title: The transmembrane segment S6 determines cation versus anion selectivity of TRPM2 and TRPM8.
abstract: TRPM2 and TRPM8, closely related members of the transient receptor potential (TRP) family, are cation channels activated by quite different mechanisms. Their transmembrane segments S5 and S6 are highly conserved. To identify common structures in S5 and S6 that govern interaction with the pore, we created a chimera in which the S5-pore-S6 region of TRPM8 was inserted into TRPM2, along with a lysine at each transition site. Currents through this chimera were induced by ADP-ribose (ADPR) in cooperation with Ca(2+). In contrast to wild-type TRPM2 channels, currents through the chimera were carried by Cl(-), as demonstrated in ion substitution experiments using the cation N-methyl-D-glucamine (NMDG) and the anion glutamate. Extracellular NMDG had no effects. The substitution of either intracellular or extracellular Cl(-) with glutamate shifted the reversal potential, decreased the current amplitude and induced a voltage-dependent block relieved by depolarization. The lysine in S6 was responsible for the anion selectivity; insertion of a lysine into corresponding sites within S6 of either TRPM2 or TRPM8 created anion channels that were activated by ADPR (TRPM2 I1045K) or by cold temperatures (TRPM8 V976K). The positive charge of the lysine was decisive for the glutamate block because the mutant TRPM2 I1045H displayed cation currents that were blocked at acidic but not alkaline intracellular pH values. We conclude that the distal part of S6 is crucial for the discrimination of charge. Because of the high homology of S6 in the whole TRP family, this new role of S6 may apply to further TRP channels.
authors.affiliation: Institute of Physiology, Medical Faculty, RWTH Aachen, D-52057 Aachen, Germany. fkuehn@physiology.rwth-aachen.de
authors.forename: Frank J P
authors.initials: FJ
authors.lastname: Kühn
authors.forename: Gabriel
authors.initials: G
authors.lastname: Knop
authors.forename: Andreas
authors.initials: A
authors.lastname: Lückhoff
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D000838:Anions; D016466:CHO Cells; D002412:Cations; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D008536:Meglumine / Q000737:chemistry; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050053:TRPM Cation Channels / Q000502:physiology*
chemical_list: D000838:Anions; D002412:Cations; D050053:TRPM Cation Channels; C121934:TRPM2 protein, human; C425966:TRPM8 protein, human; D000246:Adenosine Diphosphate Ribose; D008536:Meglumine