Activation of vanilloid receptor type I in the endoplasmic reticulum fails to activate store-operated Ca2+ entry

Biochem J. 2003 Jun 1;372(Pt 2):517-28. doi: 10.1042/BJ20021574.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Arachidonic Acids / pharmacology
  • Baculoviridae / genetics
  • Baculoviridae / metabolism*
  • Barium / metabolism
  • Boron Compounds / pharmacology
  • Calcium / physiology*
  • Calcium Channel Blockers / pharmacology
  • Calcium Channels / drug effects
  • Calcium Channels / physiology*
  • Calcium Signaling / drug effects
  • Cannabinoids / pharmacology
  • Capsaicin / pharmacology
  • Cells, Cultured / cytology
  • Cells, Cultured / drug effects
  • Cells, Cultured / metabolism
  • Diterpenes / pharmacology
  • Endocannabinoids
  • Endoplasmic Reticulum / metabolism*
  • Enzyme Inhibitors / pharmacology
  • Humans
  • Immunoblotting
  • Ion Transport
  • Microscopy, Confocal
  • Neurotoxins / pharmacology
  • Polyunsaturated Alkamides
  • Rats
  • Receptors, Drug / genetics
  • Receptors, Drug / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • TRPV Cation Channels
  • Thapsigargin / pharmacology
  • Transfection
  • Type C Phospholipases / metabolism

Substances

  • Arachidonic Acids
  • Boron Compounds
  • Calcium Channel Blockers
  • Calcium Channels
  • Cannabinoids
  • Diterpenes
  • Endocannabinoids
  • Enzyme Inhibitors
  • Neurotoxins
  • Polyunsaturated Alkamides
  • Receptors, Drug
  • Recombinant Proteins
  • TRPV Cation Channels
  • TRPV1 receptor
  • Barium
  • Thapsigargin
  • resiniferatoxin
  • 2-aminoethoxydiphenyl borate
  • Type C Phospholipases
  • Capsaicin
  • Calcium
  • anandamide