Evidence for a direct interaction of thapsigargin with voltage-dependent Ca²⁺ channel

Abstract

The effect of thapsigargin, an inhibitor of the sarco-endoplasmic reticulum Ca²⁺-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.