Alternative Splicing of a Protein Domain Indispensable for Function of Transient Receptor Potential Melastatin 3 (TRPM3) Ion Channels

Frühwald, Julia; Londoño, Julia Camacho; Dembla, Sandeep; Mannebach, Stefanie; Lis, Annette; Drews, Anna; Wissenbach, Ulrich; Oberwinkler, Johannes; Philipp, Stephan E.

doi:10.1074/jbc.m112.396663

Cited by 70 publications

(85 citation statements)

References 34 publications

(36 reference statements)

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“…Thus far, the splice variants of numerous ion channels, including TRP channels, have been described (13,14). The role of these TRP-channel splice variants in biological behavior is becoming increasingly clear; however, at present, there is little information regarding the functions of TRPM8 isoforms in the field of carcinogenesis and cancer progression.…”

Section: Discussionmentioning

confidence: 99%

Overexpression of short TRPM8 variant α promotes cell migration and invasion, and decreases starvation-induced apoptosis in prostate cancer LNCaP cells

et al. 2015

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Abstract. The aim of the present study was to investigate the function of a transient receptor potential melastatin 8 (TRPM8) splice variant, short TRMP8α (sM8α), in the androgen-dependent prostate cancer LNCaP cell line, and to evaluate the potential involvement of the mitogen-activated protein kinase (MAPK) signaling pathway. The coding DNA for sM8α was cloned and transfected into LNCaP cells to generate cells that overexpress this isoform of TRPM8. Cellular proliferation was determined by performing an MTT assay, and flow cytometry was used to analyze apoptosis and cell cycle distribution. Furthermore, cellular migration and invasion were evaluated using Transwell ® migration assays. The subcellular location of recombinant sM8α was detected by quantum dots-based immunofluorescent imaging, western blotting was performed to examine the expression levels of proteins in the MAPK signaling pathway and reverse transcription-polymerase chain reaction was used to determine the expression of sM8α mRNA transcripts. The present study demonstrated that sM8α mRNA was expressed at a low level in the LNCaP, DU145 and PC-3 prostate cancer cell lines. Additionally, the recombinant sM8α protein was located in the cytoplasm of LNCaP cells and its overexpression significantly reduced starvation-induced apoptosis in these cells (P<0.05), possibly by means of reduced activation of phosphorylated-c-Jun N-terminal kinase (p-JNK). The migration and invasion of the LNCaP cells were markedly enhanced by the overexpression of sM8α, possibly via activation of MMP-2. Furthermore, overexpression of sM8α in LNCaP cells did not alter the expression of full-length TRPM8 and had no effect on cellular proliferation. Overall, the results of the present study indicate that sM8α may be important in the regulation of prostate cancer cell migration and invasion through the activation of matrix metalloproteinase-2, as well as in the regulation of apoptosis through the activation of p-JNK in the MAPK signaling pathway.

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Section: Discussionmentioning

confidence: 99%

Overexpression of short TRPM8 variant α promotes cell migration and invasion, and decreases starvation-induced apoptosis in prostate cancer LNCaP cells

et al. 2015

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“…Different splice variants that impede on the Ca 21 permeability of the pore (e.g., the a1 splicing event) (Oberwinkler et al, 2005) or even eliminate a functional pore (Frühwald et al, 2012) further complicate the situation. In TRPM3-deficient transgenic mice, phenotypic alterations hint at a role of TRPM3 in thermal pain perception (Vriens et al, 2011), an effect that is closely mimicked by a pharmacologic block of the channel, as described in the present study.…”

Section: Discussionmentioning

confidence: 99%

“…Human embryonic kidney 293 (HEK293) cells used for transient transfection were cultured in Earle's minimum essential medium (Invitrogen/Life Technologies, Carlsbad, CA), supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 units×ml 21 penicillin, and 0.1 mg×ml 21 streptomycin. HEK293 cells stably expressing myc-tagged mouse TRPM3a2 (HEK mTRPM3 ) were obtained and maintained as described elsewhere (Frühwald et al, 2012;Straub et al, 2013). HEK293 cells stably expressing other sensory transient receptor potential (TRP) channels (HEK hTRPA1 , HEK hTRPM8:CFP , HEK rTRPV1:YFP ) were obtained by a limiting dilution method, as described recently elsewhere (Hellwig et al, 2004;Urban et al, 2012).…”

