title: Mechanisms underlying heterogeneous Ca2+ sparklet activity in arterial smooth muscle.
abstract: In arterial smooth muscle, single or small clusters of Ca(2+) channels operate in a high probability mode, creating sites of nearly continual Ca(2+) influx (called "persistent Ca(2+) sparklet" sites). Persistent Ca(2+) sparklet activity varies regionally within any given cell. At present, the molecular identity of the Ca(2+) channels underlying Ca(2+) sparklets and the mechanisms that give rise to their spatial heterogeneity remain unclear. Here, we used total internal reflection fluorescence (TIRF) microscopy to directly investigate these issues. We found that tsA-201 cells expressing L-type Cavalpha1.2 channels recapitulated the general features of Ca(2+) sparklets in cerebral arterial myocytes, including amplitude of quantal event, voltage dependencies, gating modalities, and pharmacology. Furthermore, PKCalpha activity was required for basal persistent Ca(2+) sparklet activity in arterial myocytes and tsA-201 cells. In arterial myocytes, inhibition of protein phosphatase 2A (PP2A) and 2B (PP2B; calcineurin) increased Ca(2+) influx by evoking new persistent Ca(2+) sparklet sites and by increasing the activity of previously active sites. The actions of PP2A and PP2B inhibition on Ca(2+) sparklets required PKC activity, indicating that these phosphatases opposed PKC-mediated phosphorylation. Together, these data unequivocally demonstrate that persistent Ca(2+) sparklet activity is a fundamental property of L-type Ca(2+) channels when associated with PKC. Our findings support a novel model in which the gating modality of L-type Ca(2+) channels vary regionally within a cell depending on the relative activities of nearby PKCalpha, PP2A, and PP2B.
authors.affiliation: Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, 98195, USA.
authors.forename: Manuel F
authors.initials: MF
authors.lastname: Navedo
authors.forename: Gregory C
authors.initials: GC
authors.lastname: Amberg
authors.forename: Madeline
authors.initials: M
authors.lastname: Nieves
authors.forename: Jeffery D
authors.initials: JD
authors.lastname: Molkentin
authors.forename: Luis F
authors.initials: LF
authors.lastname: Santana
mesh_terms: D000818:Animals; D002118:Calcium / Q000494:pharmacology / Q000502:physiology; D020746:Calcium Channels, L-Type / Q000502:physiology*; D002460:Cell Line; D002536:Cerebral Arteries / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D051571:Protein Kinase C-alpha / Q000172:deficiency / Q000235:genetics / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); D051571:Protein Kinase C-alpha; D002118:Calcium