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

Abstract

Engagement of α₅β₁-integrin by fibronectin (FN) acutely enhances Cav1.2 channel (Ca_L) current in rat arteriolar smooth muscle and human embryonic kidney cells (HEK293-T) expressing Ca_L. Using coimmunoprecipitation strategies, we show that coassociation of Ca_L 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 Ca_L and α₅β₁-integrin and between Ca_L and phosphorylated c-Src are also revealed and enhanced by FN treatment. Using site-directed mutagenesis of Ca_L heterologously expressed in HEK293-T cells, we identified two regions of Ca_L 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 Ca_L colocalization with β₁-integrin on the plasma membrane. Collectively, our results strongly suggest that 1) upon ligation by FN, Ca_L associates with α₅β₁-integrin in a macromolecular complex including PKA, c-Src, and potentially other protein kinases; 2) phosphorylation of Ca_L at Y²¹²² and/or S¹⁹⁰¹ is required for association of Ca_L 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 Ca_L, mediates current potentiation following α₅β₁-integrin engagement. These findings provide new evidence for how interactions between α₅β₁-integrin and FN can modulate Ca_L entry and consequently alter the physiological function of multiple types of excitable cells.

integrins are heterodimeric transmembrane proteins that facilitate adhesion and communication of cells with their surroundings through binding to extracellular matrix proteins (ECM). Extensive studies implicate an essential role for integrins in the transmission of mechanical forces at focal adhesion sites (23, 25, 30). Integrin-mediated mechanotransduction is accomplished by activation of signaling molecules, such as focal adhesion kinase (FAK) and other tyrosine kinases, including c-Src, in conjunction with the recruitment of multiple proteins to focal adhesions and reorganization of the cytoskeleton (36). Integrins thereby translate physical forces into biochemical signals that ultimately alter cellular function.

Ca²⁺ entry through the L-type voltage-gated Cav1.2 calcium channel (Ca_L) is required for stretch-induced contraction of vascular smooth muscle (VSM). Intracellular pressurization of isolated arterial myocytes leads to enhancement of Ca_L current, suggesting that Ca_L may be modulated by mechanical stress applied to the plasma membrane (22). A link between Ca_L and integrins in transducing mechanical force has also been demonstrated in arterioles. The application of peptides containing an Arg-Gly-Asp (RGD) sequence, a cryptic binding motif in ECM that becomes exposed during vascular injury (7), inhibits pressure-dependent myogenic tone, through interactions with multiple VSM and endothelial cell integrins (8). Moreover, function-blocking antibodies against α₅-, β₁-, and β₃-integrin significantly reduce the degree of myogenic constriction to pressure elevation (29). The effects of integrin antibodies on myogenic tone are mediated in part by altered Ca²⁺ entry through Ca_L because electrophysiological studies reveal selective effects of integrin ligands on Ca_L current; the engagement and clustering of α₅β₁-integrin by insoluble fibronectin (FN) or anti-α₅-integrin antibody acutely potentiates Ca_L current, whereas engagement of α_vβ₃-integrin by vitronectin or soluble ligands inhibits Ca_L current in isolated VSM cells (41). Using site-directed mutagenesis strategies, two phosphorylation sites within the COOH terminus of the α_1C-subunit of Ca_L (α_1C-Ca_L) were identified as targets of protein kinase A (PKA) and c-Src that become activated downstream from α₅β₁-integrin ligation (14).

A critical remaining issue is the extent to which α₅β₁-integrin, PKA, c-Src, and Ca_L are spatially coupled within a cell. In the present study, we tested the hypothesis that α₅β₁-integrins spatially associate with Ca_L and that this association is required to modulate Ca_L function. Using human embryonic kidney cells (HEK293-T) overexpressing the neuronal isoform of Ca_L and VSM cells from rat skeletal muscle arterioles as model systems, the interactions between endogenous α₅β₁-integrin and Ca_L and between c-Src and Ca_L were examined using immunoprecipitation (IP), immunoblotting (IB), immunofluorescence confocal microscopy, and patch-clamp methods.

METHODS

Cell culture and transient transfection.

HEK293-T/(TSA201) cells were maintained in 10% FBS (Hyclone)-DMEM (high glucose) supplemented with 2 mM glutamine, 100 units of penicillin, and 100 μg of streptomycin in 5% CO₂ at 37°C. When cells reached 70–80% confluence, wild-type (WT) or mutant neuronal Cav1.2 calcium channel (Ca_L) cDNAs composed of α_1C-c (6 μg), β_1b (3 μg), and α₂δ (2 μg) subunits, in a total volume of 14 μl, were transfected into HEK293-T cells grown in 60-mm tissue culture dishes containing 2.5 ml of DMEM with Lipofectamine 2000 (20 μl) for 18–24 h. Green fluorescent protein (GFP; 0.5–1 μg) was cotransfected with Ca_L to monitor transfection efficiency. Untransfected cells were used as a control. Cells were incubated for another 24 h posttransfection before passing and plating onto either FN (catalog no. 354403, BD BioCoat, BD Biosciences), BSA (2%), or poly-l-lysine-treated plates for 1 h, followed by protein isolation or immunofluorescence imaging. To account for variation in the expression level of α_1C-Ca_L due to variation in transfection efficiency among the different Ca_L mutants, the experiments for all Ca_L mutants were performed in parallel with WT-Ca_L transfection using the same passage of cells.

