Assessment of the role of the inositol 1,4,5-trisphosphate receptor in the activation of transient receptor potential channels and store-operated Ca2+ entry channels.
The mechanism for coupling between Ca(2+) stores and store-operated channels (SOCs) is an important but unresolved question. Although SOCs have not been molecularly identified, transient receptor potential (TRP) channels share a number of operational parameters with SOCs. The question of whether activation of SOCs and TRP channels is mediated by the inositol 1,4,5-trisphosphate receptor (InsP(3)R) was examined using the permeant InsP(3)R antagonist, 2-aminoethoxydiphenyl borate (2-APB) in both mammalian and invertebrate systems. In HEK293 cells stably transfected with human TRPC3 channels, the actions of 2-APB to block carbachol-induced InsP(3)R-mediated store release and carbachol-induced Sr(2+) entry through TRPC3 channels were both reversed at high agonist levels, suggesting InsP(3)Rs mediate TRPC3 activation. However, electroretinogram recordings of the light-induced current in Drosophila revealed that the TRP channel-mediated responses in wild-type as well as trp and trpl mutant flies were all inhibited by 2-APB. This action of 2-APB is likely InsP(3)R-independent since InsP(3)Rs are dispensable for the light response. We used triple InsP(3)R knockout DT40 chicken B-cells to further assess the role of InsP(3)Rs in SOC activation. (45)Ca(2+) flux analysis revealed that although DT40 wild-type cells retained normal InsP(3)Rs mediating 2-APB-sensitive Ca(2+) release, the DT40InsP(3)R-k/o cells were devoid of functional InsP(3)Rs. Using intact cells, all parameters of Ca(2+) store function and SOC activation were identical in DT40wt and DT40InsP(3)R-k/o cells. Moreover, in both cell lines SOC activation was completely blocked by 2-APB, and the kinetics of action of 2-APB on SOCs (time dependence and IC(50)) were identical. The results indicate that (a) the action of 2-APB on Ca(2+) entry is not mediated by the InsP(3)R and (b) the effects of 2-APB provide evidence for an important similarity in the function of invertebrate TRP channels, mammalian TRP channels, and mammalian store-operated channels.
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Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Venkatachalam
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Li
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Montell
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Kurosaki
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Patterson
R L
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Gill
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D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D030801:Animals, Genetically Modified; D001896:Boron Compounds / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002120:Calcium Channel Agonists / Q000494:pharmacology; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D002645:Chickens; D004305:Dose-Response Relationship, Drug; D004330:Drosophila; D004596:Electroretinography; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008027:Light; D018727:Muscarinic Antagonists / Q000378:metabolism; D009154:Mutation; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013324:Strontium / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
D001896:Boron Compounds; D002120:Calcium Channel Agonists; D015220:Calcium Channels; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018727:Muscarinic Antagonists; D018160:Receptors, Cytoplasmic and Nuclear; C085029:adenophostin A; D002217:Carbachol; C109986:2-aminoethoxydiphenyl borate; D000241:Adenosine; D002118:Calcium; D013324:Strontium