Spatial association of the Cav1.2 calcium channel with α5β1-integrin

Engagement of α5β1-integrin by fibronectin (FN) acutely enhances Cav1.2 channel (CaL) current in rat arteriolar smooth muscle and human embryonic kidney cells (HEK293-T) expressing CaL. Using coimmunoprecipitation strategies, we show that coassociation of CaL with α5- or β1-integrin in HEK293-T cells is specific and depends on cell adhesion to FN. In rat arteriolar smooth muscle, coassociations between CaL and α5β1-integrin and between CaL and phosphorylated c-Src are also revealed and enhanced by FN treatment. Using site-directed mutagenesis of CaL heterologously expressed in HEK293-T cells, we identified two regions of CaL required for these interactions: 1) COOH-terminal residues Ser1901 and Tyr2122, known to be phosphorylated by protein kinase A (PKA) and c-Src, respectively; and 2) two proline-rich domains (PRDs) near the middle of the COOH terminus. Immunofluorescence confocal imaging revealed a moderate degree of wild-type CaL colocalization with β1-integrin on the plasma membrane. Collectively, our results strongly suggest that 1) upon ligation by FN, CaL associates with α5β1-integrin in a macromolecular complex including PKA, c-Src, and potentially other protein kinases; 2) phosphorylation of CaL at Y2122 and/or S1901 is required for association of CaL with α5β1-integrin; and 3) c-Src, via binding to PRDs that reside in the II–III linker region and/or the COOH terminus of CaL, mediates current potentiation following α5β1-integrin engagement. These findings provide new evidence for how interactions between α5β1-integrin and FN can modulate CaL entry and consequently alter the physiological function of multiple types of excitable cells

Regulation of L-type calcium channel sparklet activity by c-Src and PKC-α.

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

Regulation of L-type calcium channel sparklet activity by c-Src and PKC-α.

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

Regulation of L-type calcium channel sparklet activity by c-Src and PKC-α

The activity of persistent Ca(2+) sparklets, which are characterized by longer and more frequent channel open events than low-activity sparklets, contributes substantially to steady-state Ca(2+) entry under physiological conditions. Here, we addressed two questions related to the regulation of Ca(2+) sparklets by PKC-α and c-Src, both of which increase whole cell Ca(v)1.2 current: 1) Does c-Src activation enhance persistent Ca(2+) sparklet activity? 2) Does PKC-α activate c-Src to produce persistent Ca(2+) sparklets? With the use of total internal reflection fluorescence microscopy, Ca(2+) sparklets were recorded from voltage-clamped tsA-201 cells coexpressing wild-type (WT) or mutant Ca(v)1.2c (the neuronal isoform of Ca(v)1.2) constructs ± active or inactive PKC-α/c-Src. Cells expressing Ca(v)1.2c exhibited both low-activity and persistent Ca(2+) sparklets. Persistent Ca(2+) sparklet activity was significantly reduced by acute application of the c-Src inhibitor PP2 or coexpression of kinase-dead c-Src. Ca(v)1.2c constructs mutated at one of two COOH-terminal residues (Y(2122)F and Y(2139)F) were used to test the effect of blocking putative phosphorylation sites for c-Src. Expression of Y(2122)F but not Y(2139)F Ca(v)1.2c abrogated the potentiating effect of c-Src on Ca(2+) sparklet activity. We could not detect a significant change in persistent Ca(2+) sparklet activity or density in cells coexpressing Ca(v)1.2c + PKC-α, regardless of whether WT or Y(2122)F Ca(v)1.2c was used, or after PP2 application, suggesting that PKC-α does not act upstream of c-Src to produce persistent Ca(2+) sparklets. However, our results indicate that persistent Ca(2+) sparklet activity is promoted by the action of c-Src on residue Y(2122) of the Ca(v)1.2c COOH terminus

Integrin receptor activation triggers converging regulation of Cav1.2 calcium channels by c-Src and protein kinase A pathways.

L-type, voltage-gated Ca 2+ channels (Ca L ) play critical roles in brain and muscle cell excitability. Here we show that currents through heterologously expressed neuronal and smooth muscle Ca L channel isoforms are acutely potentiated following 5 1 integrin activation. Only the 1C pore-forming channel subunit is critical for this process. Truncation and site-directed mutagenesis strategies reveal that regulation of Cav1.2 by 5 1 integrin requires phosphorylation of 1C C-terminal residues S1901 and Y2122. These sites are known to be phosphorylated by PKA and c-Src, respectively, and are conserved between rat neuronal (Cav1.2c) and smooth muscle (Cav1.2b) isoforms. Kinase assays are consistent with phosphorylation of these two residues by PKA and c-Src. Following 5 1 integrin activation, native Ca L channels in rat arteriolar smooth muscle exhibit potentiation that is completely blocked by combined PKA and Src inhibition. Our results demonstrate that integrin-ECM interactions are a common mechanism for the acute regulation of Ca L channels in brain and muscle. These findings are consistent with the growing recognition of the importance of integrin-channel interactions in cellular responses to injury and the acute control of synaptic and blood vessel function. Voltage-gated calcium channels play critical roles in the regulation of calcium entry across the plasma membranes of excitable cells. L-type calcium channels (Ca L ), which are highly expressed in brain and muscle, are heteromeric transmembrane proteins composed of a poreforming 1C (Cav1.2) subunit along with accessory , 2 , , and sometimes subunits (1,2). The 1C subunit contains four highly-conserved repeat regions with 24 membrane-spanning domains, in addition to a variable length N-terminus and relatively long, intracellular C-terminus. The three 1C isoforms (neuronal, Cav 1.2c; smooth muscle, Cav 1.2b; cardiac, Cav 1.2a) exhibit significant sequence differences in their N-and C-termini but all are regulated by intracellular kinases in ways that uniquely determine calcium entry and cell excitability. The regulation of Ca L channels by serinethreonine kinases has been extensively investigated. PKG phosphorylates a conserved serine reside in the cytoplasmic I-II linker (3) of all three 1C isoforms, leading to inhibition of current. PKC phosphorylates N-terminal threonine residues in cardiac and smooth muscle isoforms (4-6) leading in most cases to potentiation of current. PKA phosphorylates all three 1C isoforms at a conserved C-terminal serine, (S1901 in Cav1.2c; S1928 in Cav1.2a), thereby mediating -adrenergic potentiation of calcium current in cardiac myocytes and neurons (7-9). PKA also regulates 1C in smooth muscle, but the functional consequences on calcium current are complicated by crossover activation of PKG, which is expressed at high levels in that tissue (10)