L-type Ca2+ (CaL) channels are a primary pathway of Ca2+ entry into vascular smooth muscle cells (VSMCs). Ca L channels play a key role in establishment of vascular tone and its modulation in response to various stimuli. Of the four subclasses of Ca L channels, Cav1.2 is the subclass expressed in VSMCs. Ca v1.2 has 3 isoforms: Cav1.2a (cardiac), Cav1.2b (smooth muscle) and Cav1.2c (neuronal). Cell-attached patch clamp and total internal reflection fluorescence (TIRF) studies on CaL channels have revealed the occurrence of a high activity gating mode characterized by frequent, prolonged channel openings, in addition to the more prevalently occurring gating mode with brief and rare channel openings. In cells loaded with a Ca2+ fluorescence indicator, Ca2+ influx during the opening of a CaL channel leads to a loCaLized increase in Ca2+ fluorescence, termed a Ca2+ sparklet. In TIRF studies, two populations of CaL channels exhibiting distinct gating behaviors are classified as persistent Ca2+ sparklets and low activity Ca2+ sparklets. Low activity Ca 2+ sparklets represent stochastic gating of CaL channels with brief and rare CaL channel openings; whereas persistent Ca 2+ sparklets represent a CaL channel population with increased open probability due to membrane depolarization or channel phosphorylation. Persistent Ca2+ sparklets are thought to be primarily regulated by the VSMC isoform of protein kinase C, PKCα, and account for approximately 50% of the steady state Ca2+ entry through CaL channels. However, the mechanism of PKCα action on CaL channels has remained unclear. c-Src, another highly expressed kinase in VSMCs, potentiates CaL channel current and its action may or may not be related to PKC. Whether c-Src activation induces persistent Ca2+ sparklet activity through CaL channels is not known. The objective of this study is to understand how PKCα and c-Src regulate persistent Ca 2+ sparklet activity through CaL channels. Based on evidence that several PKC isoforms can activate c-Src, and preliminary experiments in which c-Src activity was inhibited, I hypothesized that persistent Ca2+ sparklet activity is chiefly determined by c-Src, which acts downstream of PKCα and directly phosphorylates the CaL channel at its C-terminal residue Y2122.
To test my hypothesis, I performed TIRF imaging of Ca2+ sparklets on voltage clamped HEK 293T cells co-expressing active/ inactive PKC or c-Src plus wild type (WT) or mutant CaL channels to allow or alter kinase-modulated phosphorylation. I used the neuronal isoform of the Cav1.2 channel due to availability of Cav1.2c mutant constructs suitable for this study. The cells were loaded with the Ca 2+-sensitive indicator Rhod-2 through the patch-pipette. Xestospongin C and tetracaine were used to block IP3 (inositol 1,4,5-triphosphate) and ryanodine receptors, respectively, and to minimize Ca2+ fluorescence events unrelated to Ca2+ sparklets. Ca2+ sparklets were detected using a custom program written in Interactive Data Language and Ca2+ sparklet data were analyzed using a signal mass method.
TIRF imaging of cells expressing WT Cav1.2c + c-Src revealed the frequent occurrence of persistent Ca2+ sparklets, whereas cells transfected with c-Src alone only rarely exhibited events resembling persistent Ca2+ sparklets. Treatment of cells expressing WT Cav1.2c + c-Src with 10 µM PP2 reduced the signal mass of persistent Ca2+ sparklets significantly without having an effect on the signal mass of low activity Ca2+ sparklets. Kinase-dead (kd) c-Src was used to competitively inhibit endogenous c- Src. In cells expressing WT Cav1.2c + kd c-Src, persistent Ca2+ sparklet activity was almost completely eliminated. Cells expressing c-Src plus Ca v1.2c containing a mutation of a putative c-Src phosphorylation site, Y2122F Cav1.2c, showed a significant reduction in both signal mass and density of persistent Ca2+ sparklets compared to cells expressing WT Cav1.2c + c-Src. In contrast, the signal mass and density of persistent Ca2+ sparklets observed in cells expressing c-Src and Cav1.2 containing a mutation of another likely c- Src phosphorylation site, Y2139F Cav1.2c, were not significantly different from those recorded in cells expressing WT Ca v1.2c + c-Src. (Abstract shortened by ProQuest.)
|Advisor:||Davis, Michael J.|
|Commitee:||Davis, Michael J., Gillis, kevin D., Gv, Liqun, Meininger, Gerald A.|
|School:||University of Missouri - Columbia|
|School Location:||United States -- Missouri|
|Source:||DAI-B 80/07(E), Dissertation Abstracts International|
|Subjects:||Cellular biology, Physiology|
|Keywords:||C-SRC, Fluorescence microscopy, L-type calcium channel, PKC, Total internal reflection|
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