Inhibition of voltage-gated ca 2+ channel activity in small cell lung carcinoma by the ca 2+/calmodulin-dependent protein kinase inhibitor KN-62 (1-[ n, o-bis(5-isoquinolinesulfonyl)- n-methyl-L-tyrosyl]-4-phenylpiperazine)

Abstract

Although small cell lung carcinoma (SCLC) cells express both voltage-gated Ca 2+ channels (VGCC) and second messenger-operated Ca 2+ channels (SMOCC), little is known about the factors that regulate the activity of these channels in SCLC cells. Ca 2+/calmodulin-dependent protein kinase (CaM kinase) type II has been implicated recently in regulating Ca 2+ channel activity in other cell types. Because of this, we investigated the effects of the specific CaM kinase antagonist 1-[ N, O-bis(5-isoquinolinesulfonyl)- N-methyl-L-tryosyl]-4-phenylpiperazine (KN-62) on Ca 2+ channel activity in SCLC cells. Incubation with 10 μM KN-62 for 20 min inhibited depolarization-dependent 45Ca 2+ influx by 96.1 ± 2.1% in four independent SCLC cell lines, and by 42.2 ± 6.8% in the NCI-H146 SCLC cell line. Similar inhibitory effects of KN-62 were observed when Fura-2 was used to measure depolarization-dependent Ca 2+ influx. These results indicate that KN-62 potently inhibits VGCC activity in SCLC cells. In contrast, KN-62 (10 μM, 20 min) did not inhibit significantly Ca 2+ mobilization induced by muscarinic acetylcholine receptor (mAChR) activation in SCLC cells. This indicates that SMOCC are less susceptible than VGCC to inhibition by KN-62 in SCLC cells. Because mAChR activation also inhibits VGCC activity in SCLC cells, we examined the effects of KN-62 on the mAChR-mediated inhibition of VGCC activity. To do this, we measured depolarization-dependent 45Ca 2+ influx in SCLC cells incubated with submaximal concentrations of KN-62 and the mAChR agonist carbachol. Treatment of cells with both drugs resulted in almost twice as much inhibition of VGCC activity as in cells treated with only one of the drugs. This indicates that inactivation of CaM kinase with KN-62 does not suppress the ability of mAChR agonists to inhibit VGCC activity.