The aim of the present study was to characterize the role played by different L-type Ca2+ channel subunits in [Ca2+](i) increase induced by maitotoxin (MTX). In the presence of 5 mM extracellular K+, MTX (0.01-0.5 ng/ml) induced a significant concentration-dependent increase in Fura-2-monitored [Ca2+](i) in single Chinese hamster ovary (CHO) cells expressing the alpha(1c) (CHOC alpha 9 cells) or the alpha(1c)beta(3)alpha(2)delta (CHOC alpha 9 beta 3 alpha 2/delta 4 cells) subunits of voltage-gated Ca2+ channels (VGCCs), whereas the effect was much reduced in wild-type CHO cells lacking VGCCs. In addition, MTX effect on CHOC alpha 9, CHOC alpha 9 beta 3 alpha 2/delta 4, and GH(3) cells (0.01-0.1 ng/ml) was inhibited by the selective L-type Ca2+ channel entry-blocker nimodipine (10 mu M); a nimodipine-insensitive component was still present, particularly at high (>1 ng/ml) toxin concentrations. In CHOC alpha 9 beta 3 alpha 2/delta 4 cells, depolarizing concentrations of extracellular K+ (55 mM) reinforced the [Ca2+](i) increase induced by MTX(0.1 ng/ml), and this effect was prevented by nimodipine (10 mu M). Finally, patch-clamp experiments in CHOC alpha 9 beta 3 alpha 2/delta 4 cells showed that low MTX concentrations (0.03 ng/ml) induced the occurrence of an inward current at -60 mV, which was completely prevented by Cd2+ (100 mu M) and by nimodipine (10 mu M), whereas the same dihydropyridine concentration (10 mu M) failed to prevent the electrophysiological effects of a higher toxin concentration (3 ng/ml). In conclusion, the results of the present study showed that MTX-induced [Ca2+](i) elevation involves two components: 1) an action on L-type VGCCs at the pore-forming alpha(1c) subunit level, which is responsible for the greatest rise of [Ca2+](i); and 2) a VGCC-independent mechanism that is present both in excitable and in nonexcitable cells and is responsible for a lower elevation of [Ca2+](i).
Studies on maitotoxin-induced intracellular Ca2+ elevation in Chinese hamster ovary cells stably transfected with cDNAs encoding for L-type Ca2+ channel subunits
TAGLIALATELA, Maurizio;
1999-01-01
Abstract
The aim of the present study was to characterize the role played by different L-type Ca2+ channel subunits in [Ca2+](i) increase induced by maitotoxin (MTX). In the presence of 5 mM extracellular K+, MTX (0.01-0.5 ng/ml) induced a significant concentration-dependent increase in Fura-2-monitored [Ca2+](i) in single Chinese hamster ovary (CHO) cells expressing the alpha(1c) (CHOC alpha 9 cells) or the alpha(1c)beta(3)alpha(2)delta (CHOC alpha 9 beta 3 alpha 2/delta 4 cells) subunits of voltage-gated Ca2+ channels (VGCCs), whereas the effect was much reduced in wild-type CHO cells lacking VGCCs. In addition, MTX effect on CHOC alpha 9, CHOC alpha 9 beta 3 alpha 2/delta 4, and GH(3) cells (0.01-0.1 ng/ml) was inhibited by the selective L-type Ca2+ channel entry-blocker nimodipine (10 mu M); a nimodipine-insensitive component was still present, particularly at high (>1 ng/ml) toxin concentrations. In CHOC alpha 9 beta 3 alpha 2/delta 4 cells, depolarizing concentrations of extracellular K+ (55 mM) reinforced the [Ca2+](i) increase induced by MTX(0.1 ng/ml), and this effect was prevented by nimodipine (10 mu M). Finally, patch-clamp experiments in CHOC alpha 9 beta 3 alpha 2/delta 4 cells showed that low MTX concentrations (0.03 ng/ml) induced the occurrence of an inward current at -60 mV, which was completely prevented by Cd2+ (100 mu M) and by nimodipine (10 mu M), whereas the same dihydropyridine concentration (10 mu M) failed to prevent the electrophysiological effects of a higher toxin concentration (3 ng/ml). In conclusion, the results of the present study showed that MTX-induced [Ca2+](i) elevation involves two components: 1) an action on L-type VGCCs at the pore-forming alpha(1c) subunit level, which is responsible for the greatest rise of [Ca2+](i); and 2) a VGCC-independent mechanism that is present both in excitable and in nonexcitable cells and is responsible for a lower elevation of [Ca2+](i).I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
