Regulation of L-type calcium channel sparklet activity by PKC and C-src

Ca2+ sparklets are elementary fluorescence events associated with Ca2+ entry through L-type Ca2+ channels (Cav1.2) channels and are classified as persistent and low activity Ca2+ sparklets. Persistent Ca2+ sparklets are characterized by longer and more frequent channel open events and account for approximately 50 percent of the steady state Ca2+ entry through Cav1.2 channels. Previous studies suggest that the alpha isoform of protein kinase C (PKCalpha]) underlies persistent Ca2+ sparklet activity, but the mechanism of PKC[alpha] action on Cav1.2 channels is unclear. c-Src, another highly expressed kinase in vascular smooth muscle, phosphorylates Cav1.2 to increase whole-cell Ba2+ current (IBa) but it remains unknown if c-Src induces persistent Ca2+ sparklet activity through Cav1.2 channels. Here, I addressed two questions: 1) Does c-Src produce persistent Ca2+ sparklets through Cav1.2c (the neuronal isoform of Cav1.2)? 2) Does PKC[alpha] activate c-Src to produce persistent Ca2+ sparklets? TIRF microscopy was used to record Ca2+ sparklets from voltage-clamped HEK 293T cells co-expressing wild type (WT) or mutant Cav1.2c + active or inactive PKC[alpha]/c-Src. The results indicate that c-Src produces persistent Ca2+ sparklet activity, which is significantly reduced in the presence of the c-Src inhibitor, PP2, or with overexpression of kinase-dead c-Src. I tested two potential c-Src phosphorylation sites (Y2122F and Y2139F) on Cav1.2c for their role in production of persistent Ca2+sparklets. The Y2122F mutation significantly reduced persistent Ca2+sparklet activity while the Y2139F mutation was without any effect, indicating that c-Src phosphorylates Cav1.2c at Y2122 to induce persistent Ca2+ sparklets. Y2122F and Y2139F mutations did not have any significant effect on persistent Ca2+ sparklets in cells expressing Cav1.2c + PKC[alpha], indicating that PKC[alpha] does not act upstream of c-Src to produce persistent Ca2+sparklets. Whether PKC[alpha] phosphorylates S1901, the classical PKA phosphorylation site, to produce persistent Ca2+sparklet activity remains to be resolved.