Different mechanisms regulate lysophosphatidic acid (LPA)-dependent versus phorbol ester-dependent internalization of the LPA1 receptor

Lysophosphatidic acid (LPA) stimulates cells by activation of five G-protein-coupled receptors, termed LPA 1-5. The LPA 1 receptor is the most widely expressed and is a major regulator of cell migration. In this study, we show that phorbol ester (PMA)-induced internalization of the LPA(1) receptor requires clathrin AP-2 complexes, protein kinase C, and a distal dileucine motif (amino acids 352 and 353) in the cytoplasmic tail but not beta-arrestin. Agonist-dependent internalization of LPA 1, however, requires a cluster of serine residues (amino acids 341-347) located proximal to the dileucine motif, beta-arrestin, and to a lesser extent clathrin AP-2. The serine cluster of LPA 1 is required for beta-arrestin2-GFP translocation to the plasma membrane and signal desensitization. In contrast, the dileucine motif (IL) is required for both basal and PMA-induced internalization. Evidence for the beta-arrestin independence of PMA-induced internalization of LPA 1 comes from the observations that beta-arrestin2-GFP is not recruited to the plasma membrane upon PMA treatment and that LPA 1 is readily internalized in beta-arrestin1/2 knock-out mouse embryonic fibroblasts. These results indicate that distinct molecular mechanisms regulate agonist-dependent and PMA-dependent internalization of the LPA 1 receptor.     

Modulation of KCa3.1 Channels by Eicosanoids,Omega-3 Fatty Acids,and Molecular Determinants

Background

Cytochrome P450- and ω-hydrolase products (epoxyeicosatrienoic acids (EETs), hydroxyeicosatetraeonic acid (20-HETE)), natural omega-3 fatty acids (ω3), and pentacyclic triterpenes have been proposed to contribute to a wide range of vaso-protective and anti-fibrotic/anti-cancer signaling pathways including the modula-tion of membrane ion channels. Here we studied the modulation of intermediate-conductance Ca²⁺/calmodulin-regulated K⁺ channels (K_Ca3.1) by EETs, 20-HETE, ω3, and pentacyclic triterpenes and the structural requirements of these fatty acids to exert channel blockade.

Methodology/Principal Findings

We studied modulation of cloned human hK_Ca3.1 and the mutant hK_Ca3.1^V275A in HEK-293 cells, of rK_Ca3.1 in aortic endothelial cells, and of mK_Ca3.1 in 3T3-fibroblasts by inside-out and whole-cell patch-clamp experiments, respectively. In inside-out patches, Ca²⁺-activated hK_Ca3.1 were inhibited by the ω3, DHA and α-LA, and the ω6, AA, in the lower µmolar range and with similar potencies. 5,6-EET, 8,9-EET, 5,6-DiHETE, and saturated arachidic acid, had no appreciable effects. In contrast, 14,15-EET, its stable derivative, 14,15-EEZE, and 20-HETE produced channel inhibition. 11,12-EET displayed less inhibitory activity. The K_Ca3.1^V275A mutant channel was insensitive to any of the blocking EETs. Non-blocking 5,6-EET antagonized the inhibition caused by AA and augmented cloned hK_Ca3.1 and rK_Ca3.1 whole-cell currents. Pentacyclic triterpenes did not modulate K_Ca3.1 currents.

Conclusions/Significance

Inhibition of K_Ca3.1 by EETs (14,15-EET), 20-HETE, and ω3 critically depended on the presence of electron double bonds and hydrophobicity within the 10 carbons preceding the carboxyl-head of the molecules. From the physiological perspective, metabolism of AA to non-blocking 5,6,- and 8,9-EET may cause AA-de-blockade and contribute to cellular signal transduction processes influenced by these fatty acids.     

