Isoform-specific inhibition of TRPC4 channel by phosphatidylinositol 4,5-bisphosphate.
Full-length transient receptor potential (TRP) cation channel TRPC4alpha and shorter TRPC4beta lacking 84 amino acids in the cytosolic C terminus are expressed in smooth muscle and endothelial cells where they regulate membrane potential and Ca(2+) influx. In common with other "classical" TRPCs, TRPC4 is activated by G(q)/phospholipase C-coupled receptors, but the underlying mechanism remains elusive. Little is also known about any isoform-specific channel regulation. Here we show that TRPC4alpha but not TRPC4beta was strongly inhibited by intracellularly applied phosphatidylinositol 4,5-bisphosphate (PIP(2)). In contrast, several other phosphoinositides (PI), including PI(3,4)P(2), PI(3,5)P(2), and PI(3,4,5)P(3), had no effect or even potentiated TRPC4alpha indicating that PIP(2) inhibits TRPC4alpha in a highly selective manner. We show that PIP(2) binds to the C terminus of TRPC4alpha but not that of TRPC4beta in vitro. Its inhibitory action was dependent on the association of TRPC4alpha with actin cytoskeleton as it was prevented by cytochalasin D treatment or by the deletion of the C-terminal PDZ-binding motif (Thr-Thr-Arg-Leu) that links TRPC4 to F-actin through the sodium-hydrogen exchanger regulatory factor and ezrin. PIP(2) breakdown appears to be a required step in TRPC4alpha channel activation as PIP(2) depletion alone was insufficient for channel opening, which additionally required Ca(2+) and pertussis toxin-sensitive G(i/o) proteins. Thus, TRPC4 channels integrate a variety of G-protein-dependent stimuli, including a PIP(2)/cytoskeleton dependence reminiscent of the TRPC4-like muscarinic agonist-activated cation channels in ileal myocytes.
Otsuguro
Ken-ichi
K
Cardiovascular Biomedical Research Centre, School of Medicine and Dentistry, Queen's University Belfast, Belfast, United Kingdom.
Tang
Jisen
J
Tang
Yufang
Y
Xiao
Rui
R
Freichel
Marc
M
Tsvilovskyy
Volodymyr
V
Ito
Shigeo
S
Flockerzi
Veit
V
Zhu
Michael X
MX
Zholos
Alexander V
AV
D000199:Actins / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000235:genetics / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007082:Ileum / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D032342:Muscle Cells / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism; D019384:Nucleic Acid Synthesis Inhibitors; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000494:pharmacology*; D018129:Phosphatidylinositol Phosphates / Q000494:pharmacology; D011485:Protein Binding / Q000502:physiology; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D050052:TRPC Cation Channels / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism
D000199:Actins; D019384:Nucleic Acid Synthesis Inhibitors; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D020033:Protein Isoforms; D050052:TRPC Cation Channels; C489571:TRPC4 ion channel; D015638:Cytochalasin D; D010738:Type C Phospholipases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium