Characterisation of recombinant rat TRPM2 and a TRPM2-like conductance in cultured rat striatal neurones.
TRPM2, a member of the TRP ion channel family, is expressed both in the brain and immune cells of the monocyte lineage. Functionally, it is unique in its activation by intracellular ADP-ribose and both oxidative and nitrosative stress. To date studies of this channel have concentrated on human recombinant channels and rodent native preparations. This provides the potential for cross-species complications in the interpretation of native tissue observations based on recombinant channel phenotype. Consequently, we have cloned and heterologously expressed rat TRPM2 (rTRPM2) in HEK293 cells. We find that, like hTRPM2, it responds to intracellular ADP-ribose in a manner dependent on extracellular Ca(2+). At the single channel level rTRPM2 is a slow gating, large conductance (84pS) channel that rapidly runs down in isolated membrane patches. Pharmacologically, rTRPM2 is rapidly and irreversibly blocked by clotrimazole (10muM), thus resembling hTRPM2 but not the TRPM2-like current of the rat-derived insulinoma CRI-G1, which exhibits reversible inhibition by this agent. We show that cultured rat striatal neurones exhibit an ADP-ribose-activated conductance at both the whole cell and single channel level. Pharmacologically this neuronal current can be irreversibly inhibited by clotrimazole. It is also sensitive to removal of extracellular Ca(2+), suggesting that it is mediated by TRPM2-containing channels. These data provide a functional characterisation of heterologously expressed rTRPM2 and demonstrate that, in addition to the previous descriptions in immune cells, microglia and insulinomas, a TRPM2-like conductance can be found in neurones derived from the rodent CNS.
Hill
Kerstin
K
Neurology, GlaxoSmithKline Research and Development Ltd, New Frontiers Science Park (North), Harlow, Essex, UK.
Tigue
Natalie J
NJ
Kelsell
Rosemary E
RE
Benham
Christopher D
CD
McNulty
Shaun
S
Schaefer
Michael
M
Randall
Andrew D
AD
D000246:Adenosine Diphosphate Ribose / Q000494:pharmacology; D000818:Animals; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D004594:Electrophysiology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007202:Indicators and Reagents; D008856:Microscopy, Fluorescence; D017072:Neostriatum / Q000166:cytology / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D018384:Oxidative Stress / Q000502:physiology; D018408:Patch-Clamp Techniques; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism*; D050053:TRPM Cation Channels / Q000187:drug effects / Q000378:metabolism*; D014162:Transfection
D007202:Indicators and Reagents; D011994:Recombinant Proteins; D050053:TRPM Cation Channels; D000246:Adenosine Diphosphate Ribose; D004247:DNA; D006861:Hydrogen Peroxide