Adaptor Protein2 (AP2) Orchestrates CXCR2‐Mediated Cell Migration

The chemokine receptor CXCR2 is vital for inflammation, wound healing, angiogenesis, cancer progression and metastasis. Adaptor protein 2 (AP2), a clathrin binding heterotetrameric protein comprised of α, β2, μ2 and σ2 subunits, facilitates clathrin‐mediated endocytosis. Mutation of the LLKIL motif in the CXCR2 carboxyl‐terminal domain (CTD) results in loss of AP2 binding to the receptor and loss of ligand‐mediated receptor internalization and chemotaxis. AP2 knockdown also results in diminished ligand‐mediated CXCR2 internalization, polarization and chemotaxis. Using knockdown/rescue approaches with AP2‐μ2 mutants, the binding domains were characterized in reference to CXCR2 internalization and chemotaxis. When in an open conformation, μ2 Patch 1 and Patch 2 domains bind tightly to membrane PIP2 phospholipids. When AP2‐μ2, is replaced with μ2 mutated in Patch 1 and/or Patch 2 domains, ligand‐mediated receptor binding and internalization are not lost. However, chemotaxis requires AP2‐μ2 Patch 1, but not Patch 2. AP2‐σ2 has been demonstrated to bind dileucine motifs to facilitate internalization. Expression of AP2‐σ2 V88D and V98S dominant negative mutants resulted in loss of CXCR2 mediated chemotaxis. Thus, AP2 binding to both membrane phosphatidylinositol phospholipids and dileucine motifs is crucial for directional migration or chemotaxis. Moreover, AP2‐mediated receptor internalization can be dissociated from AP2‐mediated chemotaxis.      

Niflumic acid blocks native and recombinant T-type channels

Voltage‐dependent calcium channels are widely distributed in animal cells, including spermatozoa. Calcium is fundamental in many sperm functions such as: motility, capacitation, and the acrosome reaction (AR), all essential for fertilization. Pharmacological evidence has suggested T‐type calcium channels participate in the AR. Niflumic acid (NA), a non‐steroidal anti‐inflammatory drug commonly used as chloride channel blocker, blocks T‐currents in mouse spermatogenic cells and Cl^? channels in testicular sperm. Here we examine the mechanism of NA blockade and explore if it can be used to separate the contribution of different Ca_V3 members previously detected in these cells. Electrophysiological patch‐clamp recordings were performed in isolated mouse spermatogenic cells and in HEK cells heterologously expressing Ca_V3 channels. NA blocks mouse spermatogenic cell T‐type currents with an IC₅₀ of 73.5 µM, without major voltage‐dependent effects. The NA blockade is more potent in the open and in the inactivated state than in the closed state of the T‐type channels. Interestingly, we found that heterologously expressed Ca_V3.1 and Ca_V3.3 channels were more sensitive to NA than Ca_V3.2 channels, and this drug substantially slowed the recovery from inactivation of the three isoforms. Molecular docking modeling of drug‐channel binding predicts that NA binds preferentially to the extracellular face of Ca_V3.1 channels. The biophysical characteristics of mouse spermatogenic cell T‐type currents more closely resemble those from heterologously expressed Ca_V3.1 channels, including their sensitivity to NA. As Ca_V3.1 null mice maintain their spermatogenic cell T‐currents, it is likely that a novel Ca_V3.2 isoform is responsible for them. J. Cell. Physiol. 227: 2542–2555, 2012. © 2011 Wiley Periodicals, Inc.     

Intermediate conductance Ca2+-activated potassium channel (KCa3.1) in the inner mitochondrial membrane of human colon cancer cells

Patch–clamping mitoplasts isolated from human colon carcinoma 116 cells has allowed the identification and characterization of the intermediate conductance Ca²⁺-activated K⁺-selective channel K_Ca3.1, previously studied only in the plasma membrane of various cell types. Its identity has been established by its biophysical and pharmacological properties. Its localisation in the inner membrane of mitochondria is indicated by Western blots of subcellular fractions, by recording of its activity in mitochondria made fluorescent by a mitochondria-targeted fluorescent protein and by the co-presence of channels considered to be markers of the inner membrane. Moderate increases of mitochondrial matrix [Ca²⁺] will cause mtK_Ca3.1 opening, thus linking inner membrane K⁺ permeability and transmembrane potential to Ca²⁺ signalling.     

