Activation of vanilloid receptor type I in the endoplasmic reticulum fails to activate store-operated Ca2+ entry.
To evaluate interaction of vanilloid receptor type 1 (TRPV1) with endogenous Ca(2+) signalling mechanisms, TRPV1 was expressed in Spodoptera frugiperda (Sf 9) insect cells using recombinant baculovirus. Stimulation of TRPV1-expressing cells, but not control Sf 9 cells, with resiniferatoxin (RTX), capsaicin or anandamide, produced an increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)), with EC(50) values of 166 pM, 24.5 nM and 3.89 microM respectively. In the absence of extracellular Ca(2+), both capsaicin and RTX caused an increase in [Ca(2+)](i) with EC(50) values of approx. 10 microM and 10 nM respectively. This TRPV1-induced release of Ca(2+) from intracellular stores was not blocked by U73122, suggesting that phospholipase C was not involved. Substantial overlap was found between the thapsigargin- and RTX-sensitive internal Ca(2+) pools, and confocal imaging showed that intracellular TRPV1 immunofluorescence co-localized with the endoplasmic reticulum targeting motif KDEL. To determine if TRPV1-induced mobilization of intracellular Ca(2+) activates endogenous store-operated Ca(2+) entry, the effect of 2-aminoethoxydiphenyl borate (2-APB) on Ba(2+) influx was examined. 2-APB blocked thapsigargin-induced Ba(2+) influx, but not RTX-induced Ba(2+) entry. In the combined presence of thapsigargin and a store-releasing concentration of RTX, the 2-APB-sensitive component was essentially identical with the thapsigargin-induced component. Similar results were obtained in HEK-293 cells stably expressing TRPV1. These results suggest that TRPV1 forms agonist-sensitive channels in the endoplasmic reticulum, which when activated, release Ca(2+) from internal stores, but fail to activate endogenous store-operated Ca(2+) entry. Selective activation of intracellular TRPV1, without concomitant involvement of plasmalemmal Ca(2+) influx mechanisms, could play an important role in Ca(2+) signalling within specific subcellular microdomains.
Wisnoskey
Brian J
BJ
Rammelkamp Center for Education and Research, MetroHealth Medical Center, Cleveland, OH 44109, USA.
Sinkins
William G
WG
Schilling
William P
WP
D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism*; D001464:Barium / Q000378:metabolism; D001896:Boron Compounds / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D020013:Calcium Signaling / Q000187:drug effects; D002186:Cannabinoids / Q000494:pharmacology; D002211:Capsaicin / Q000494:pharmacology; D002478:Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004224:Diterpenes / Q000494:pharmacology; D063388:Endocannabinoids; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D017136:Ion Transport; D018613:Microscopy, Confocal; D009498:Neurotoxins / Q000494:pharmacology; D053284:Polyunsaturated Alkamides; D051381:Rats; D011955:Receptors, Drug / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D050916:TRPV Cation Channels; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
D001095:Arachidonic Acids; D001896:Boron Compounds; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D002186:Cannabinoids; D004224:Diterpenes; D063388:Endocannabinoids; D004791:Enzyme Inhibitors; D009498:Neurotoxins; D053284:Polyunsaturated Alkamides; D011955:Receptors, Drug; D011994:Recombinant Proteins; D050916:TRPV Cation Channels; C494484:TRPV1 receptor; D001464:Barium; D019284:Thapsigargin; C024353:resiniferatoxin; C109986:2-aminoethoxydiphenyl borate; D010738:Type C Phospholipases; D002211:Capsaicin; D002118:Calcium; C078814:anandamide