The calcium-conducting ion channel transient receptor potential canonical 6 is involved in macrophage inflammatory protein-2-induced migration of mouse neutrophils.

AIM: The role of the calcium-conducting ion channel transient receptor potential canonical 6 (TRPC6) in macrophage inflammatory protein-2 (MIP-2) induced migration of mouse neutrophils was investigated. METHODS: Neutrophil granulocytes isolated from murine bone marrow of wild-type (TRPC6+/+) and TRPC6 knockout (TRPC6)/)) mice were tested for the presence of TRPC6 channel expression using quantitative real-time polymerase chain reactions and immunocytochemistry. The effect of different stimuli (e.g. MIP-2, 1-oleoyl-2-acetyl-sn-glycerol, formyl-methionyl-leucyl-phenylalanin) on migration of isolated neutrophils was tested by two-dimensional (2D) migration assays, phalloidin staining and intracellular calcium imaging. RESULTS: We found that neutrophil granulocytes express TRPC6 channels. MIP-2 induced fast cell migration of isolated neutrophils in a 2D celltracking system. Strikingly, MIP-2 was less potent in neutrophils derived from TRPC6)/) mice. These cells showed less phalloidin-coupled fluorescence and the pattern of cytosolic calcium transients was altered. CONCLUSIONS: We describe in this paper for the first time a role for transient receptor potential (TRP) channels in migration of native lymphocytes as a new paradigm for the universal functional role of TRPs. Our data give strong evidence that TRPC6 operates downstream to CXC-type Gq-protein-coupled chemokine receptors upon stimulation with MIP-2 and is crucial for the arrangement of filamentous actin in migrating neutrophils. This is a novel cell function of TRP channel beyond their well-recognized role as universal cell sensors.

TRPM8.

Originally cloned as a prostate-specific protein, TRPM8 is now best known as a cold- and menthol-activated channel implicated in thermosensation. In this chapter we provide a brief review of current knowledge concerning the biophysical properties, gating mechanisms, pharmacology and (patho)physiology of this TRP channel.

A novel function of capsaicin-sensitive TRPV1 channels: involvement in cell migration.

Cell migration relies on a tight temporal and spatial regulation of the intracellular Ca2+ concentration ([Ca2+]i). [Ca2+]i in turn depends on Ca2+ influx via channels in the plasma membrane whose molecular nature is still largely unknown for migrating cells. A mechanosensitive component of the Ca2+ influx pathway was suggested. We show here that the capsaicin-sensitive transient receptor potential channel TRPV1, that plays an important role in pain transduction, is one of the Ca2+ influx channels involved in cell migration. Activating TRPV1 channels with capsaicin leads to an acceleration of human hepatoblastoma (HepG2) cells pretreated with hepatocyte growth factor (HGF). The speed rises by up to 50% and the displacement is doubled. Patch clamp experiments revealed the presence of capsaicin and resiniferatoxin (RTX)-sensitive currents. In contrast, HepG2 cells kept in the absence of HGF are not accelerated by capsaicin and express no capsaicin- or RTX-sensitive current. The TRPV1 antagonist capsazepine prevents the stimulation of migration and inhibits capsaicin-sensitive currents. Finally, we compared the contribution of capsaicin-sensitive TRPV1 channels to cell migration with that of mechanosensitive TRPV4 channels that are also expressed in HepG2 cells. A specific TRPV4 agonist, 4alpha-phorbol 12,13-didecanoate, does not increase the displacement. In summary, we assigned a novel role to capsaicin-sensitive TRPV1 channels. They are important Ca2+ influx channels required for cell migration.

Structure-function relationship of the TRP channel superfamily.

Transient receptor potential (TRP) channels are involved in the perception of a wide range of physical and chemical stimuli, including temperature and osmolarity changes, light, pain, touch, taste and pheromones, and in the initiation of cellular responses thereupon. Since the last decade, rapid progress has been made in the identification and characterization of new members of the TRP superfamily. They constitute a large superfamily of cation channels that are expressed in almost all cell types in both invertebrates and vertebrates. This review summarizes and discusses the current knowledge on the TRP protein structure and its impact on the regulation of the channel function.

Modulation of the Ca2 permeable cation channel TRPV4 by cytochrome P450 epoxygenases in vascular endothelium.

