Alternative splicing switches the divalent cation selectivity of TRPM3 channels.
TRPM3 is a poorly understood member of the large family of transient receptor potential (TRP) ion channels. Here we describe five novel splice variants of TRPM3, TRPM3alpha1-5. These variants are characterized by a previously unknown amino terminus of 61 residues. The differences between the five variants arise through splice events at three different sites. One of these splice sites might be located in the pore region of the channel as indicated by sequence alignment with other, better-characterized TRP channels. We selected two splice variants, TRPM3alpha1 and TRPM3alpha2, that differ only in this presumed pore region and analyzed their biophysical characteristics after heterologous expression in human embryonic kidney 293 cells. TRPM3alpha1 as well as TRPM3alpha2 induced a novel, outwardly rectifying cationic conductance that was tightly regulated by intracellular Mg(2+). However, these two variants are highly different in their ionic selectivity. Whereas TRPM3alpha1-encoded channels are poorly permeable for divalent cations, TRPM3alpha2-encoded channels are well permeated by Ca(2+) and Mg(2+). Additionally, we found that currents through TRPM3alpha2 are blocked by extracellular monovalent cations, whereas currents through TRPM3alpha1 are not. These differences unambiguously show that TRPM3 proteins constitute a pore-forming channel subunit and localize the position of the ion-conducting pore within the TRPM3 protein. Although the ionic selectivity of ion channels has traditionally been regarded as rather constant for a given channel-encoding gene, our results show that alternative splicing can be a mechanism to produce channels with very different selectivity profiles.
Oberwinkler
Johannes
J
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Universität des Saarlandes, Gebäude 46, D-66421 Homburg, Germany.
Lis
Annette
A
Giehl
Klaus M
KM
Flockerzi
Veit
V
Philipp
Stephan E
SE
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015152:Blotting, Northern; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry* / Q000378:metabolism; D002412:Cations / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D017403:In Situ Hybridization; D007477:Ions; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008564:Membrane Potentials; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D050053:TRPM Cation Channels; D013997:Time Factors; D014018:Tissue Distribution; D014162:Transfection
D015220:Calcium Channels; D002412:Cations; D018076:DNA, Complementary; D007477:Ions; D050053:TRPM Cation Channels; C475667:TRPM3 protein, human; D049452:Green Fluorescent Proteins; D008274:Magnesium; D002118:Calcium