Subcellular distribution of calcium-sensitive potassium channels (IK1) in migrating cells

Cell migration is crucial for wound healing, immune defense, or formation of tumor metastases. In addition to the cytoskeleton, Ca2+ sensitive K+ channels (IK1) are also part of the cellular "migration machinery." We showed that Ca2+ sensitive K+ channels support the retraction of the rear part of migrating MDCK-F cells by inducing a localized shrinkage at this cell pole. So far the molecular nature and in particular the subcellular distribution of these channels in MDCK-F cells is unknown. We compared the effect of IK1 channel blockers and activators on the current of a cloned IK1 channel from MDCK-F cells (cIK1) and the migratory behavior of these cells. Using IK1 channels labeled with a HA-tag or the enhanced green fluorescent protein we studied the subcellular distribution of the canine (cIK1) and the human (hIK1) channel protein in different migrating cells. The functional impact of cIK1 channel activity at the front or rear part of MDCK-F cells was assessed with a local superfusion technique and a detailed morphometric analysis. We show that it is cIK1 whose activity is required for migration of MDCK-F cells. IK1 channels are found in the entire plasma membrane, but they are concentrated at the cell front. This is in part due to membrane ruffling at this cell pole. However, there appears to be only little cIK1 channel activity at the front of MDCK-F cells. In our view this apparent discrepancy can be explained by differential regulation of IK1 channels at the front and rear part of migrating cells.     

Intracellular Ca(2+) oscillations induced by over-expressed Ca(V)3.1 T-type Ca(2+) channels in NG108-15 cells

T-type Ca²⁺ channel family includes three subunits Ca_V3.1, Ca_V3.2 and Ca_V3.3 and have been shown to control burst firing and intracellular Ca²⁺ concentration ([Ca²⁺]_i) in neurons. Here, we investigated whether Ca_V3.1 channels could generate a pacemaker current and contribute to cell excitability. Ca_V3.1 clones were over-expressed in the neuronal cell line NG108-15. Ca_V3.1 channel expression induced repetitive action potentials, generating spontaneous membrane potential oscillations (MPOs) and concomitant [Ca²⁺]_i oscillations. These oscillations were inhibited by T-type channels antagonists and were present only if the membrane potential was around −61 mV. [Ca²⁺]_i oscillations were critically dependent on Ca²⁺ influx through Ca_V3.1 channels and did not involve Ca²⁺ release from the endoplasmic reticulum. The waveform and frequency of the MPOs are constrained by electrophysiological properties of the Ca_V3.1 channels. The trigger of the oscillations was the Ca_V3.1 window current. This current induced continuous [Ca²⁺]_i increase at −60 mV that depolarized the cells and triggered MPOs. Shifting the Ca_V3.1 window current potential range by increasing the external Ca²⁺ concentration resulted in a corresponding shift of the MPOs threshold. The hyperpolarization-activated cation current (I_h) was not required to induce MPOs, but when expressed together with Ca_V3.1 channels, it broadened the membrane potential range over which MPOs were observed. Overall, the data demonstrate that the Ca_V3.1 window current is critical in triggering intrinsic electrical and [Ca²⁺]_i oscillations.     

Selective Activation of KCa3.1 and CRAC Channels by P2Y2 Receptors Promotes Ca2+ Signaling,Store Refilling and Migration of Rat Microglial Cells

