Modulation of recombinant transient-receptor-potential-like (TRPL) channels by cytosolic Ca2+.
Whole-cell current recordings were used to examine the involvement of intracellular Ca2+ in the modulation of recombinant transient-receptor-potential like (TRPL) channels of Drosophila photoreceptor cells. TRPL was stably transfected in Chinese hamster ovary (CHO) cells and the expression of a calmodulin-binding protein with a molecular mass that corresponded to TRPL was demonstrated using calmodulin overlays. In cells expressing TRPL, ionic currents that were prominently outwardly rectifying were detected prior to activation of intracellular signalling pathways. The outwardly rectifying currents reversed close to 0 mV and did not occur after removal of permeant cations from the intracellular space. This suggests that TRPL forms non-selective cationic channels that appear to be constitutively active in mammalian cell lines. The TRPL channel currents were enhanced by manoeuvres that activate the phospholipase C (PLC) signalling pathway. These included activation of membrane receptors by thrombin, activation of G proteins by cell dialysis with guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma-S]) and release of Ca2+ from intracellular stores by dialysis with inositol 1,4,5-trisphosphate (IP3). After complete depletion of Ca2+ stores, IP3 had no effect on TRPL currents, suggesting that IP3 does not activate recombinant TRPL channels directly. However, thapsigargin, which induces a rise of cytosolic Ca2+, increased TRPL channel currents. Cell dialysis with solutions containing various concentrations of Ca2+ enhanced TRPL currents in a dose-dependent manner (EC50=450 nM Ca2+). Conversely, chelation of cytosolic Ca2+ abolished TRPL channel currents. The present results indicate that the activity of recombinant TRPL channels expressed in mammalian cell lines is up-regulated by a rise of cytosolic Ca2+.
Zimmer
S
S
Institut für Pharmakologie und Toxikologie, Universität des Saarlandes, Homburg/Saar, Germany.
Trost
C
C
Wissenbach
U
U
Philipp
S
S
Freichel
M
M
Flockerzi
V
V
Cavalié
A
A
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D004330:Drosophila; D029721:Drosophila Proteins; D004594:Electrophysiology; D015870:Gene Expression / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015290:Second Messenger Systems / Q000502:physiology; D014162:Transfection; D050051:Transient Receptor Potential Channels; D014785:Vision, Ocular / Q000502:physiology
D002148:Calmodulin-Binding Proteins; D029721:Drosophila Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D050051:Transient Receptor Potential Channels; C074192:trpl protein, Drosophila; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D015544:Inositol 1,4,5-Trisphosphate; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium