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dc.contributor.advisorDavis, Michael J., 1953 Nov.-eng
dc.contributor.authorGulia, Jyote, 1981-eng
dc.date.issued2010eng
dc.date.submitted2010 Falleng
dc.descriptionTitle from PDF of title page (University of Missouri--Columbia, viewed on April 21, 2014).eng
dc.description.abstractCa2+ 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% 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.eng
dc.format.extentxiii, 136 pageseng
dc.identifier.urihttps://hdl.handle.net/10355/41906
dc.identifier.urihttps://doi.org/10.32469/10355/41906eng
dc.languageEnglisheng
dc.publisherUniversity of Missouri--Columbiaeng
dc.relation.ispartofcommunityUniversity of Missouri--Columbia. Graduate School. Theses and Dissertationseng
dc.rightsOpenAccess.eng
dc.rights.licenseThis work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.
dc.sourceSubmitted by University of Missouri--Columbia Graduate School.eng
dc.subjectL-type calcium channeleng
dc.subjectTotal internal reflection fluorescence microscopyeng
dc.subjectCa2+ sparkletseng
dc.subject.FASTVascular smooth muscleeng
dc.subject.FASTCalcium channelseng
dc.subject.FASTProtein kinaseseng
dc.subject.FASTMuscle cellseng
dc.titleRegulation of L-type calcium channel sparklet activity by PKC and C-srceng
dc.typeThesiseng
thesis.degree.disciplineBiological engineering (MU)eng
thesis.degree.grantorUniversity of Missouri--Columbiaeng
thesis.degree.levelDoctoraleng
thesis.degree.namePh. D.eng


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