RESUMEN
TRPM3 channels play important roles in the detection of noxious heat and in inflammatory thermal hyperalgesia. The activity of these ion channels in somatosensory neurons is tightly regulated by µ-opioid receptors through the signaling of Gßγ proteins, thereby reducing TRPM3-mediated pain. We show here that Gßγ directly binds to a domain of 10 amino acids in TRPM3 and solve a cocrystal structure of this domain together with Gßγ. Using these data and mutational analysis of full-length proteins, we pinpoint three amino acids in TRPM3 and their interacting partners in Gß1 that are individually necessary for TRPM3 inhibition by Gßγ. The 10-amino-acid Gßγ-interacting domain in TRPM3 is subject to alternative splicing. Its inclusion in or exclusion from TRPM3 channel proteins therefore provides a mechanism for switching on or off the inhibitory action that Gßγ proteins exert on TRPM3 channels.
Asunto(s)
Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/farmacología , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/farmacología , Canales Catiónicos TRPM/química , Canales Catiónicos TRPM/efectos de los fármacos , Canales Catiónicos TRPM/metabolismo , Sitios de Unión , Calcio/metabolismo , Subunidades beta de la Proteína de Unión al GTP/química , Subunidades gamma de la Proteína de Unión al GTP/química , Células HEK293 , Humanos , Hiperalgesia/metabolismo , Modelos Moleculares , Mutación , Neuronas/metabolismo , Dolor/metabolismo , Receptores Opioides/metabolismo , Canales Catiónicos TRPM/genéticaRESUMEN
TRPM3 channels form ionotropic steroid receptors in the plasma membrane of pancreatic ß and dorsal root ganglion cells and link steroid hormone signaling to insulin release and pain perception, respectively. We identified and compared the function of a number of TRPM3 splice variants present in mouse, rat and human tissues. We found that variants lacking a region of 18 amino acid residues display neither Ca(2+) entry nor ionic currents when expressed alone. Hence, splicing removes a region that is indispensable for channel function, which is called the ICF region. TRPM3 variants devoid of this region (TRPM3ΔICF), are ubiquitously present in different tissues and cell types where their transcripts constitute up to 15% of the TRPM3 isoforms. The ICF region is conserved throughout the TRPM family, and its presence in TRPM8 proteins is also necessary for function. Within the ICF region, 10 amino acid residues form a domain essential for the formation of operative TRPM3 channels. TRPM3ΔICF variants showed reduced interaction with other TRPM3 isoforms, and their occurrence at the cell membrane was diminished. Correspondingly, coexpression of ΔICF proteins with functional TRPM3 subunits not only reduced the number of channels but also impaired TRPM3-mediated Ca(2+) entry. We conclude that TRPM3ΔICF variants are regulatory channel subunits fine-tuning TRPM3 channel activity.
Asunto(s)
Empalme Alternativo , Canales Catiónicos TRPM/genética , Secuencia de Aminoácidos , Animales , Señalización del Calcio , Secuencia Conservada , Exones , Células HEK293 , Humanos , Inmunoprecipitación , Potenciales de la Membrana , Ratones , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sitios de Empalme de ARN , Ratas , Homología de Secuencia de Aminoácido , Canales Catiónicos TRPM/química , Canales Catiónicos TRPM/metabolismoRESUMEN
N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenyl propionamide is a newly-designed pain killer selectively activating G-protein-coupled mu-opioid receptors (MOR) in acidic injured tissues, and therefore devoid of central side effects which are typically elicited at normal pH values in healthy tissues. However, the neuronal mechanisms underlying NFEPP's antinociceptive effects were not examined in detail so far. Voltage-dependent Ca2+ channels (VDCCs) in nociceptive neurons play a major role in the generation and inhibition of pain. In this study, we focused on the effects of NFEPP on calcium currents in rat dorsal root ganglion (DRG) neurons. The inhibitory role of the G-protein subunits Gi/o and Gßγ on VDCCs was investigated using the blockers pertussis toxin and gallein, respectively. GTPγS binding, calcium signals and MOR phosphorylation were also investigated. All experiments were performed at acidic and normal pH values using NFEPP in comparison to the conventional opioid agonist fentanyl. At low pH, NFEPP produced more efficient G-protein activation in transfected HEK293 cells and significantly reduced VDCCs in depolarized DRG neurons. The latter effect was mediated by Gßγ subunits, and NFEPP-mediated MOR phosphorylation was pH-dependent. Fentanyl's responses were not affected by pH changes. Our data indicate that NFEPP-induced MOR signaling is more effective at low pH and that the inhibition of calcium channels in DRG neurons underlies NFEPP's antinociceptive actions.