For electrophysiological experiments, HEK293-T cells were transfected using calcium phosphate, after which patch-clamp recordings were performed 48–72 h posttransfection as previously described (14). Only single cells expressing GFP were used for electrophysiological protocols.

The various mutations in the α_1C-COOH terminus were generated using site-directed mutagenesis as described previously (14) and as illustrated in Fig. 1.

Fig. 1.

Schematic depicting the domain structure of wild-type (WT) L-type voltage-gated Cav1.2 calcium channel (Ca_L) α_1C-subunit and mutant α_1C-Ca_L constructs used in this study. I–IV, the four transmembrane repeat of α_1C. EF and DCT, EF hand motif and the distal COOH terminus inhibitory domain; P, proline-rich domain; truncated α_1C-Ca_L, WT α_1C-Ca_L missing the distal COOH-terminal 280 amino acids; ΔP1/ΔP2 α_1C-Ca_L, WT α_1C-Ca_L missing the two proline-rich domains (aa 1955–1959; aa 1967–1973); S¹⁹⁰¹A/Y²¹²²F α_1C-Ca_L, two single-point mutations at serine (S¹⁹⁰¹A) and tyrosine (Y²¹²²F) in full-length neuronal WT-Ca_L.

Isolation of skeletal muscle arterioles.

The isolation of first- and second-order arterioles from rat cremaster muscle was performed as described previously (21, 40, 41), except for addition of 1× Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific) into the dissection chamber immediately before dissection. All animal protocols were approved by the University of Missouri Animal Care and Use Committee and conformed to the Public Health Service Policy for the Humane Care and Use of Laboratory Animals (PHS Policy, 1996).

Electrophysiological recording.

Patch-clamp recordings were made using an EPC9 amplifier under the control of Pulse software (HEKA Instruments) in the conventional whole cell mode, as previously described (14). Pipettes were filled with Cs⁺ pipette solution containing (in mM) 110 CsCl, 20 TEA chloride, 10 EGTA, 2 MgCl₂, 10 HEPES, and 1 CaCl₂ (pH 7.2 with CsOH). Ba²⁺ was used as the charge carrier to increase the magnitude of the inward current and to minimize calcium-dependent current inactivation. Cs⁺ was used to block endogenous K⁺ current.

Immunoprecipitation and immunoblotting.

