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

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.