RESUMEN
TRPM3 proteins assemble to Ca2+-permeable cation channels in the plasma membrane, which act as nociceptors of noxious heat and mediators of insulin and cytokine release. Here we show that TRPM3 channel activity is strongly dependent on intracellular Ca2+. Conceivably, this effect is attributed to the Ca2+ binding protein calmodulin, which binds to TRPM3 in a Ca2+-dependent manner. We identified five calmodulin binding sites within the amino terminus of TRPM3, which displayed different binding affinities in dependence of Ca2+. Mutations of lysine residues in calmodulin binding site 2 strongly reduced calmodulin binding and TRPM3 activity indicating the importance of this domain for TRPM3-mediated Ca2+ signaling. Our data show that TRPM3 channels are regulated by intracellular Ca2+ and provide the basis for a mechanistic understanding of the regulation of TRPM3 by calmodulin.
Asunto(s)
Calcio/metabolismo , Calmodulina/metabolismo , Canales Catiónicos TRPM/metabolismo , Secuencia de Aminoácidos , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Calmodulina/genética , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Fotólisis/efectos de los fármacos , Canales Catiónicos TRPM/genéticaRESUMEN
Opioids, agonists of µ-opioid receptors (µORs), are the strongest pain killers clinically available. Their action includes a strong central component, which also causes important adverse effects. However, µORs are also found on the peripheral endings of nociceptors and their activation there produces meaningful analgesia. The cellular mechanisms downstream of peripheral µORs are not well understood. Here, we show in neurons of murine dorsal root ganglia that pro-nociceptive TRPM3 channels, present in the peripheral parts of nociceptors, are strongly inhibited by µOR activation, much more than other TRP channels in the same compartment, like TRPV1 and TRPA1. Inhibition of TRPM3 channels occurs via a short signaling cascade involving Gßγ proteins, which form a complex with TRPM3. Accordingly, activation of peripheral µORs in vivo strongly attenuates TRPM3-dependent pain. Our data establish TRPM3 inhibition as important consequence of peripheral µOR activation indicating that pharmacologically antagonizing TRPM3 may be a useful analgesic strategy.
Asunto(s)
Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/farmacología , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/farmacología , Receptores Opioides mu/metabolismo , Canales Catiónicos TRPM/efectos de los fármacos , Analgésicos Opioides/agonistas , Animales , Escala de Evaluación de la Conducta , Calcio/metabolismo , Señalización del Calcio/fisiología , Ganglios Espinales/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Nociceptores/fisiología , Dolor/metabolismo , Receptores Opioides/metabolismo , Canal Catiónico TRPA1/metabolismo , Canales Catiónicos TRPV/metabolismoRESUMEN
Transient receptor potential A1 channels are well-known as chemosensors in neuronal cells. However, recent studies also point to non-neuronal functions in epithelia. Here, we show that TRPA1 channels are expressed in epithelial MDCK II cells and contribute to Ca(2+) influx and whole-cell currents after stimulation with AITC. Stimulation of TRPA1 channels induced an immediate reduction of the transepithelial resistance of MDCK II cell layers that was blocked by the TRPA1 antagonist HC-030031. The present data provide strong evidence for a new role of TRPA1 channels in regulating the tightness of epithelial cell barriers.
Asunto(s)
Células Epiteliales/metabolismo , Canales de Potencial de Receptor Transitorio/genética , Canales de Potencial de Receptor Transitorio/metabolismo , Animales , Calcio/metabolismo , Perros , Isotiocianatos/farmacología , Células de Riñón Canino Madin DarbyRESUMEN
BACKGROUND AND PURPOSE: Signalling through phospholipase C (PLC) controls many cellular processes. Much information on the relevance of this important pathway has been derived from pharmacological inhibition of the enzymatic activity of PLC. We found that the most frequently employed PLC inhibitor, U73122, activates endogenous ionic currents in widely used cell lines. Given the extensive use of U73122 in research, we set out to identify these U73122-sensitive ion channels. EXPERIMENTAL APPROACH: We performed detailed biophysical analysis of the U73122-induced currents in frequently used cell lines. KEY RESULTS: At concentrations required to inhibit PLC, U73122 modulated the activity of transient receptor potential melastatin (TRPM) channels through covalent modification. U73122 was shown to be a potent agonist of ubiquitously expressed TRPM4 channels and activated endogenous TRPM4 channels in CHO cells independently of PLC and of the downstream second messengers PI(4,5)P2 and Ca(2+) . U73122 also potentiated Ca(2) (+) -dependent TRPM4 currents in human Jurkat T-cells, endogenous TRPM4 in HEK293T cells and recombinant human TRPM4. In contrast to TRPM4, TRPM3 channels were inhibited whereas the closely related TRPM5 channels were insensitive to U73122, showing that U73122 exhibits high specificity within the TRPM channel family. CONCLUSIONS AND IMPLICATIONS: Given the widespread expression of TRPM4 and TRPM3 channels, these actions of U73122 must be considered when interpreting its effects on cell function. U73122 may also be useful for identifying and characterizing TRPM channels in native tissue, thus facilitating the analysis of their physiology.