Functional regulation of L-type calcium channels via protein kinase A- mediated phosphorylation of the β2 subunit

Abstract

Activation of protein kinase A (PKA) through the β-adrenergic receptor pathway is crucial for the positive regulation of cardiac L-type currents; however it is still unclear which phosphorylation events cause the robust regulation of channel function. In order to study whether or not the recently identified PKA phosphorylation sites on the β2 subunit are of functional significance, we coexpressed wild-type (WT) or mutant β2 subunits in tsA- 201 cells together with an α(1C) subunit, α(1C)Δ1905, that lacked the C- terminal 265 amino acids, including the only identified PKA site at Ser-1928. This truncated α(1C) subunit was similar to the truncated α(1C) subunit isolated from cardiac tissue not only in size (~190 kDa), but also with respect to its failure to serve as a PKA substrate. In cells transfected with the WT β2 subunit, voltage-activated Ba2+ currents were significantly increased when purified PKA was included in the patch pipette. Furthermore, mutations of Ser-478 and Ser-479 to Ala, but not Ser-459 to Ala, on the β2 subunit, completely abolished the PKA-induced increase of currents. The data indicate that the PKA-mediated stimulation of cardiac L-type Ca2+ currents may be at least partially caused by phosphorylation of the β2 subunit at Ser-478 and Ser-479.