Proteins from HEK293-T cells were isolated as described by Ling et al. (27) with minor modifications. The lysis buffer was composed of 1% Triton X-100 containing the following: 2.5 mM Tris, pH 7.4, 13.8 mM NaCl, 0.3 mM KCl, 2 mM EDTA, 1 mM EGTA, 10 mM NaF, 1 mM benzamidine, 1 mM Na₃VO₄, 4 mM Pefabloc, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 10 μg/ml of calpain I and II, and 10 μg/ml pepstatin A. Isolation of total proteins in arterioles was performed as described above except for using 0.25% Triton X-100, along with 2% n-octyl-β-d-glucopyranoside to lyse arterioles. Arterioles (1st order and 2nd order) were snap-frozen in liquid nitrogen after dissection. Vessels were cut into smaller pieces and pulverized with a plastic pestle in 0.65-ml centrifuge tubes in the presence of an optimal amount of lysis buffer. Tubes containing arterioles were frequently dipped into liquid nitrogen and kept on dry ice during pulverization. Three hundred to four hundred micrograms of total protein (150–250 μg for arterioles) were precleared with protein A/G agarose beads (catalog no. SC-2003; Santa Cruz) in the presence of either rabbit or mouse IgG (catalog no. PP64 and PP54, respectively; Millipore), followed by overnight IP in lysis buffer at 4°C with one of the following antibodies: 1) either rabbit anti-Cav1.2 polyclonal antibody (Ab) (3.2 μg; catalog no. AB5156, Millipore) or pan Calcium Channel Ab (catalog no. Ab6298, Abcam), recognizing the α_1C-subunit of Ca_L (α_1C-Ca_L); 2) mouse anti-human β₁-integrin monoclonal Ab (5 μg, clone B3B11, catalog no. MAB2251Z, Millipore); 3) purified mouse anti-human α₅-integrin Ab (5 μg, clone 1/CD49e, catalog no. 610634, BD Biosciences); 4) mouse anti-pp60c-Src (4 μg, clone GD11, catalog no. 05-184, Millipore); or 5) anti-phosphorylated-Tyr Ab (5 μg, clone 4G10, catalog no. 05-321, Millipore). The immune complex was captured using either protein A (catalog no. SC-2001, Santa Cruz) or protein G agarose beads (catalog no. 17088601, Amersham) prior to SDS-PAGE (6%), followed by overnight transfer to nitrocellulose membrane (0.45 nm) and IB. To confirm binding specificity, the whole cell lysate was also immunoprecipitated with 1) control IgG for the appropriate host species and captured by protein A or protein G beads, or 2) protein A or protein G beads alone. Equal loading of total protein was confirmed by Ponceau staining (Sigma) before IB. Ca_L was detected using rabbit anti-Ca_L α_1C-subunit Ab (catalog no. AB5156, 1:6,500 dilution) for HEK293-T cells or pan Calcium Channel Ab (1:2,500 dilution) for rat arterioles. Endogenous β₁-integrin was detected using mouse anti-human β₁-integrin Ab or rabbit anti-rat β₁-integrin Ab (catalog no. MAB2251Z, 1:1,400 dilution and catalog no. AB1952P, 1:1,500 dilution, respectively; Millipore). c-Src or phosphorylated c-Src was detected using anti-pp60c-Src (catalog no. 05-184, 1:1,200 dilution, Millipore) or rabbit anti-rat phospho-Src family (Tyr416) Ab (catalog no. 2101S, 1:1,000 dilution, Cell Signaling Technology), respectively. The immune complexes were incubated with either goat anti-rabbit IgG-horseradish peroxidase (HRP)-conjugated antibody (H&L, 1:120,000 dilution, catalog no. AP132P, Millipore) or donkey anti-mouse-IgG-HRP-conjugated antibodies (H&L, 1:60,000 dilution, catalog no. 715-035-150, Jackson Research Laboratory) followed by detection with Pierce SuperSignal extended Dura or Femto substrate (Thermo Scientific). After probing, the blot was stripped with Restore Western blot stripping buffer (Thermo Scientific) and reprobed with either anti-α_1C-Ca_L, anti-β₁-integrin, or anti-c-Src Ab to assess the relative loading. For detection of the immune complex from arterioles, the blot was processed as described above except that SuperSignal Western Blot Enhancer (catalog no. 46641, Thermo Scientific) was used according to the manufacturer's instruction before IB. The results were captured by exposure to Kodak Biomax MR, MS, or Light film (Sigma), and the band intensity for the protein of interest was quantified using Bio-Rad Quantity One. Quantification of co-IP was determined as described by Cherubini et al. (4). The ratio of immunoprecipitated α_1C-Ca_L to β₁-integrin in WT-α_1C-Ca_L-expressing cells was set as 1. The ratio of immunoprecipitated α_1C-Ca_L to β₁-integrin in mutant α_1C-Ca_L-expressing cells was expressed relative to WT-α_1C-Ca_L.

Immunofluorescence confocal imaging.

To determine the distribution and expression of α_1C-Ca_L and β₁-integrin, cells were plated on poly-l-lysine or FN-coated tissue culture dishes with coverglass bottoms (Fluorodish, catalog no. FD35-100, WPI) for 1 h after 24 h of transfection, followed by fixation with 2% paraformaldehyde and staining as described previously (40). α_1C-Ca_L was determined using the same antibody for IP, followed by donkey anti-rabbit IgG conjugated to Alexa 488 (1:1,600 dilution, catalog no. A21206, Invitrogen). β₁-Integrin was detected using mouse anti-human β₁-integrin Ab (1:75 dilution, clone HUTS 21, catalog no. 556037, BD Biosciences), followed by goat anti-mouse IgG conjugated with Alexa 555 (1:1,400 dilution, catalog no. A21424, Invitrogen). Counterstaining of nuclei was performed using TO-PRO-3 (0.5 μM, catalog no. T3605, Invitrogen). Staining of paxillin or vinculin was determined using rabbit anti-paxillin polyclonal Ab (1:300 dilution, catalog no. AB3794, Millipore) or mouse anti-vinculin antibody (clone V284, 1:80 dilution; catalog no. 05-386, Millipore). Cells were incubated with donkey anti-rabbit IgG conjugated with Alexa 488 alone or goat anti-mouse IgG conjugated to Alexa 555 alone, or the combination of both as a negative control to confirm the specificity of staining. ProLong Gold Antifade Reagent (catalog no. P36934, Invitrogen) was applied to the sample after staining, followed by storage at −30°C until images were captured.

