Actomyosin contractility regulates various cell biological processes including cytokinesis, adhesion and migration. While in lower eukaryotes, a-kinases control actomyosin relaxation, a similar role for mammalian a-kinases has yet to be established. Here, we examined whether TRPM7, a cation channel fused to an a-kinase, can affect actomyosin function. We demonstrate that activation of TRPM7 by bradykinin leads to a Ca 2 þ -and kinase-dependent interaction with the actomyosin cytoskeleton. Moreover, TRPM7 phosphorylates the myosin IIA heavy chain. Accordingly, low overexpression of TRPM7 increases intracellular Ca 2 þ levels accompanied by cell spreading, adhesion and the formation of focal adhesions. Activation of TRPM7 induces the transformation of these focal adhesions into podosomes by a kinase-dependent mechanism, an effect that can be mimicked by pharmacological inhibition of myosin II. Collectively, our results demonstrate that regulation of cell adhesion by TRPM7 is the combined effect of kinase-dependent and -independent pathways on actomyosin contractility.

SUMMARY Worldwide, acute and chronic pain affects 20% of the adult population and represents an enormous financial and emotional burden. Using genome-wide neuronal-specific RNAi knock-down in Drosophila, we report a global screen for an innate behavior and identify hundreds of novel genes implicated in heat nociception, including the α2δ-family calcium channel subunit straightjacket (stj). Mice mutant for the stj ortholog CACNA2D3 (α2δ3) also exhibit impaired behavioral heat pain sensitivity. In addition, in humans, α2δ3 SNP variants associate with reduced sensitivity to acute noxious heat and chronic back pain. Functional imaging in α2δ3 mutant mice revealed impaired transmission of thermal pain evoked signals from the thalamus to higher order pain centers. Intriguingly, in α2δ3 mutant mice thermal pain and tactile stimulation triggered strong cross-activation or synesthesia of brain regions involved in vision, olfaction, and hearing.

Imaging of fluorescence resonance energy transfer (FRET) between suitable fluorophores is increasingly being used to study cellular processes with high spatiotemporal resolution. The genetically encoded Cyan (CFP) and Yellow (YFP) variants of Green Fluorescent Protein have become the most popular donor and acceptor pair in cell biology. FRET between these fluorophores can be imaged by detecting sensitized emission. This technique, for which CFP is excited and transfer is detected as emission of YFP, is sensitive, fast, and straightforward, provided that proper corrections are made. In this study, the detection of sensitized emission between CFP and YFP by confocal microscopy is optimized. It is shown that this FRET pair is best excited at 430 nm. We identify major sources of error and variability in confocal FRET acquisition including chromatic aberrations and instability of the excitation sources. We demonstrate that a novel correction algorithm that employs online corrective measurements yields reliable estimates of FRET efficiency, and it is also shown how the effect of other error sources can be minimized.

