Mammalian homologues of the Drosophila transient receptor potential (TRP) channel gene encode a family of at least 20 ion channel proteins. They are widely distributed in mammalian tissues, but their specific physiological functions are largely unknown. A common theme that links the TRP channels is their activation or modulation by phosphatidylinositol signal transduction pathways. The channel subunits have six transmembrane domains that most probably assemble into tetramers to form non-selective cationic channels, which allow for the influx of calcium ions into cells. Three subgroups comprise the TRP channel family; the best understood of these mediates responses to painful stimuli. Other proposed functions include repletion of intracellular calcium stores, receptor-mediated excitation and modulation of the cell cycle.

We cloned and characterized a protein kinase and ion channel, TRP-PLIK. As part of the long transient receptor potential channel subfamily implicated in control of cell division, it is a protein that is both an ion channel and a protein kinase. TRP-PLIK phosphorylated itself, displayed a wide tissue distribution, and, when expressed in CHO-K1 cells, constituted a nonselective, calcium-permeant, 105-picosiemen, steeply outwardly rectifying conductance. The zinc finger containing alpha-kinase domain was functional. Inactivation of the kinase activity by site-directed mutagenesis and the channel's dependence on intracellular adenosine triphosphate (ATP) demonstrated that the channel's kinase activity is essential for channel function.

TRPM7 (ChaK1, TRP-PLIK, LTRPC7) is a ubiquitous, calcium-permeant ion channel that is unique in being both an ion channel and a serine/threonine kinase. The kinase domain of TRPM7 directly associates with the C2 domain of phospholipase C (PLC). Here, we show that in native cardiac cells and heterologous expression systems, G alpha q-linked receptors or tyrosine kinase receptors that activate PLC potently inhibit channel activity. Numerous experimental approaches demonstrated that phosphatidylinositol 4,5-bisphosphate (PIP(2)), the substrate of PLC, is a key regulator of TRPM7. We conclude that receptor-mediated activation of PLC results in the hydrolysis of localized PIP(2), leading to inactivation of the TRPM7 channel.

Fluorescence homotransfer (electronic energy transfer between identical fluorophores) has the potential to quantitate the number of subunits in membrane protein oligomers. Homotransfer strongly depolarizes fluorescence emission as a result of intermolecular excitation energy exchange between an initially excited, oriented molecule and a randomly oriented neighbor. We have theoretically treated fluorescein labeled subunits in an oligomer as a cluster of molecules that can exchange excitation energy back and forth among the subunits within that group. We find that the larger the number of subunits, the more depolarized is the emission. The general equations to calculate the expected anisotropy for complexes composed of varying numbers of labeled subunits are presented. Self-quenching of fluorophores, orientation, and changes in lifetime are also discussed and/or considered. To test this theory, we have specifically labeled melittin on its N-terminal with fluorescein and monitored its monomer to tetramer equilibrium both in solution and in lipid bilayers. The calculated anisotropies are close to the experimental values when non-fluorescent fluorescein dimers are taken into account. Our results show that homotransfer may be a promising method to study membrane-protein oligomerization.