Microglial activation involves Ca²⁺ signaling, and numerous receptors can evoke elevation of intracellular Ca²⁺. ATP released from damaged brain cells can activate ionotropic and metabotropic purinergic receptors, and act as a chemoattractant for microglia. Metabotropic P₂Y receptors evoke a Ca²⁺ rise through release from intracellular Ca²⁺ stores and store-operated Ca²⁺ entry, and some have been implicated in microglial migration. This Ca²⁺ rise is expected to activate small-conductance Ca²⁺-dependent K⁺ (SK) channels, if present. We previously found that SK3 (KCa2.3) and KCa3.1 (SK4/IK1) are expressed in rat microglia and contribute to LPS-mediated activation and neurotoxicity. However, neither current has been studied by elevating Ca²⁺ during whole-cell recordings. We hypothesized that, rather than responding only to Ca²⁺, each channel type might be coupled to different receptor-mediated pathways. Here, our objective was to determine whether the channels are differentially activated by P₂Y receptors, and, if so, whether they play differing roles. We used primary rat microglia and a rat microglial cell line (MLS-9) in which riluzole robustly activates both SK3 and KCa3.1 currents. Using electrophysiological, Ca²⁺ imaging and pharmacological approaches, we show selective functional coupling of KCa3.1 to UTP-mediated P₂Y₂ receptor activation. KCa3.1 current is activated by Ca²⁺ entry through Ca²⁺-release-activated Ca²⁺ (CRAC/Orai1) channels, and both CRAC/Orai1 and KCa3.1 channels facilitate refilling of Ca²⁺ stores. The Ca²⁺ dependence of KCa3.1 channel activation was skewed to abnormally high concentrations, and we present evidence for a close physical association of the two channel types. Finally, migration of primary rat microglia was stimulated by UTP and inhibited by blocking either KCa3.1 or CRAC/Orai1 channels. This is the first report of selective coupling of one type of SK channel to purinergic stimulation of microglia, transactivation of KCa3.1 channels by CRAC/Orai1, and coordinated roles for both channels in store refilling, Ca²⁺ signaling and microglial migration.     

Regulation of Neuronal Cav3.1 Channels by Cyclin-Dependent Kinase 5 (Cdk5)

Low voltage-activated (LVA) T-type Ca²⁺ channels activate in response to subthreshold membrane depolarizations and therefore represent an important source of Ca²⁺ influx near the resting membrane potential. In neurons, these proteins significantly contribute to control relevant physiological processes including neuronal excitability, pacemaking and post-inhibitory rebound burst firing. Three subtypes of T-type channels (Ca_v3.1 to Ca_v3.3) have been identified, and using functional expression of recombinant channels diverse studies have validated the notion that T-type Ca²⁺ channels can be modulated by various endogenous ligands as well as by second messenger pathways. In this context, the present study reveals a previously unrecognized role for cyclin-dependent kinase 5 (Cdk5) in the regulation of native T-type channels in N1E-115 neuroblastoma cells, as well as recombinant Ca_v3.1channels heterologously expressed in HEK-293 cells. Cdk5 and its co-activators play critical roles in the regulation of neuronal differentiation, cortical lamination, neuronal cell migration and axon outgrowth. Our results show that overexpression of Cdk5 causes a significant increase in whole cell patch clamp currents through T-type channels in N1E-115 cells, while siRNA knockdown of Cdk5 greatly reduced these currents. Consistent with this, overexpression of Cdk5 in HEK-293 cells stably expressing Ca_v3.1channels upregulates macroscopic currents. Furthermore, using site-directed mutagenesis we identified a major phosphorylation site at serine 2234 within the C-terminal region of the Ca_v3.1subunit. These results highlight a novel role for Cdk5 in the regulation of T-type Ca²⁺ channels.     

MAL regulates clathrin-mediated endocytosis at the apical surface of Madin-Darby canine kidney cells

MAL is an integral protein component of the machinery for apical transport in epithelial Madin-Darby canine kidney (MDCK) cells. To maintain its distribution, MAL cycles continuously between the plasma membrane and the Golgi complex. The clathrin-mediated route for apical internalization is known to differ from that at the basolateral surface. Herein, we report that MAL depends on the clathrin pathway for apical internalization. Apically internalized polymeric Ig receptor (pIgR), which uses clathrin for endocytosis, colocalized with internalized MAL in the same apical vesicles. Time-lapse confocal microscopic analysis revealed cotransport of pIgR and MAL in the same endocytic structures. Immunoelectron microscopic analysis evidenced colabeling of MAL with apically labeled pIgR in pits and clathrin-coated vesicles. Apical internalization of pIgR was abrogated in cells with reduced levels of MAL, whereas this did not occur either with its basolateral entry or the apical internalization of glycosylphosphatidylinositol-anchored proteins, which does not involve clathrin. Therefore, MAL is critical for efficient clathrin-mediated endocytosis at the apical surface in MDCK cells.     

