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Abstract: Objective: Src has been proposed to activate L-type calcium channel activity by binding to the α1c subunit. In the II–III linker region of this subunit there is a novel consensus sequence for Src binding. We have examined whether this site is a functional Src interaction site and investigated the effect displacing Src from this region has on calcium channel activity. Methods: In vitro binding assays were performed to map α1 subunit interaction sites. Cardiac myocytes were isolated enzymatically from rat ventricles. Whole cell patch-clamp technique was used to record Ca2+ channel currents in cells that had been loaded with the Src inhibitor PP1 and/or peptides with amino acid sequence corresponding to the hypothesized Src docking site. Co-immunoprecipitation and pull-down studies were undertaken to identify proteins co-complexing with the α1 subunit. Results: Peptides corresponding to the II–III linker region and C-terminal tail of the α1c subunit, but not scrambled peptide controls, were found to inhibit Src SH3 domain binding to the channel and significantly reduced the channel current amplitude. The II–III linker region peptide shifted the inactivation curve to the left whereas the C-terminal tail region peptide shifted the activation curve to the right when compared to scramble peptide controls. PP1-pre-treatment of myocytes also reduced the current amplitude, decreased the V 1/2 for channel inactivation and abolished any further effect on currents by Src binding peptides. The tyrosine kinase PYK2 was found to co-associate with Src and the channel, but PP1 pre-treatment reduced this co-association. Conclusions: Src binds to both the II–III linker and C-terminal tail regions of the α1c subunit to differentially modulate channel activity. PYK2 is also able to co-complex with Src when bound to this region of the channel but only when Src is catalytically active. Together the two kinases may synergistically regulate channel activity. [Copyright &y& Elsevier]

Carboxysomes are anabolic bacterial microcompartments that play an essential role in carbon fixation in cyanobacteria. This self-assembling proteinaceous organelle encapsulates the key CO 2 -fixing enzymes, Rubisco and carbonic anhydrase, using a polyhedral shell constructed by hundreds of shell protein paralogs. Deciphering the precise arrangement and structural organization of Rubisco enzymes within carboxysomes is crucial for understanding the formation process and overall functionality of carboxysomes. Here, we employed cryo-electron tomography and subtomogram averaging to delineate the three-dimensional packaging of Rubiscos within β-carboxysomes in the freshwater cyanobacterium Synechococcus elongatus PCC7942 that were grown under low light. Our results revealed that Rubiscos are arranged in multiple concentric layers parallel to the shell within the β-carboxysome lumen. We also identified the binding of Rubisco with the scaffolding protein CcmM in β-carboxysomes, which is instrumental for Rubisco encapsulation and β-carboxysome assembly. Using QconCAT-based quantitative mass spectrometry, we further determined the absolute stoichiometric composition of the entire β-carboxysome. This study and recent findings on the β-carboxysome structure provide insights into the assembly principles and structural variation of β-carboxysomes, which will aid in the rational design and repurposing of carboxysome nanostructures for diverse bioengineering applications.

Objective: Src has been proposed to activate L-type calcium channel activity by binding to the alpha1c subunit. In the II-III linker region of this subunit there is a novel consensus sequence for Src binding. We have examined whether this site is a functional Src interaction site and investigated the effect displacing Src from this region has on calcium channel activity., Methods: In vitro binding assays were performed to map alpha1 subunit interaction sites. Cardiac myocytes were isolated enzymatically from rat ventricles. Whole cell patch-clamp technique was used to record Ca(2+) channel currents in cells that had been loaded with the Src inhibitor PP1 and/or peptides with amino acid sequence corresponding to the hypothesized Src docking site. Co-immunoprecipitation and pull-down studies were undertaken to identify proteins co-complexing with the alpha1 subunit., Results: Peptides corresponding to the II-III linker region and C-terminal tail of the alpha1c subunit, but not scrambled peptide controls, were found to inhibit Src SH3 domain binding to the channel and significantly reduced the channel current amplitude. The II-III linker region peptide shifted the inactivation curve to the left whereas the C-terminal tail region peptide shifted the activation curve to the right when compared to scramble peptide controls. PP1-pre-treatment of myocytes also reduced the current amplitude, decreased the V(1/2) for channel inactivation and abolished any further effect on currents by Src binding peptides. The tyrosine kinase PYK2 was found to co-associate with Src and the channel, but PP1 pre-treatment reduced this co-association., Conclusions: Src binds to both the II-III linker and C-terminal tail regions of the alpha1c subunit to differentially modulate channel activity. PYK2 is also able to co-complex with Src when bound to this region of the channel but only when Src is catalytically active. Together the two kinases may synergistically regulate channel activity.