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The Rho family of small GTPases, including Rho, Rac, and Cdc42 isoforms, regulates different aspects of cytoskeletal organization, which are coordinated in the process of cell migration (1). Of these, Rac is involved in the protrusion of lamellipodia, which occur principally at the leading edge of migrating cells but also emerge from around newly adherent cells to mediate cell spreading (2, 3). Rac also regulates gene transcription, cell cycle progression, and transformation in vitro (4 -6) and is implicated in tumor initiation and progression in vivo (7). Rac is activated in response to various stimuli, including growth factors and adhesion to the extracellular matrix. However, how these stimuli ultimately result in Rac activation is poorly understood.The principal regulators of Rac activation are the guanine nucleotide exchange factors (GEFs) 1 and GTPase activating proteins. GEFs induce activation by exchanging GDP for GTP, whereas GTPase activating proteins enhance the intrinsic rate of hydrolysis of bound GTP to GDP, resulting in inactivation. In cells, Rac exists predominantly in its inactive GDP-bound form in a complex with RhoGDI (8). RhoGDI binds and masks the hydrophobic C-terminal region of Rac, the same region that is responsible for targeting Rac to the plasma membrane (9). Thus RhoGDI maintains Rac in the cytoplasm and must dissociate to allow Rac to translocate to the membrane and interact with membrane-associated activators (10 -12). It was shown recently (13, 14) that integrin signals disrupt the Rac-RhoGDI interaction, enabling Rac to target to regions of cell-matrix interaction and activate an adhesion-dependent signaling pathway. Thus appropriate localization, as well as activation, is necessary for Rac to carry out its functions. Increased intracellular calcium [Ca 2ϩ ] i represents a ubiquitous second messenger system in cells, linking receptor activation to downstream signaling pathways. Previous studies (15-18) have described relationships between intracellular calcium and the activation and function of Rho family GTPases in processes including muscle contraction and the exocytic response. Intracellular calcium is also required for thrombin-and collagen-induced Rac activation in platelets (19). In neutrophils, however, chemoattractant-induced Rac activation is independent of intracellular calcium (20), suggesting that the relationship between calcium and Rac signaling is dependent on the cell type and/or the growth factor receptor involved. Ras-GRF 1 and 2, exchange factors specific for both Ras and Rac (21,22), harbor a calcium-calmodulin binding site (23) whereas the Rac exchange factor, Tiam1, is phosphorylated by calcium-calmodulin-dependent protein kinase II, which leads to increased nucleotide exchange on Rac (24). These findings suggest that nucleotide exchange on Rac may be regulated by changes in intracellular calcium. Various studies have implicated protein kinase C (PKC) in the activation of Rac. In Swiss 3T3 cells, phorbol ester treatment induces membrane ruffling, which is...

TRPM7 is a ubiquitously expressed nonspecific cation channel that has been implicated in cellular Mg2؉ homeostasis. We have recently shown that moderate overexpression of TRPM7 in neuroblastoma N1E-115 cells elevates cytosolic Ca 2؉ levels and enhances cell-matrix adhesion. Furthermore, activation of TRPM7 by phospholipase C (PLC)-coupled receptor agonists caused a further increase in intracellular Ca 2؉ levels and augmented cell adhesion and spreading in a Ca 2؉ -dependent manner (1). Regulation of the TRPM7 channel is not well understood, although it has been reported that PIP 2 hydrolysis closes the channel. Here we have examined the regulation of TRPM7 by PLC-coupled receptor agonists such as bradykinin, lysophosphatidic acid, and thrombin. Using FRET assays for second messengers, we have shown that the TRPM7-dependent Ca 2؉ increase closely correlates with activation of PLC. Under noninvasive "perforated patch clamp" conditions, we have found similar activation of TRPM7 by PLC-coupled receptor agonists. Although we could confirm that, under whole-cell conditions, the TRPM7 currents were significantly inhibited following PLC activation, this PLC-dependent inhibition was only observed when [Mg 2؉ ] i was reduced below physiological levels. Thus, under physiological ionic conditions, TRPM7 currents were activated rather than inhibited by PLC-activating receptor agonists.TRPM7 is a ubiquitously expressed nonspecific cation channel that, intriguingly, contains a C-terminal serine-threonine kinase domain. It belongs to the transient receptor potential melastatin-related (TRPM) 2 subfamily of TRP channels that transduce sensory signals (2). TRPM7 functioning appears essential for life in that both knock-out and overexpression of the channels cause growth arrest, loss of cell adhesion, and rapid cell death (3-5). We recently discovered that low overexpression of TRPM7 induces cell spreading and adhesion and that its activation by PIP 2 -hydrolyzing receptor agonists leads to the formation of adhesion complexes in a kinase-dependent manner (1).Currents carried by TRPM7 channels exogenously expressed in mammalian cells have been analyzed by several groups. In physiological solutions, the channel conducts mainly Ca 2ϩ and Mg 2ϩ (6), but in the absence of these divalent cations, K ϩ and Na ϩ (3, 7) permeate efficiently. A characteristic feature is the inhibition of TRPM7 currents by physiological (1-2 mM) intracellular Mg 2ϩ levels; in whole-cell patch clamp experiments, large outwardly rectifying TRPM7 currents (3,7,8) are evoked by perfusion with Mg 2ϩ -free pipette solutions. Furthermore, MgATP and MgGTP also inhibit the channels (3, 9), although some controversy was raised on this issue (10). TRPM7 currents have been termed MagNuM (for Mg 2ϩ nucleotide-regulated metal ion (3)) or MIC (for Mg 2ϩ -inhibited cation (10)) currents. These terms will here be used interchangeably to reflect whole-cell currents evoked by internal Mg 2ϩ depletion. MIC/MagNuM currents revert at about 0 mV and lack voltageand time-depend...