Lipopolysaccharide induces cell volume increase and migration of dendritic cells

Migration of dendritic cells (DCs) plays an important role in T‐cell‐mediated adaptive immune responses. Lipopolysaccharide (LPS) sensed by Toll‐like receptor 4 (TLR4) serves as a signal for DC migration. We analyzed LPS‐induced DC volume changes preceding the directed movement towards chemoattractants. Treatment with LPS resulted in rapid, prolonged cell swelling in wild‐type (WT), but not in TLR4^?/? bone marrow‐derived (BM) DCs indicating that TLR4 signaling is essential for LPS‐induced swelling. As a consequence, LPS‐treatment enhanced the migratory activity along a chemokine (CCL21)‐gradient in WT, but not in TLR4‐deficient BMDCs suggesting that the LPS/TLR4‐induced swelling response facilitates DC migration. Moreover, the role of calcium‐activated potassium channels (K_Ca3.1) as putative regulators of immune cell volume regulation and migration was analyzed in LPS‐challenged BMDCs. We found that the LPS‐induced swelling of K_Ca3.1‐deficient DCs was impaired when compared to WT DCs. Accordingly, the LPS‐induced increase in [Ca²⁺]_i detected in WT DCs was reduced in K_Ca3.1‐deficient DCs. Finally, directed migration of LPS‐challenged K_Ca3.1‐deficient DCs was low compared to WT DCs indicating that activation of K_Ca3.1 is involved in LPS‐induced DC migration. These findings suggest that both TLR4 and K_Ca3.1 contribute to the migration of LPS‐activated DCs as an important feature of the adaptive immune response.

     

A Screen for Endocytic Motifs

Sorting signals for cargo selection into coated vesicles are usually in the form of short linear motifs. Three motifs for clathrin‐mediated endocytosis have been identified: YXXΦ, [D/E]XXXL[L/I] and FXNPXY. To search for new endocytic motifs, we made a library of CD8 chimeras with random sequences in their cytoplasmic tails, and used a novel fluorescence‐activated cell sorting (FACS)‐based assay to select for endocytosed constructs. Out of the five tails that were most efficiently internalized, only one was found to contain a conventional motif. Two contain dileucine‐like sequences that appear to be variations on the [D/E]XXXL[L/I] motif. Another contains a novel internalization signal, YXXXΦN, which is able to function in cells expressing a mutant µ2 that cannot bind YXXΦ, indicating that it is not a variation on the YXXΦ motif. Similar sequences are present in endogenous proteins, including a functional YXXXΦN (in addition to a classical YXXΦ) in cytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4). Thus, the repertoire of endocytic motifs is more extensive than the three well‐characterized sorting signals.     

Potential role for a novel AP180-related protein during endocytosis in MDCK cells

Clathrin assembly protein, AP180, was originally identified as a brain-specific protein localized to the presynaptic junction. AP180 acts to limit vesicle size and maintain a pool of releasable synaptic vesicles during rapid recycling. In this study, we show that polarized epithelial Madin-Darby canine kidney (MDCK) cells express two AP180-related proteins: the ubiquitously expressed 62-kDa clathrin assembly lymphoid myeloid leukemia (CALM, AP180-2) protein and a novel high-molecular-weight homolog that we have named AP180-3. Sequence analysis of AP180-3 expressed in MDCK cells shows high homology to AP180 from rat brain. AP180-3 contains conserved motifs found in brain-specific AP180, including the epsin NH₂-terminal homology (ENTH) domain, the binding site for the -subunit of AP-2, and DLL repeats. Our studies show that AP180-3 from MDCK cells forms complexes with AP-2 and clathrin and that membrane recruitment of these complexes is modulated by phosphorylation. We demonstrate by immunohistochemistry that AP180-3 is localized to cytoplasmic vesicles in MDCK cells and is also present in tubule epithelial cells from mouse kidney. We observed by immunodetection that a high-molecular-weight AP180-related protein is expressed in numerous cells in addition to MDCK cells. clathrin assembly lympoid myeloid leukemia; kidney epithelial cells; epsin NH₂-terminal homology domain; DLL repeats; clathrin; AP-2     

