title: The selectivity filter of the cation channel TRPM4.
abstract: Transient receptor potential channel melastatin subfamily (TRPM) 4 and its close homologue, TRPM5, are the only two members of the large transient receptor potential superfamily of cation channels that are impermeable to Ca(2+). In this study, we located the TRPM4 selectivity filter and investigated possible structural elements that render it Ca(2+)-impermeable. Based on homology with known cation channel pores, we identified an acidic stretch of six amino acids in the loop between transmembrane helices TM5 and TM6 ((981)EDMDVA(986)) as a potential selectivity filter. Substitution of this six-amino acid stretch with the selectivity filter of TRPV6 (TIIDGP) resulted in a functional channel that combined the gating hallmarks of TRPM4 (activation by Ca(2+), voltage dependence) with TRPV6-like sensitivity to block by extracellular Ca(2+) and Mg(2+) as well as Ca(2+) permeation. Neutralization of Glu(981) resulted in a channel with normal permeability properties but a strongly reduced sensitivity to block by intracellular spermine. Neutralization of Asp(982) yielded a functional channel that exhibited extremely fast desensitization (tau < 5 s), possibly indicating destabilization of the pore. Neutralization of Asp(984) resulted in a non-functional channel with a dominant negative phenotype when coexpressed with wild type TRPM4. Combined neutralization of all three acidic residues resulted in a functional channel whose voltage dependence was shifted toward very positive potentials. Substitution of Gln(977) by a glutamate, the corresponding residue in divalent cation-permeable TRPM channels, altered the monovalent cation permeability sequence and resulted in a pore with moderate Ca(2+) permeability. Our findings delineate the selectivity filter of TRPM channels and provide the first insight into the molecular basis of monovalent cation selectivity.
authors.affiliation: Department of Physiology, Campus Gasthuisberg, KU Leuven, Belgium. bernd.nilius@med.kuleuven.ac.be
authors.forename: Bernd
authors.initials: B
authors.lastname: Nilius
authors.forename: Jean
authors.initials: J
authors.lastname: Prenen
authors.forename: Annelies
authors.initials: A
authors.lastname: Janssens
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Owsianik
authors.forename: Chunbo
authors.initials: C
authors.lastname: Wang
authors.forename: Michael X
authors.initials: MX
authors.lastname: Zhu
authors.forename: Thomas
authors.initials: T
authors.lastname: Voets
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D019921:Biotinylation; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry*; D027682:Cation Transport Proteins / Q000737:chemistry*; D002412:Cations; D002460:Cell Line; D004594:Electrophysiology; D017353:Gene Deletion; D005799:Genes, Dominant; D018698:Glutamic Acid / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D008274:Magnesium / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D050053:TRPM Cation Channels; D013997:Time Factors
chemical_list: D015220:Calcium Channels; D027682:Cation Transport Proteins; D002412:Cations; D008565:Membrane Proteins; D050053:TRPM Cation Channels; C437888:TRPM4 protein, human; C404409:TRPM5 protein, human; D049452:Green Fluorescent Proteins; D018698:Glutamic Acid; D008274:Magnesium; D000409:Alanine; D002118:Calcium