Negatively charged amino acids near and in transient receptor potential (TRP) domain of TRPM4 channel are one determinant of its Ca2+ sensitivity.
Transient receptor potential (TRP) channel melastatin subfamily member 4 (TRPM4) is a broadly expressed nonselective monovalent cation channel. TRPM4 is activated by membrane depolarization and intracellular Ca(2+), which is essential for the activation. The Ca(2+) sensitivity is known to be regulated by calmodulin and membrane phosphoinositides, such as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Although these regulators must play important roles in controlling TRPM4 activity, mutation analyses of the calmodulin-binding sites have suggested that Ca(2+) binds to TRPM4 directly. However, the intrinsic binding sites in TRPM4 remain to be elucidated. Here, by using patch clamp and molecular biological techniques, we show that there are at least two functionally different divalent cation-binding sites, and the negatively charged amino acids near and in the TRP domain in the C-terminal tail of TRPM4 (Asp-1049 and Glu-1062 of rat TRPM4) are required for maintaining the normal Ca(2+) sensitivity of one of the binding sites. Applications of Co(2+), Mn(2+), or Ni(2+) to the cytosolic side potentiated TRPM4 currents, increased the Ca(2+) sensitivity, but were unable to evoke TRPM4 currents without Ca(2+). Mutations of the acidic amino acids near and in the TRP domain, which are conserved in TRPM2, TRPM5, and TRPM8, deteriorated the Ca(2+) sensitivity in the presence of Co(2+) or PI(4,5)P2 but hardly affected the sensitivity to Co(2+) and PI(4,5)P2. These results suggest a novel role of the TRP domain in TRPM4 as a site responsible for maintaining the normal Ca(2+) sensitivity. These findings provide more insights into the molecular mechanisms of the regulation of TRPM4 by Ca(2+).
From the Laboratory of Pharmacology, Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido, 060-0818 and the Department of Molecular Physiology, Niigata University School of Medicine, Niigata 951-8510, Japan souya@vetmed.hokudai.ac.jp.
Soichiro
S
Yamaguchi
From the Laboratory of Pharmacology, Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido, 060-0818 and.
Akira
A
Tanimoto
From the Laboratory of Pharmacology, Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido, 060-0818 and.
Ken-ichi
K
Otsuguro
the Department of Molecular Physiology, Niigata University School of Medicine, Niigata 951-8510, Japan.
Hiroshi
H
Hibino
From the Laboratory of Pharmacology, Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido, 060-0818 and.
Shigeo
S
Ito
D000818:Animals; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002413:Cations, Divalent / Q000378:metabolism / Q000494:pharmacology; D003035:Cobalt / Q000378:metabolism / Q000494:pharmacology; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D009154:Mutation; D018408:Patch-Clamp Techniques; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism / Q000494:pharmacology; D011914:Rats, Inbred BN; D050053:TRPM Cation Channels / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014162:Transfection
D002413:Cations, Divalent; D019269:Phosphatidylinositol 4,5-Diphosphate; D050053:TRPM Cation Channels; C500239:TRPM4 protein, rat; D001224:Aspartic Acid; D003035:Cobalt; D018698:Glutamic Acid; D002118:Calcium
Calcium; Electrophysiology; Ion Channel; Manganese; Molecular Biology; Phosphoinositide; Physiology; Site-directed Mutagenesis; Transient Receptor Potential Channels (TRP Channels)