The dileucine motif within the tail of MPR46 is required for sorting of the receptor in endosomes

The cytoplasmic tail of MPR46 carries a leucine-based motif that is required for the sorting of lysosomal enzymes by the receptor. In addition, it is one of three independent, but functionally redundant, internalization signals present in the cytoplasmic tail of MPR46. We have analyzed a mutant of MPR46, in which the dileucine pair was replaced by alanines (MPR46 LL/AA) with respect to its intracellular distribution and trafficking. Ultrastructural analysis of cells expressing the MPR46 LL/AA mutant revealed that the substitution of the dileucine pair causes a shift of the receptor distribution from the TGN, where it is packaged into AP1-containing vesicles, to vesicular structures distributed throughout the cytoplasm. The vesicles could be identified as early endosomes with internalized BSA-gold and rab5 as markers. By analyzing the receptor trafficking biochemically, we found that return of the LL/AA mutant receptor from the plasma membrane/endosome pool back to the TGN was impaired, while recycling from endosomes to the plasma membrane was enhanced. In conclusion, our data indicate that the dileucine motif in the MPR46 tail is required for a sorting event in endosomes.     

Basolateral sorting of chloride channel 2 is mediated by interactions between a dileucine motif and the clathrin adaptor AP-1

In spite of the many key cellular functions of chloride channels, the mechanisms that mediate their subcellular localization are largely unknown. ClC-2 is a ubiquitous chloride channel usually localized to the basolateral domain of epithelia that regulates cell volume, ion transport, and acid–base balance; mice knocked out for ClC-2 are blind and sterile. Previous work suggested that CLC-2 is sorted basolaterally by TIFS⁸¹²LL, a dileucine motif in CLC-2''s C-terminal domain. However, our in silico modeling of ClC-2 suggested that this motif was buried within the channel''s dimerization interface and identified two cytoplasmically exposed dileucine motifs, ESMI⁶²³LL and QVVA⁶³⁵LL, as candidate sorting signals. Alanine mutagenesis and trafficking assays support a scenario in which ESMI⁶²³LL acts as the authentic basolateral signal of ClC-2. Silencing experiments and yeast three-hybrid assays demonstrated that both ubiquitous (AP-1A) and epithelium-specific (AP-1B) forms of the tetrameric clathrin adaptor AP-1 are capable of carrying out basolateral sorting of ClC-2 through interactions of ESMI⁶²³LL with a highly conserved pocket in their γ1-σ1A hemicomplex.     

An Acidic Cluster in the Cytosolic Domain of Human Cytomegalovirus Glycoprotein B Is a Signal for Endocytosis from the Plasma Membrane

We previously reported that human cytomegalovirus (CMV) glycoprotein B (gB) is transported to apical membranes in CMV-infected polarized retinal pigment epithelial (ARPE-19) cells and in Madin-Darby canine kidney (MDCK) epithelial cells constitutively expressing gB. The cytosolic domain of gB contains a cluster of acidic amino acids, a motif that plays a pivotal role in vectorial trafficking in polarized epithelial cells and may also function as a signal for entry into the endocytic pathway. Here we compared gB internalization and recycling to the plasma membrane in CMV-infected human fibroblasts (HF) and ARPE-19 cells by using antibody-internalization experiments. Immunofluorescence and quantitative assays showed that gB was internalized from the cell surface into clathrin-coated transport vesicles and then recycled to the plasma membrane. gB colocalized with clathrin-coated vesicles containing the transferrin receptor in the early endocytic/recycling pathway, indicating that gB traffics in this pathway. The specific role of the acidic cluster in regulating the sorting of gB-containing vesicles in the early endocytic/recycling pathway was examined in MDCK cells expressing mutated gB derivatives. Immunofluorescence assays showed that derivatives lacking the acidic cluster were impaired in internalization and failed to recycle. These findings, together with our earlier observation that the acidic cluster is a key determinant for targeting gB molecules to apical membranes in epithelial cells, establish that this signal is recognized by cellular proteins that participate in polarized sorting and transport in the early endocytic/recycling pathway.     