m-Calpain is a protease implicated in the control of cell adhesion through focal adhesion disassembly. The mechanism by which the enzyme is spatially and temporally controlled is not well understood, particularly because the dependence of calpain on calcium exceeds the submicromolar concentrations normally observed in cells. Here we show that the channel kinase TRPM7 localizes to peripheral adhesion complexes with m-calpain, where it regulates cell adhesion by controlling the activity of the protease. Our research revealed that overexpression of TRPM7 in cells caused cell rounding with a concomitant loss of cell adhesion that is dependent upon the channel of the protein but not its kinase activities. Knockdown of m-calpain blocked TRPM7-induced cell rounding and cell detachment. Silencing of TRPM7 by RNA interference, however, strengthened cell adhesion and increased the number of peripheral adhesion complexes in the cells. Together, our results suggest that the ion channel TRPM7 regulates cell adhesion through m-calpain by mediating the local influx of calcium into peripheral adhesion complexes.TRPM7 is one of only two ion channels to possess its own kinase domain (1). It is a member of the transient receptor potential ion channel family with the closest similarity to its bifunctional homologue TRPM6 as well as to melastatin (TRPM1), whose reduced expression has been used as a prognosis marker for metastasis in patients with localized melanoma (2-6). TRPM7 is also distinctive in its ion permeability, allowing Ca 2ϩ as well as Mg 2ϩ and other cations to compose its inward current (7,8). The channel kinase is a member of the recently discovered ␣-kinase family (9, 10). Annexin I has been identified as a substrate for the kinase, but the functional significance of annexin I phosphorylation by TRPM7 is not yet understood (11). Autophosphorylation of the channel does not alter channel activity (12). However, phospholipase C inactivates TRPM7 channel activity through hydrolysis of phosphatidylinositol 4,5-bisphosphate, which is presumably gating the channel (13,14). Magnesium ions block channel activity (8,(15)(16)(17), and, more recently, TRPM7 current has been shown to be potentiated by protons (18). Despite these recent advances in understanding TRPM7 channel regulation, the physiological role of this unique bifunctional protein still remains unclear.The passage of Mg 2ϩ by TRPM7 has linked it to the regulation of magnesium homeostasis in mammalian cells (19). Its capacity to carry calcium, in contrast, has been associated with calcium overload during anoxic cell death (20), calcium-dependent regulation of the cell cycle (21), and most recently, skeletogenesis and kidney stone formation in zebrafish (22). An early study by Nadler et al. (8) showed that overexpression of TRPM7 caused HEK-293 cells to detach and die, suggesting that the channel may have a role in controlling cell adhesion.Here we present evidence that TRPM7 is a potent regulator of m-calpain. Fourteen distinct members of the mammalian calpain famil...

The Formin proteins are central players in mediating cytoskeletal reorganization and are epistatically positioned in a pathway downstream of Rho activation. These proteins exist in the cytoplasm in an autoinhibited state, which is mediated by intramolecular interactions between the amino-terminal GTPase binding domain (GBD) that encompasses the diaphanous inhibitory domain (DID) and the carboxyl-terminal diaphanous autoregulatory domain (DAD). It Dishevelled ͉ Wnt ͉ Rho D irectional cell migration is required for the development of an organism with proper polarity including dorsoventral, anterior-posterior, and left-right symmetry. Examples of these cell movements include those of gastrulation and neural fold closure. These cell movements are tightly regulated by secreted ligands (1, 2). One of these signaling pathways required for cell movements is the noncanonical Wnt pathway (3-5).Noncanonical Wnt signaling, also termed the planar cell polarity pathway, regulates cell movements through modification of the actin cytoskeleton (1,3,4,6). A number of molecular components for this pathway have been identified including Wnt11, Fz, Dvl, Daam1, Rho, Rac, JNK, Strabismus, and Prickle (reviewed in ref. 5). For noncanonical Wnt signaling, the binding of Wnt to the Frizzled (Fz) receptor stimulates a signal that is transduced to the cytoplasmic phosphoprotein Dishevelled (Dvl). At the level of Dvl, two independent and parallel pathways lead to the activation of the small GTPases Rho and Rac. The first pathway signaling to the small GTPase Rho occurs through the molecule Daam1 (7). This Rho pathway leads to the activation of the Rho-associated kinase Rock and mediates cytoskeletal reorganization (5, 8). The second activates another small GTPase of the Rho-family, Rac, which in turns stimulates JNK activity (9-11). Daam1 is a Formin protein family and has been shown to regulate gastrulation; however, how Daam1 is activated for its function remains unknown.The Formin proteins are central players in regulating cytoskeletal reorganization in mammalian cells (12). The Formin proteins contain three major domains termed the GTPase binding domain (GBD), Formin homology 1 (FH1) domain, and Formin homology 2 (FH2) domain (13). These proteins are proposed to exist in the cytoplasm in an autoinhibited state, which is mediated by a domain termed the diaphanous autoinhibitory domain (DAD) (12). This DAD found in the carboxyl terminus mediates interaction with the amino terminus of the protein and serves to ''lock'' the protein in a folded or closed conformation (12). It is proposed that Rho activation allows for Rho-GTP to bind to the GBD and release this molecule from autoinhibition. The FH1 and FH2 can then bind to effectors to mediate effects on the cytoskeleton. Intriguingly, the FH2 domain has recently been shown to be capable of nucleating actin filaments by itself in vitro, suggesting a complex interplay between the FH1 and FH2 domains along with their effectors for actin polymerization (12). However, it remains unclear how th...