The biolipid sphingosine-1-phosphate (S1P) is an essential modulator of innate immunity, cell migration, and wound healing. It is released locally upon acute tissue injury from endothelial cells and activated thrombocytes and, therefore, may give rise to acute posttraumatic pain sensation via a yet elusive molecular mechanism. We have used an interdisciplinary approach to address this question, and we find that intradermal injection of S1P induced significant licking and flinching behavior in wild-type mice and a dose-dependent flare reaction in human skin as a sign of acute activation of nociceptive nerve terminals. Notably, S1P evoked a small excitatory ionic current that resulted in nociceptor depolarization and action potential firing. This ionic current was preserved in "cation-free" solution and blocked by the nonspecific Cl Ϫ channel inhibitor niflumic acid and by preincubation with the G-protein inhibitor GDP-␤-S. Notably, S1P 3 receptor was detected in virtually all neurons in human and mouse DRG. In line with this finding, S1P-induced neuronal responses and spontaneous pain behavior in vivo were substantially reduced in S1P 3 Ϫ/Ϫ mice, whereas in control S1P 1 floxed (S1P 1 fl/fl ) mice and mice with a nociceptor-specific deletion of S1P 1 Ϫ/Ϫ receptor (SNS-S1P 1 Ϫ/Ϫ ), neither the S1P-induced responses in vitro nor the S1P-evoked pain-like behavior was altered. Therefore, these findings indicate that S1P evokes significant nociception via G-proteindependent activation of an excitatory Cl Ϫ conductance that is largely mediated by S1P 3 receptors present in nociceptors, and point to these receptors as valuable therapeutic targets for post-traumatic pain.

Low extracellular calcium (Ca 2ϩ ) promotes release of parathyroid hormone (PTH), which acts on multiple organs to maintain overall Ca 2ϩ balance. In the distal part of the nephron, PTH stimulates active Ca 2ϩ reabsorption via the adenylyl cyclase-cAMP-protein kinase A (PKA) pathway, but the molecular target of this pathway is unknown. The transient receptor potential vanilloid 5 (TRPV5) channel constitutes the luminal gate for Ca 2ϩ entry in the distal convoluted tubule and has several putative PKA phosphorylation sites. Here, we investigated the effect of PTH-induced cAMP signaling on TRPV5 activity. Using fluorescence resonance energy transfer, we studied cAMP and Ca 2ϩ dynamics during PTH stimulation of HEK293 cells that coexpressed the PTH receptor and TRPV5. PTH increased cAMP levels, followed by a rise in TRPV5-mediated Ca 2ϩ influx. PTH (1 to 31) and forskolin, which activate the cAMP pathway, mimicked the stimulation of TRPV5 activity. Remarkably, TRPV5 activation was limited to conditions of strong intracellular Ca 2ϩ buffering. Cell surface biotinylation studies demonstrated that forskolin did not affect TRPV5 expression on the cell surface, suggesting that it alters the single-channel activity of a fixed number of TRPV5 channels. Application of the PKA catalytic subunit, which phosphorylated TRPV5, directly increased TRPV5 channel open probability. Alanine substitution of threonine-709 abolished both in vitro phosphorylation and PTH-mediated stimulation of TRPV5. In summary, PTH activates the cAMP-PKA signaling cascade, which rapidly phosphorylates threonine-709 of TRPV5, increasing the channel's open probability and promoting Ca 2ϩ reabsorption in the distal nephron.