Effect of tetramethylpyrazine (TMP) on Ca2+ signal transduction and cell viability in a model of renal tubular cells

Tetramethylpyrazine (TMP) is a compound purified from herb. Its effect on Ca²⁺ concentrations ([Ca²⁺]_i) in renal cells is unclear. This study examined whether TMP altered Ca²⁺ signaling in Madin‐Darby canine kidney (MDCK) cells. TMP at 100–800 μM induced [Ca²⁺]_i rises, which were reduced by Ca²⁺ removal. TMP induced Mn²⁺ influx implicating Ca²⁺ entry. TMP‐induced Ca²⁺ entry was inhibited by 30% by modulators of protein kinase C (PKC) and store‐operated Ca²⁺ channels. Treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5‐di‐tert‐butylhydroquinone (BHQ) inhibited 93% of TMP‐evoked [Ca²⁺]_i rises. Treatment with TMP abolished BHQ‐evoked [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) abolished TMP‐induced responses. TMP at 200–1000 μM decreased viability, which was not reversed by pretreatment with the Ca²⁺ chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid‐acetoxymethyl ester. Together, in MDCK cells, TMP induced [Ca²⁺]_i rises by evoking PLC‐dependent Ca²⁺ release from endoplasmic reticulum and Ca²⁺ entry via PKC‐sensitive store‐operated Ca²⁺ entry. TMP also caused Ca²⁺‐independent cell death.     

Structural Determinants of the High Affinity Extracellular Zinc Binding Site on Cav3.2 T-type Calcium Channels

Ca_v3.2 T-type channels contain a high affinity metal binding site for trace metals such as copper and zinc. This site is occupied at physiologically relevant concentrations of these metals, leading to decreased channel activity and pain transmission. A histidine at position 191 was recently identified as a critical determinant for both trace metal block of Ca_v3.2 and modulation by redox agents. His¹⁹¹ is found on the extracellular face of the Ca_v3.2 channel on the IS3-S4 linker and is not conserved in other Ca_v3 channels. Mutation of the corresponding residue in Ca_v3.1 to histidine, Gln¹⁷², significantly enhances trace metal inhibition, but not to the level observed in wild-type Ca_v3.2, implying that other residues also contribute to the metal binding site. The goal of the present study is to identify these other residues using a series of chimeric channels. The key findings of the study are that the metal binding site is composed of a Asp-Gly-His motif in IS3–S4 and a second aspartate residue in IS2. These results suggest that metal binding stabilizes the closed conformation of the voltage-sensor paddle in repeat I, and thereby inhibits channel opening. These studies provide insight into the structure of T-type channels, and identify an extracellular motif that could be targeted for drug development.     

An N-terminal dileucine motif directs two-pore channels to the tonoplast of plant cells

Two‐pore channels (TPCs) constitute a family of endolysosomal cation channels with functions in Ca²⁺ signaling. We used a mutational analysis to investigate the role of channel domains for the trafficking of the Arabidopsis TPC1 to the tonoplast, a process that is generally not well understood in plants. The results show that the soluble C‐terminus was not essential for targeting but for channel function, while further C‐terminal truncations of two or more transmembrane domains impaired protein trafficking. An N‐terminal dileucine motif (EDPLI) proved to be critical for vacuolar targeting of TPC1, which was independent of the adaptor protein AP‐3. Deletion or mutation of this sorting motif, which is conserved among TPCs caused redirection of the protein transport to the plasma membrane. An N‐terminal region with a predicted α‐helical structure was shown to support efficient vacuolar trafficking and was essential for TPC1 function. Similar to their localization in mammalian endosomes and lysosomes, MmTPC1 and MmTPC2 were targeted to small organelles and the membrane of the lytic vacuole, respectively, when expressed in plant cells. These results shed new light on the largely uncharacterized sorting signals of plant tonoplast proteins and reveal similarities between the targeting machinery of plants and mammals.