Localization and Functional Analysis of the Calcium Permeable Melastatin-like Channel TRPM3

TRPM3 is a highly conserved melastatin-like transient receptor potential (TRP) gene with direct orthologs in all chordates and close homologs in all bilateral animals. So far, little is known about its biological role, activation mechanism, expression pattern and the functional role of its domains....

Ausführliche Beschreibung

Gespeichert in:
1. Verfasser: Chen, Xiaodi
Beteiligte: Plant, Tim (Prof. Dr.) (BetreuerIn (Doktorarbeit))
Format: Dissertation
Sprache:Deutsch
Veröffentlicht: Philipps-Universität Marburg 2009
Pharmakologie und Toxikologie
Schlagworte:
Online Zugang:PDF-Volltext
Tags: Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
Zusammenfassung:TRPM3 is a highly conserved melastatin-like transient receptor potential (TRP) gene with direct orthologs in all chordates and close homologs in all bilateral animals. So far, little is known about its biological role, activation mechanism, expression pattern and the functional role of its domains. The aim of this work was to clone a cDNA of mouse TRPM3, to express it in in vitro overexpression models and evaluate its function, to raise antisera against TRPM3 in order to determine its cellular expression profile in the mouse and, finally, to obtain a knock-out allele in the mouse in order to assess its biological role. 1. In this work, we cloned two cDNAs of mTRPM3, both starting with the unique starting exon we had identifed in a 5-RACE study. One variant is 1337 amino acids long and has a shorter C-terminal domain than the other, which has 1719 amino acid residues. Both splice variants are readily expressable in HEK-293 cells. Plasma membrane localization was greatly augmented by stable inducible expression in differentiated, polarized MDCK cells, where mTRPM31719 resided in the basolateral compartment. When overexpressed in HEK-293 cells, TRPM3 was unresponsive to hypo- or hyperosmolar stimuli, D-erythro-sphingosine and protocols used to elicit store-operated calcium entry. Instead, we observed a robust constitutive calcium entry through TRPM3, which was seen with both splice variants and abolished by point mutations in the pore domain. 2. We confirmed by means of fluorescence resonance energy transfer (FRET) and co-immunoprecipitation that TRPM3, like other TRP channels, forms multimeric channel complexes. The C-terminus of TRPM3 was found to attach to the plasma membrane through a palmitoylation of a dual-cysteine motif, a modification that quantitatively affected calcium entry through TRPM3. In a systematic yeast two hybrid screen of a kidney library against the C-terminus of TRPM3, we identified a number of candidate interaction partners of TRPM3, including alpha-B crystallin and protein kinase inhibitor gamma (PKIG). 3. We investigated the TRPM3 expression pattern by a combination of Northern blots, Western blots and lacZ stainings in a mouse model based on the GeneTrap approach. We found TRPM3 to be mainly enriched in eye and brain tissues, but, unlike human TRPM3, very low levels in kidney. Using lacZ staining of cryostat sections, TRPM3 was detected mainly in sensory tissues like the bipolar and gangion cell layers of the eye, the embryonic ear bubble and dorsal root ganglia. In the brain, a considerable enrichment of TRPM3 gene activity was seen, among others, in various neuron populations of the hippocampus formation (dentate gyrus, CA1, CA3), the Purkinje cells layer, and the neocortex. Thus, TRPM3 is mainly expressed in cells of ectodermal origin. 4. We characterized a mouse model in which TRPM3 had been targeted by a gene trap insertion. We confirmed the gene trap insertion by means of PCR, we confirmed the absence of the protein in eye tissues by means of a Western blot using a newly made peptide rabbit antiserum, and we isogenized the genetical background for skin coloration markers using Mendelian crosses. A basic study of TRPM3s phenotype showed the absence of morphological and overall behavioural defects and complete viability of the mutant. Further assays addressing the visual abilities of the TRPM3-/- mouse showed no signs of blindness. In summary, we cloned cDNAs of TRPM3, expressed them in vitro and found that TRPM3 is calcium-permeable and constitutively active. TRPM3 is palmitoylated on its C-terminus and interacts with a variety of proteins we identified by a yeast two hybrid approach, many of them novel in this regard. TRPM3 is highly enriched in sensory and central nervous tissues. The TRPM3-/- mouse is viable without any major neurological or sensory defect as far as tested. Further investigation on tissues which will have to address the specific function of TRPM3 where it is enriched.