRPM7, as seen by total internal reflection fluorescence microscopy, closely correlated with increases in TRPM7 current. Both endogenous and heterologously expressed TRPM7 was found in tubulovesicular structures that were translocated to the region of the plasma membrane on induction of shear stress. In vascular smooth muscle cells, but not in several types of endothelial cells, fluid flow increased endogenous native TRPM7 current amplitude. We hypothesize that TRPM7 plays a role in pathological response to vessel wall injury.

TRPM1 forms ion channels associated with melanin content in melanocytes.

Newly identified TRPM1 isoforms that mediate current are highly conserved, present intracellularly, and associated with melanin content. A Melanocyte Channel TRPM1 (also known as melastatin), a protein that is primarily found in cells that produce the pigment melanin, has a molecular structure resembling that of other members of the transient receptor potential (TRP) family of cation channels. TRPM1 has not previously been shown to carry current, however, and its function remains unknown. Oancea et al. identified two previously unrecognized TRPM1 splice variants and showed that they mediate current when expressed in human melanoma cells. Moreover, they found endogenous TRPM1-mediated currents in melanocytes and melanoma cells. When fluorescently labeled TRPM1 was heterologously expressed in human embryonic kidney or melanoma cell lines it primarily localized to intracellular vesicular structures, suggesting that its major function may be intracellular. Intriguingly, TRPM1 expression in melanocytes correlated with melanin content, leading the authors to postulate that it may play a role in melanocyte pigmentation. TRPM1 (melastatin), which encodes the founding member of the TRPM family of transient receptor potential (TRP) ion channels, was first identified by its reduced expression in a highly metastatic mouse melanoma cell line. Clinically, TRPM1 is used as a predictor of melanoma progression in humans because of its reduced abundance in more aggressive forms of melanoma. Although TRPM1 is found primarily in melanin-producing cells and has the molecular architecture of an ion channel, its function is unknown. Here we describe an endogenous current in primary human neonatal epidermal melanocytes and mouse melanoma cells that was abrogated by expression of microRNA directed against TRPM1. Messenger RNA analysis showed that at least five human ion channel–forming isoforms of TRPM1 could be present in melanocytes, melanoma, brain, and retina. Two of these isoforms are encoded by highly conserved splice variants that are generated by previously uncharacterized exons. Expression of these two splice variants in human melanoma cells generated an ionic current similar to endogenous TRPM1 current. In melanoma cells, TRPM1 is prevalent in highly dynamic intracellular vesicular structures. Plasma membrane TRPM1 currents are small, raising the possibility that their primary function is intracellular, or restricted to specific regions of the plasma membrane. In neonatal human epidermal melanocytes, TRPM1 expression correlates with melanin content. We propose that TRPM1 is an ion channel whose function is critical to normal melanocyte pigmentation and is thus a potential target for pigmentation disorders.

Calbindin-D28K dynamically controls TRPV5-mediated Ca2+ transport

In Ca2+‐transporting epithelia, calbindin‐D28K (CaBP28K) facilitates Ca2+ diffusion from the luminal Ca2+ entry side of the cell to the basolateral side, where Ca2+ is extruded into the extracellular compartment. Simultaneously, CaBP28K provides protection against toxic high Ca2+ levels by buffering the cytosolic Ca2+ concentration ([Ca2+]i) during high Ca2+ influx. CaBP28K consistently colocalizes with the epithelial Ca2+ channel TRPV5, which constitutes the apical entry step in renal Ca2+‐transporting epithelial cells. Here, we demonstrate using protein‐binding analysis, subcellular fractionation and evanescent‐field microscopy that CaBP28K translocates towards the plasma membrane and directly associates with TRPV5 at a low [Ca2+]i. 45Ca2+ uptake measurements, electrophysiological recordings and transcellular Ca2+ transport assays of lentivirus‐infected primary rabbit connecting tubule/distal convolute tubule cells revealed that associated CaBP28K tightly buffers the flux of Ca2+ entering the cell via TRPV5, facilitating high Ca2+ transport rates by preventing channel inactivation. In summary, CaBP28K acts in Ca2+‐transporting epithelia as a dynamic Ca2+ buffer, regulating [Ca2+] in close vicinity to the TRPV5 pore by direct association with the channel.

UV light phototransduction activates transient receptor potential A1 ion channels in human melanocytes

Human skin is constantly exposed to solar ultraviolet radiation (UVR), the most prevalent environmental carcinogen. Humans have the unique ability among mammals to respond to UVR by increasing their skin pigmentation, a protective process driven by melanin synthesis in epidermal melanocytes. The molecular mechanisms used by melanocytes to detect and respond to long-wavelength UVR (UVA) are not well understood. We recently identified a UVA phototransduction pathway in melanocytes that is mediated by G protein-coupled receptors and leads to rapid calcium mobilization. Here we report that in human epidermal melanocytes physiological doses of UVR activate a retinal-dependent current mediated by transient receptor potential A1 (TRPA1) ion channels. The TRPA1 photocurrent is UVA-specific and requires G protein and phospholipase C signaling, thus contributing to UVA-induced calcium responses to mediate downstream cellular effects and providing evidence for TRPA1 function in mammalian phototransduction. Remarkably, TRPA1 activation is required for the UVR-induced and retinal-dependent early increase in cellular melanin. Our results show that TRPA1 is essential for a unique extraocular phototransduction pathway in human melanocytes that is activated by physiological doses of UVR and results in early melanin synthesis.