TRPV4 is a broadly expressed Ca2+-permeable cation channel in the vanilloid subfamily of transient receptor potential channels. TRPV4 gates in response to a large variety of stimuli, including cell swelling, warm temperatures, the synthetic phorbol ester 4alpha-phorbol 12,13-didecanoate (4alpha-PDD), and the endogenous lipid arachidonic acid (AA). Activation by cell swelling and AA requires cytochrome P450 (CYP) epoxygenase activity to convert AA to epoxyeicosatrienoic acids (EETs) such as 5,6-EET, 8,9-EET, which both act as direct TRPV4 agonists. To evaluate the role of TRPV4 and its modulation by the CYP pathway in vascular endothelial cells, we performed Ca2+ imaging and patch-clamp measurements on mouse aortic endothelial cells (MAECs) isolated from wild-type and TRPV4(-/-) mice. All TRPV4-activating stimuli induced robust Ca2+ responses in wild-type MAECs but not in MAECs isolated from TRPV4(-/-) mice. Upregulation of CYP2C expression by preincubation with nifedipine enhanced the responses to AA and cell swelling in wild-type MAECs, whereas responses to other stimuli remained unaffected. Conversely, inhibition of CYP2C9 activity with sulfaphenazole abolished the responses to AA and hypotonic solution (HTS). Moreover, suppression of EET hydrolysis using 1-adamantyl-3-cyclo-hexylurea or indomethacin, inhibitors of soluble epoxide hydrolases (sEHs), and cyclooxygenases, respectively, enhanced the TRPV4-dependent responses to AA, HTS, and EETs but not those to 4alpha-PDD or heat. Together, our data establish that CYP-derived EETs modulate the activity of TRPV4 channels in endothelial cells and shows the unraveling of novel modulatory pathways via CYP2C modulation and sEH inhibition.

Casein kinase I-dependent phosphorylation and stability of the yeast multidrug transporter Pdr5p.

The pleiotropic drug resistance protein, Pdr5p, is an ATP-binding cassette transporter of the plasma membrane of Saccharomyces cerevisiae. Overexpression of Pdr5p results in increased cell resistance to a variety of cytotoxic compounds, a phenotype reminiscent of the multiple drug resistance seen in tumor cells. Pdr5p and two other yeast ATP-binding cassette transporters, Snq2p and Yor1p, were found to be phosphorylated on serine residues in vitro. Mutations in the plasma membrane-bound casein kinase I isoforms, Yck1p and Yck2p, abolished Pdr5p phosphorylation and modified the multiple drug resistance profile. We showed Pdr5p to be ubiquitylated when overexpressed. However, instability of Pdr5p was only seen in Yck1p- and Yck2p-deficient strains, in which it was degraded in the vacuole via a Pep4p-dependent mechanism. Our results suggest that casein kinase I activity is required for membrane trafficking of Pdr5p to the cell surface. In the absence of functional Yck1p and Yck2p, Pdr5p is transported to the vacuole for degradation.

Isolation of three contiguous genes, ACR1, ACR2 and ACR3, involved in resistance to arsenic compounds in the yeast Saccharomyces cerevisiae.

A 4.2 kb region from Saccharomyces cerevisiae chromosome XVI was isolated as a yeast fragment conferring resistance to 7 mM-sodium arsenite (NaAsO2), when put on a multicopy plasmid. Homology searches revealed a cluster of three new open reading frames named ACR1, ACR2 and ACR3. The hypothetical product of the ACR1 gene is similar to the transcriptional regulatory proteins, encoded by YAP1, and YAP2 genes from S. cerevisiae. Disruption of the ACR1 gene conduces to an arsenite and arsenate hypersensitivity phenotype. The ACR2 gene is indispensable for arsenate but not for arsenite resistance. The hypothetical product of the ACR3 gene shows high similarity to the hypothetical membrane protein encoded by Bacillus subtilis ORF1 of the skin element and weak similarity to the ArsB membrane protein of the Staphylococcus aureus arsenical-resistance operon. Overexpression of the ACR3 gene confers an arsenite- but not an arsenate-resistance phenotype. The presence of ACR3 together with ACR2 on a multicopy plasmid expands the resistance phenotype into arsenate. These findings suggest that all three novel genes: ACR1, ACR2 and ACR3 are involved in the arsenical-resistance phenomenon in S. cerevisiae.