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Persistent Calcium Sparklet Activity of L-Type Calcium Channels: Link Between PKC and c-Src

2010; Elsevier BV; Volume: 98; Issue: 3 Linguagem: Inglês

10.1016/j.bpj.2009.12.1597

1542-0086

Jyoti Gulia, Manuel F. Navedo, Peichun Gui, Jun-Tzu Chao, Luis F. Santana, Michael J. Davis,

Neuroscience and Neuropharmacology Research

Ca2+ sparklets are a fluorescence signal associated with Ca2+ entry through L-type calcium (Cav1.2) channels, which are primary Ca2+ entry pathway in many excitable cells. Ca2+ sparklets are quantal in nature (q = 34 nM Δ[Ca2+]i) and their activity is categorized as low (0<nPs 0.2), where n = no. of quantal levels and Ps = active sparklet probability. Both types of Ca2+ sparklets are present in vascular smooth muscle cells (VSMCs) but only low activity Ca2+ sparklets are present in heterologously expressed Cav1.2 channels unless PKCα is activated. The latter effect presumably requires Cav1.2 phosphorylation, perhaps at the canonical PKA phosphorylation site, S1901, yet the exact phosphorylation site remains unclear. Using TIRF microscopy in conjunction with whole cell voltage clamp, we detected persistent Ca2+ sparklet activity in HEK 293 cells co-expressing S1901A Cav1.2c and PKCα, indicating that PKCα does not phosphorylate S1901. Also, persistent Ca2+ sparklets were detectable in cells expressing WT Cav1.2c in the absence of PKCα if c-Src was co-expressed. Furthermore, Ca2+ sparklet activity was reduced in cells expressing WT Cav1.2c and kinase dead c-Src (7/9 cells). To test if phosphorylation of a previously identified Cav1.2c tyrosine phosphorylation site (Y2122) by c-Src mediated persistent Ca2+ sparklet activity, we co-expressed Y2122F Cav1.2c and c-Src in HEK cells. Persistent Ca2+ sparklet activity was present under these conditions; however, Ca2+ sparklet activity was reduced in cells co-expressing Y2139FCav1.2c and c-Src. These data suggest that c-Src may phosphorylate Cav1.2c at Y2139 under basal conditions to produce persistent Ca2+ sparklet activity. Future experiments on HEK cells expressing Y2139FCav1.2c and PKCα will allow us to determine if persistent Ca2+ sparklet activity in VSMCs is evoked by a common mechanism involving PKCα and c-Src.