Involvement of phosphorylation of β-subunit in cAMP-dependent activation of L-type Ca 2+ channel in aortic smooth muscle-derived A7r5 cells

Abstract

We investigated the effect of intracellular cAMP on the gating kinetics of L-type Ca 2+ channel in an A7r5 smooth muscle-derived cell line using the whole-cell patch-clamp technique. Application of dibutyryl cyclic AMP (db-cAMP) to the cell increased the magnitude of Ca 2+ currents through L-type Ca 2+ 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 before 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 activation curves, were −7.0 ± 0.8 mV before and −10.8 ± 1.0 mV after application of db-cAMP ( P < 0.05). In cells pretreated with 10 μM Rp-cAMPS (a specific inhibitor of PKA), db-cAMP affected neither the I– V curve nor the activation curve for I Ca. In cells pretreated with the antisense oligonucleotide for the β-subunit of L-type Ca 2+ channel, db-cAMP failed to enhance I Ca or alter the activation curve. On the other hand, in the cells pretreated with the nonsense oligonucleotide, application of db-cAMP caused an increase in magnitude of I Ca and shifted the activation curve to the left. Western blot analysis revealed that the pretreatment of cells with antisense oligonucleotide but nonsense oligonucleotide reduced the expression of the β-subunit of the L-type Ca 2+ channel. We conclude that the cAMP-dependent phosphorylation of the β-subunit potentiates the voltage dependency of the activation kinetics of the L-type Ca 2+ channel in A7r5 cells.