UVA Phototransduction Drives Early Melanin Synthesis in Human Melanocytes

Exposure of human skin to solar ultraviolet radiation (UVR), a powerful carcinogen [1] comprising ~95% ultraviolet A (UVA) and ~5% ultraviolet B (UVB) at the Earths surface, promotes melanin synthesis in epidermal melanocytes [2, 3], which protects skin from DNA damage [4, 5]. UVB causes DNA lesions [6] that lead to transcriptional activation of melanin-producing enzymes, resulting in delayed skin pigmentation within days [7]. In contrast, UVA causes primarily oxidative damage [8] and leads to immediate pigment darkening (IPD) within minutes, via an unknown mechanism [9, 10]. No receptor protein directly mediating phototransduction in skin has been identified. Here we demonstrate that exposure of primary human epidermal melanocytes (HEMs) to UVA causes calcium mobilization and early melanin synthesis. Calcium responses were abolished by treatment with G protein or phospholipase C (PLC) inhibitors or by depletion of intracellular calcium stores. We show that the visual photopigment rhodopsin [11] is expressed in HEMs and contributes to UVR phototransduction. Upon UVR exposure, significant melanin production was measured within one hour; cellular melanin continued to increase in a retinal- and calcium-dependent manner up to 5-fold after 24 hr. Our findings identify a novel UVA-sensitive signaling pathway in melanocytes that leads to calcium mobilization and melanin synthesis and may underlie the mechanism of IPD in human skin.

Extracellular pH dynamically controls cell surface delivery of functional TRPV5 channels.

ABSTRACT Extracellular pH has long been known to affect the rate and magnitude of ion transport processes among others via regulation of ion channel activity. The Ca2+-selective transient receptor potential vanilloid 5 (TRPV5) channel constitutes the apical entry gate in Ca2+-transporting cells, contributing significantly to the overall Ca2+ balance. Here, we demonstrate that extracellular pH determines the cell surface expression of TRPV5 via a unique mechanism. By a comprehensive approach using total internal reflection fluorescence microscopy, cell surface protein labeling, electrophysiology, 45Ca2+ uptake assays, and functional channel recovery after chemobleaching, this study shows that upon extracellular alkalinization, a pool of TRPV5-containing vesicles is rapidly recruited to the cell surface without collapsing into the plasma membrane. These vesicles contain functional TRPV5 channels since extracellular alkalinization is accompanied by increased TRPV5 activity. Conversely, upon subsequent extracellular acidification, vesicles are retrieved from the plasma membrane, simultaneously resulting in decreased TRPV5 activity. Thus, TRPV5 accesses the extracellular compartment via transient openings of vesicles, suggesting that rapid responses of constitutive active TRP channels to physiological stimuli rely on vesicular “kiss and linger” interactions with the plasma membrane.

Mechanism of Persistent Protein Kinase D1 Translocation and Activation

The specificity of many signal transduction pathways relies on the spatiotemporal features of each signaling step. G protein-coupled receptor-mediated activation of protein kinases leads to diverse cellular effects. Upon receptor activation, PKD1 and several C-type protein kinases (PKCs), translocate to the plasma membrane and become catalytically active. Here we show that, unlike PKCs, PKD1 remains active at the membrane for hours. The two DAG binding C1 domains of PKD1 have distinct functional roles in targeting and maintaining PKD1 at the plasma membrane. C1A achieves fast, maximal, and reversible translocation, while C1B translocates partially, but persistently, to the plasma membrane. The persistent localization requires the C1B domain of PKD1, which binds Galphaq. We incorporate the kinetics of PKD1 translocation into a three-state model that suggests how PKD1 binding to DAG and Galphaq uniquely encodes frequency-dependent PKD1 signaling.

Reversible Desensitization of Inositol Trisphosphate-induced Calcium Release Provides a Mechanism for Repetitive Calcium Spikes

Repetitive transient increases in cytosolic calcium concentration (calcium spikes or calcium oscillations) are a common mode of signal transduction in receptor-mediated cell activation. Repetitive calcium spikes are initiated by phospholipase C-mediated production of inositol 1,4,5-trisphosphate (InsP3) and are thought to be generated by a positive feedback mechanism in which calcium potentiates its own release, a negative feedback mechanism by which calcium release is terminated, and a slow recovery process that defines the time interval between calcium spikes. The molecular mechanisms that terminate each calcium spike and define the spike frequency are not yet known. Here we show, in intact rat basophilic leukemia cells, that calcium responses induced by InsP3 are diminished for a period of 30-60 s following an InsP3-induced calcium spike. The sensitivity of calcium release for InsP3 was probed by UV laser-mediated photorelease of InsP3, and calcium responses were monitored by fluorescence calcium imaging. A maximal loss in sensitivity (desensitization) was observed for InsP3 increases that resulted in a near maximal calcium spike and was expressed as an 80-100% reduction in the calcium response to an equal amount of InsP3, released 10 s after the first UV pulse. When the amount of released InsP3 in the second pulse was increased 2-3-fold, desensitization was overcome and a second calcium response of equal amplitude to the first was produced. A power dependence of 3.2 was measured between the amount of released InsP3 and the amplitude of the triggered calcium response, explaining how a small decrease in InsP3 sensitivity can lead to a nearly complete reduction in the calcium response. Desensitization was abolished by the addition of the calcium buffers BAPTA and EGTA and could be induced by microinjection of calcium, suggesting that it is a calcium-dependent process. Half-maximal desensitization was observed at a free calcium concentration of 290 nM and increased with a power of 3.7 with peak calcium concentration. These studies suggest that reversible desensitization of InsP3-induced calcium release serves as a “saw-tooth” parameter that controls the termination of each spike and the frequency of calcium spikes.