The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a major protein kinase C (PKC) substrate in many different cell types. MARCKS is bound to the plasma membrane, and several recent studies suggest that this binding requires both hydrophobic insertion of its myristate chain into the bilayer and electrostatic interaction of its cluster of basic residues with acidic lipids. Phosphorylation of MARCKS by PKC introduces negative charges into the basic cluster, reducing its electrostatic interaction with acidic lipids and producing translocation of MARCKS from membrane to cytoplasm. The present study shows that physiological concentrations of MARCKS (<10 microM) inhibit phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in phospholipid vesicles. A peptide corresponding to the basic cluster, MARCKS(151-175), produces a similar inhibition, which was observed with both PLC-delta1 and -beta1. Direct fluorescence microscopy observations demonstrate that the MARCKS peptide forms lateral domains enriched in the acidic lipids phosphatidylserine and PIP2 but not PLC, which accounts for the observed inhibition of PIP2 hydrolysis. Phosphorylation of MARCKS(151-175) by PKC releases the inhibition and allows PLC to produce a burst of inositol 1,4, 5-trisphosphate and diacylglycerol.

The channel kinases TRPM6 and TRPM7 have recently been discovered to play important roles in Mg 2؉ and Ca 2؉ homeostasis, which is critical to both human health and cell viability. However, the molecular basis underlying these channels' unique Mg 2؉ and Ca 2؉ permeability and pH sensitivity remains unknown. Here we have created a series of amino acid substitutions in the putative pore of TRPM7 to evaluate the origin of the permeability of the channel and its regulation by pH. Two mutants of TRPM7, E1047Q and E1052Q, produced dramatic changes in channel properties. The I-V relations of E1052Q and E1047Q were significantly different from WT TRPM7, with the inward currents of 8-and 12-fold larger than TRPM7, respectively. permeation, rendering TRPM7 a monovalent selective channel. In addition, the ability of protons to potentiate inward currents was lost in E1047Q, indicating that E1047 is critical to Ca 2؉ and Mg 2؉ permeability of TRPM7, and its pH sensitivity. Mutation of the corresponding residues in the pore of TRPM6, E1024Q and E1029Q, produced nearly identical changes to the channel properties of TRPM6. Our results indicate that these two glutamates are key determinants of both channels' divalent selectivity and pH sensitivity. These findings reveal the molecular mechanisms underpinning physiological/pathological functions of TRPM6 and TRPM7, and will extend our understanding of the pore structures of TRPM channels.TRPM6 and TRPM7 belong to the TRP channel superfamily (1-5) and are distinguished from other known ion channels by virtue of having both ion channel and protein kinase activities (6 -11). In addition, TRPM6 and TRPM7 uniquely exhibit strong outward rectification, permeation to Ca 2ϩ , Mg 2ϩ , monovalent cations, and a wide array of trace metals (6 -8, 11, 12 (8,20,21,23), whereas TRPM7 is ubiquitously expressed, with highest expression in the kidney and heart (5, 6). In addition to these channels' regulation of Mg 2ϩ homeostasis, several studies have suggested multiple cellular and physiology functions for TRPM7, including anoxic neuronal death (24), cell adhesion and actomyosin contractility (25, 26), and skeletogenesis (27). Although the mechanisms by which TRPM6 and TRPM7 exert their physiological and/or pathological functions are not yet completely understood, it is clear that permeation of Ca 2ϩ and Mg 2ϩ contributes substantially to the known functions of these channels (7, 20 -22, 24, 25, 27). Moreover, a recent study demonstrated that the sensitivity of TRPM7 to external pH may contribute to controlling neurotransmitter release (28). Therefore, it is essential to understand the molecular mechanisms underlying the Ca 2ϩ and Mg 2ϩ permeability of TRPM6 and TRPM7, as well as their sensitivities to changes in pH.The aim of the present study was to identify the amino acid residues that determine Mg 2ϩ and Ca 2ϩ permeation of TRPM6 and TRPM7. We previously demonstrated that external protons significantly enhance TRPM6 and TRPM7 inward currents (11,19) by decreasing the divalent affinity to t...