Section: Methodsmentioning

confidence: 99%

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Flavanones That Selectively Inhibit TRPM3 Attenuate Thermal Nociception In Vivo

et al. 2013

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Transient receptor potential melastatin 3 (TRPM3) is a calciumpermeable nonselective cation channel that is expressed in a subset of dorsal root (DRG) and trigeminal ganglia sensory neurons. TRPM3 can be activated by the neurosteroid pregnenolone sulfate (PregS) and heat. TRPM3 2/2 mice display an impaired sensation of noxious heat and thermal hyperalgesia. We have previously shown that TRPM3 is blocked by the citrus fruit flavanones hesperetin, naringenin, and eriodictyol as well as by ononetin, a deoxybenzoin from Ononis spinosa. To further improve the tolerability, potency, and selectivity of TRPM3 blockers, we conducted a hit optimization procedure by rescreening a focused library that was composed of chemically related compounds. Within newly identified TRPM3 blockers, isosakuranetin and liquiritigenin displayed favorable properties with respect to their inhibitory potency and a selective mode of action. Isosakuranetin, a flavanone whose glycoside is contained in blood oranges and grapefruits, displayed an IC 50 of 50 nM and is to our knowledge the most potent inhibitor of TRPM3 identified so far. Both compounds exhibited a marked specificity for TRPM3 compared with other sensory TRP channels, and blocked PregS-induced intracellular free Ca 21 concentration signals and ionic currents in freshly isolated DRG neurons. Furthermore, isosakuranetin and previously identified hesperetin significantly reduced the sensitivity of mice to noxious heat and PregS-induced chemical pain. Because the physiologic functions of TRPM3 channels are still poorly defined, the development and validation of potent and selective blockers is expected to contribute to clarifying the role of TRPM3 in vivo.

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“…Exon 1 and exon 2 appear to be expressed in a mutually exclusive way, as no cDNA clones have been described having both of these exons. The different promoters may regulate expression of different isoforms called TRPM3α (α1-α5) starting with exon 1 and lacking exon 2 (Oberwinkler et al 2005), isoforms called TRPM3β (β1-β 17) starting with exon 2 (Frühwald et al 2012;Grimm et al 2003), and finally isoforms starting with a ATG codon located at the very end of exon 4 which is the start codon for transcripts starting with exon 3 (Fig. 1b, c; Lee et al 2003).…”

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confidence: 99%

Trpm3

Oberwinkler

Philipp

2014

Handbook of Experimental Pharmacology

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108

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Like most other members of the TRP family, the Trpm3 gene encodes proteins that form cation-permeable ion channels on the plasma membrane. However, TRPM3 proteins have several unique features that set them apart from the other members of this diverse family. The Trpm3 gene encodes for a surprisingly large number of isoforms generated mainly by alternative splicing. Only for two of the (at least) eight sites at which sequence diversity is generated the functional consequences have been elucidated, one leading to nonfunctional channels, the other one profoundly affecting the ionic selectivity. In the Trpm3 gene an intronic microRNA (miR-204) is co-transcribed with Trpm3. By regulating the expression of a multitude of genes, miR-204 increases the functional complexity of the Trpm3 locus. Over the past years, important progress has been made in discovering pharmacological tools to manipulate TRPM3 channel activity. These substances have facilitated the identification of endogenously expressed functional TRPM3 channels in nociceptive neurons, pancreatic beta cells, and vascular smooth muscle cells, among others. TRPM3 channels, which themselves are temperature sensitive, thus have been implicated in sensing noxious heat, in modulating insulin release, and in secretion of inflammatory cytokines. However, in many tissues where TRPM3 proteins are known to be expressed, no functional role has been identified for these channels so far. Because of the availability of adequate pharmacological and genetic tools, it is expected that future investigations on TRPM3 channels will unravel important new aspects and functions of these channels.

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Alternative Splicing of a Protein Domain Indispensable for Function of Transient Receptor Potential Melastatin 3 (TRPM3) Ion Channels

Cited by 70 publications

References 34 publications

Overexpression of short TRPM8 variant α promotes cell migration and invasion, and decreases starvation-induced apoptosis in prostate cancer LNCaP cells

Overexpression of short TRPM8 variant α promotes cell migration and invasion, and decreases starvation-induced apoptosis in prostate cancer LNCaP cells

Flavanones That Selectively Inhibit TRPM3 Attenuate Thermal Nociception In Vivo

Trpm3

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