Evidence for a direct interaction of thapsigargin with voltage-dependent Ca2+ channel.
The effect of thapsigargin, an inhibitor of the sarco-endoplasmic reticulum Ca(2+)-ATPase, on voltage-dependent Ca2+ channels has been investigated in the A7r5 cell line and in membrane preparations from rat aorta, heart and brain. Patch-clamp technique showed that, at micromolar concentrations, thapsigargin inhibited the L-type Ca2+ channel current in A7r5 cells. It depressed the current at all voltages without change in the steady state inactivation curve. The rates of inactivation of the Ca2+ current were highly variable among the cells suggesting that more than one component of L-type Ca2+ current coexist in A7r5 cells, differing in the kinetics of inactivation. Thapsigargin appeared to be more potent on the slower-inactivating Ca2+ current than on the faster-inactivating one. In the same range of concentrations, thapsigargin inhibited the specific binding of 3H(+)-isradipine in intact cells while 45Ca2+ uptake in intracellular stores of skinned cells was inhibited at nanomolar concentrations. The equilibrium dissociation constant of 3H(+)-isradipine was increased in the presence of thapsigargin as a result of an increase of the dissociation rate constant indicating that the inhibitory effect of the antagonist cannot be attributed to a simple competitive interaction with the dihydropyridine binding site. Maximum binding capacity was unaffected. A similar pattern of inhibition of 3H(+)-isradipine binding was observed in membrane preparations from rat aorta, heart and brain. Those results indicate that, at micromolar concentrations, thapsigargin inhibits the voltage-dependent Ca2+ current by a direct interaction with the L-type Ca2+ channels.
Buryi
V
V
Laboratoire de Pharmacologie, Université Catholique de Louvain, Bruxelles, Belgium.
Morel
N
N
Salomone
S
S
Kerger
S
S
Godfraind
T
T
D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002460:Cell Line; D006321:Heart / Q000187:drug effects; D017275:Isradipine; D008564:Membrane Potentials / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology; D009206:Myocardium / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin
D002121:Calcium Channel Blockers; D013729:Terpenes; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D017275:Isradipine