The activity of persistent Ca²⁺ sparklets, which are characterized by longer and more frequent channel open events than low-activity sparklets, contributes substantially to steady-state Ca²⁺ entry under physiological conditions. Here, we addressed two questions related to the regulation of Ca²⁺ sparklets by PKC-α and c-Src, both of which increase whole cell Cav1.2 current: 1) Does c-Src activation enhance persistent Ca²⁺ sparklet activity? 2) Does PKC-α activate c-Src to produce persistent Ca²⁺ sparklets? With the use of total internal reflection fluorescence microscopy, Ca²⁺ sparklets were recorded from voltage-clamped tsA-201 cells coexpressing wild-type (WT) or mutant Cav1.2c (the neuronal isoform of Cav1.2) constructs ± active or inactive PKC-α/c-Src. Cells expressing Cav1.2c exhibited both low-activity and persistent Ca²⁺ sparklets. Persistent Ca²⁺ sparklet activity was significantly reduced by acute application of the c-Src inhibitor PP2 or coexpression of kinase-dead c-Src. Cav1.2c constructs mutated at one of two COOH-terminal residues (Y²¹²²F and Y²¹³⁹F) were used to test the effect of blocking putative phosphorylation sites for c-Src. Expression of Y²¹²²F but not Y²¹³⁹F Cav1.2c abrogated the potentiating effect of c-Src on Ca²⁺ sparklet activity. We could not detect a significant change in persistent Ca²⁺ sparklet activity or density in cells coexpressing Cav1.2c + PKC-α, regardless of whether WT or Y²¹²²F Cav1.2c was used, or after PP2 application, suggesting that PKC-α does not act upstream of c-Src to produce persistent Ca²⁺ sparklets. However, our results indicate that persistent Ca²⁺ sparklet activity is promoted by the action of c-Src on residue Y²¹²² of the Cav1.2c COOH terminus.
Objective Changes in smooth muscle cell (SMC) membrane potential (Em) are critical to vasomotor responses. As a fluorescent indicator approach would lessen limitations of glass electrodes in contracting preparations, we aimed to develop a Forster (or fluorescence) resonance energy transfer (FRET)-based measurement for Em. Methods The FRET pair used in this study (donor CC2-DMPE [excitation 405 nm] and acceptor DisBAC4(3)) provide rapid measurements at a sensitivity not achievable with many ratiometric indicators. The method also combined measurement changes in Ca2+i using fluo-4 and excitation at 490 nm. Results After establishing loading conditions, a linear relationship was demonstrated between Em and fluorescence signal in FRET dye-loaded HEK cells held under voltage clamp. Over the voltage range from −70 to +30 mV, slope (of FRET signal vs. voltage, m) = 0.49 ± 0.07, r2 = 0.96 ± 0.025. Similar data were obtained in cerebral artery SMCs, slope (m) = 0.30 ± 0.02, r2 = 0.98 ± 0.02. Change in FRET emission ratio over the holding potential of −70 to +30 mV was 41.7 ± 4.9% for HEK cells and 30.0 ± 2.3% for arterial SMCs. The FRET signal was also shown to be modulated by KCl-induced depolarization in a concentration-dependent manner. Further, in isolated arterial SMCs, KCl-induced depolarization (60 mM) measurements occurred with increased fluo-4 fluorescence emission (62 ± 9%) and contraction (−27 ± 4.2%). Conclusions The data support the FRET-based approach for measuring changes in Em in arterial SMCs. Further, image-based measurements of Em can be combined with analysis of temporal changes in Ca2+i and contraction.
The L‐type calcium channel (Cav1.2) is regulated by multiple kinases, including PKA and c‐Src, which phosphorylate the Cav1.2 C‐terminus at residues S1901 and Y2122 (Cav1.2c), respectively, to enhance Ca2+ entry. PKC also enhances Cav1.2 current under some conditions and promotes persistent Cav1.2 Ca2+ sparklet activity (quantal Ca2+ entry events); however, the PKC phosphorylation site is unclear. We addressed two questions using TIRF microscopy to measure Ca2+ sparklets in patch clamped HEK cells expressing Cav1.2c. 1) Does PKC produce persistent calcium sparklet activity through phosphorylation of S1901? 2) Is c‐Src involved in the production of persistent sparklet activity?Persistent sparklets (indicating a prolonged channel opening state) were defined as nPs > 0.2, where n= # of quantal levels and Ps= probability that a given sparklet site was active. Our results suggest that both PKC and c‐Src promote persistent calcium sparklet activity, possibly by phosphorylating residue Y2122 rather than S1901 on Cav1.2c.
Based on the consistent demonstrations that the Ab peptide of Alzheimer's disease forms calcium permeant channels in artificial membranes, we have proposed that the intracellular calcium increase observed in cells exposed to Ab is initiated by calcium fluxes through Ab channels. We have found that a small four histidine peptide, NAHis04, potently inhibits the Ab-induced calcium channel currents in artificial lipids membrane. Here we report that NaHis04 also potently blocks the intracellular calcium increase which is observed in cells exposed to Ab. PC12 cells loaded with Fura 2AM show a rapid increase in fluorescence with rapidly return to base line after Ab is added to the medium. This fluorescence change occurs even when the medium contains nitrendipine, a voltage-gated calcium channel blocker, but fails to occur when application of Ab is preceded by addition of NAHis04. Steep dose response curves of percentage of responding cells and cell viability show that NAHis04 inhibits in the mm range in an apparently cooperative manner. We have developed numerous models of Ab pores in which the first part of the Ab sequence forms a large beta barrel ending at His13. We have modeled how up to four NAHis04 peptides may block these types of pores by binding to side chains of Ab residues Glu 11, His 13, and His 14.
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