title: The function of the two-pore channel TPC1 depends on dimerization of its carboxy-terminal helix.
abstract: Two-pore channels (TPCs) constitute a family of intracellular cation channels with diverse permeation properties and functions in animals and plants. In the model plant Arabidopsis, the vacuolar cation channel TPC1 is involved in propagation of calcium waves and in cation homeostasis. Here, we discovered that the dimerization of a predicted helix within the carboxyl-terminus (CTH) is essential for the activity of TPC1. Bimolecular fluorescence complementation and co-immunoprecipitation demonstrated the interaction of the two C-termini and pointed towards the involvement of the CTH in this process. Synthetic CTH peptides dimerized with a dissociation constant of 3.9 µM. Disruption of this domain in TPC1 either by deletion or point mutations impeded the dimerization and cation transport. The homo-dimerization of the CTH was analyzed in silico using coarse-grained molecular dynamics (MD) simulations for the study of aggregation, followed by atomistic MD simulations. The simulations revealed that the helical region of the wild type, but not a mutated CTH forms a highly stable, antiparallel dimer with characteristics of a coiled-coil. We propose that the voltage- and Ca(2+)-sensitive conformation of TPC1 depends on C-terminal dimerization, adding an additional layer to the complex regulation of two-pore cation channels.
authors.affiliation: Molecular Plant Physiology, Department of Biology, University of Erlangen-Nürnberg, Staudtstrasse 5, 91058, Erlangen, Germany.
authors.forename: Nina
authors.initials: N
authors.lastname: Larisch
authors.affiliation: Computational Biology, Department of Biology, University of Erlangen-Nürnberg, Staudtstrasse 5, 91058, Erlangen, Germany.
authors.forename: Sonja A
authors.initials: SA
authors.lastname: Kirsch
authors.affiliation: Developmental Biology, Department of Biology, University of Erlangen-Nürnberg, Staudtstrasse 5, 91058, Erlangen, Germany.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Schambony
authors.affiliation: Molecular Plant Physiology, Department of Biology, University of Erlangen-Nürnberg, Staudtstrasse 5, 91058, Erlangen, Germany.
authors.forename: Tanja
authors.initials: T
authors.lastname: Studtrucker
authors.affiliation: Computational Biology, Department of Biology, University of Erlangen-Nürnberg, Staudtstrasse 5, 91058, Erlangen, Germany.
authors.forename: Rainer A
authors.initials: RA
authors.lastname: Böckmann
authors.affiliation: Molecular Plant Physiology, Department of Biology, University of Erlangen-Nürnberg, Staudtstrasse 5, 91058, Erlangen, Germany. petra.dietrich@fau.de.
authors.forename: Petra
authors.identifier: 0000-0002-9209-8089
authors.initials: P
authors.lastname: Dietrich
mesh_terms: D017360:Arabidopsis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling; D057809:HEK293 Cells; D006801:Humans; D056004:Molecular Dynamics Simulation; D017354:Point Mutation; D000072756:Protein Conformation, alpha-Helical; D055503:Protein Multimerization; D000097903:Two-Pore Channels
chemical_list: D029681:Arabidopsis Proteins; D015220:Calcium Channels; C498240:TPC1 protein, Arabidopsis; D000097903:Two-Pore Channels
keywords: Calcium signaling; MD simulation; Microscale thermophoresis; Mutation; Patch-clamp; Vacuole