The selectivity filter of the cation channel TRPM4

J Biol Chem. 2005 Jun 17;280(24):22899-906. doi: 10.1074/jbc.M501686200. Epub 2005 Apr 21.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alanine / chemistry
  • Amino Acid Sequence
  • Biotinylation
  • Calcium / chemistry
  • Calcium / metabolism
  • Calcium Channels / chemistry*
  • Cation Transport Proteins / chemistry*
  • Cations
  • Cell Line
  • Electrophysiology
  • Gene Deletion
  • Genes, Dominant
  • Glutamic Acid / chemistry
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Inhibitory Concentration 50
  • Magnesium / chemistry
  • Membrane Proteins / chemistry
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation
  • Phenotype
  • Protein Conformation
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Sequence Homology, Amino Acid
  • TRPM Cation Channels
  • Time Factors

Substances

  • Calcium Channels
  • Cation Transport Proteins
  • Cations
  • Membrane Proteins
  • TRPM Cation Channels
  • TRPM4 protein, human
  • TRPM5 protein, human
  • Green Fluorescent Proteins
  • Glutamic Acid
  • Magnesium
  • Alanine
  • Calcium