Involvement of phosphorylation of beta-subunit in cAMP-dependent activation of L-type Ca2+ channel in aortic smooth muscle-derived A7r5 cells

We investigated the effect of intracellular cAMP on the gating kinetics of L-type Ca2+ channel in an A7r5 smooth muscle-derived cell line using the wh ole-cell patch-clamp technique. Application of dibutyryl cyclic AMP (db-cAM P) to the cell increased the magnitude of Ca2+ currents through L-type Ca2 channels (I-Ca), and shifted the current-voltage relationship (I-V curve) for I-Ca to the left. The magnitudes of maximum I-Ca were 14.1 +/- 0.7 befo re and 16.0 +/- 1.1 pA/pF after application of 1 mM db-cAMP (P < 0.05). The values of the half-activation potential (V-1/2) of I-Ca, estimated from ac tivation curves, were -7.0 +/- 0.8 mV before and -10.8 +/- 1.0 mV after app lication of db-cAMP (P < 0.05). In cells pretreated with 10 mu M Rp-cAMPS ( a specific inhibitor of PKA), db-cAMP affected neither the I-V curve nor th e activation curve for I-Ca. In cells pretreated with the antisense oligonu cleotide for the beta-subunit of L-type Ca2+ channel, db-cAMP failed to enh ance I-Ca or alter the activation curve. On the other hand, in the cells pr etreated with the nonsense oligonucleotide, application of db-cAMP caused a n increase in magnitude of Ic, and shifted the activation curve to the left . Western blot analysis revealed that the pretreatment of cells with antise nse oligonucleotide but nonsense oligonucleotide reduced the expression of the beta-subunit of the L-type Ca2+ channel. We conclude that the cAMP-depe ndent phosphorylation of the beta-subunit potentiates the voltage dependenc y of the activation kinetics of the L-type Ca2+ channel in A7r5 cells. (C) 2000 Elsevier Science Inc. All rights reserved.