Immunofluorescence images were taken using a laser-scanning confocal system (Leica TCS SP5 Microsystems) attached to an upright microscope (Leica DM 6000 CS) and were collected using a ×63 oil objective (numerical aperture 1.42). Excitation of the fluorophores was achieved using argon 488, He/Ne 543, or He/Ne 633 nm laser combinations with pinhole size set at 1 airy disk. Images were taken at 512 × 512 pixels with a step size of 0.2 μm and a zoom factor of 5. The image size was ∼49.2 μm².

To prevent bleed-through of different wavelengths that might interfere with colocalization analysis, all the images were taken using a sequential acquisition procedure at wavelengths of 488/519 nm (excitation/emission) for the α_1C-Ca_L-subunit; 543/569 nm (excitation/emission) for β₁-integrin, and 633/656 nm (excitation/emission) for nuclear staining.

Quantification of α_1C-Ca_L association with β₁-integrin.

The association of α_1C-Ca_L with β₁-integrin was analyzed with ImageJ software (version 1.39d, National Institutes of Health, Bethesda, MD). The degree of α_1C-Ca_L association with β₁-integrin was quantified using Pearson's coefficient, Mander's coefficient, and intensity correlation analysis (ICA) (24, 33). The advantage of using ICA analysis is that it avoids taking the overlap of randomly distributed proteins into account for colocalization if the intensity of the two target proteins does not vary coincidently (24). ICA values are distributed between −0.5 and +0.5. ICA values close to 0 indicate random staining, values −0.5 ≤ ICA < 0 represent segregated staining, and values 0 < ICA ≤ +0.5 represent interdependent staining. The degree of α_1C-Ca_L association with β₁-integrin was normalized to the average ICA value for the coassociation of paxillin with vinculin (0.25), which was shown previously to be very strong (37).

Statistical analysis.

Results are presented as means ± SE from at least three independent experiments for IP or four independent experiments for immunofluorescence confocal imaging (IF). Statistical differences were analyzed using GraphPad InStat or Prism (version 3.06 and version 5.0, respectively; GraphPad, San Diego, CA) with either analysis of variance or unpaired t-tests. P < 0.05 was considered significant.

RESULTS

Ca_L association with α₅β₁-integrin depends on adhesion to fibronectin.

To determine whether α_1C-Ca_L associated with α₅β₁-integrin, IP of whole cell lysates from HEK293-T cells expressing the neuronal Ca_L using either anti-β₁- or anti-α₅-integrin antibody was followed by IB to probe for the presence of α_1C-Ca_L. The neuronal Ca_L isoform was used as a model system because α_1C-Ca_L mutants had been previously generated by site-directed mutagenesis strategies; neuronal and smooth muscle (SM) cell (SMC) Ca_L isoform share 90% homology in amino acid sequence, and we previously demonstrated that heterologously expressed rat neuronal and SMC isoforms (Cav1.2c and Cav1.2b, respectively) showed a similar degree of current potentiation following α₅β₁-integrin ligation (14). We first determined the association between α_1C-Ca_L and β₁-integrin in cells plated on poly-l-lysine-treated dishes. As shown in Fig. 2A, no appreciable α_1C-Ca_L was observed after IP using anti-β₁-integrin Ab in either control (untransfected cells, lane 2) or cells expressing WT α_1C-Ca_L (lane 7) when plated on poly-l-lysine-treated dishes. However, when cells expressing WT α_1C-Ca_L were plated on FN (Fig. 2B), α_1C-Ca_L was detected after IP of the lysate using either anti-α₅- (lane 4) or anti-β₁-integrin antibody (lane 3). No specific α_1C-Ca_L band was observed in cells expressing α_1C-Ca_L plated on BSA-coated dishes after IP using anti-β₁-integrin or anti-c-Src Ab (Supplemental Fig. S1A, lanes 6 and 7, respectively) or after IP of the lysate using mouse IgG or protein-G (Fig. 2B, lanes 2, 5, and 9). These results indicate that the association of α_1C-Ca_L with α₅ or β₁-integrin in HEK293-T cells is specific and depends on cell adhesion to FN.

Fig. 2.

Ca_L association with α₅- or β₁-integrin depends on adhesion of human embryonic kidney cells (HEK293-T) to fibronectin (FN). A: representative blot showing coimmunoprecipitation (co-IP) protocol on protein lysed from control (untransfected) or neuronal WT-Ca_L-expressing HEK293-T cells plated onto poly-l-lysine-treated dishes. No α_1C-Ca_L appears to be pulled down by IP with anti-β₁-integrin antibody (Ab) in either control (untransfected) or WT Ca_L-expressing cells (lanes 2 and 7, respectively). Expression of α_1C-Ca_L was confirmed in cells expressing WT-Ca_L by immunoblotting (IB) with anti-α_1C-Ca_L Ab, showing a specific band at molecular mass 190 to 240 kDa (lane 4). B: representative blot showing pull-down of α_1C by anti-α₅ or anti-β₁-integrin Ab (lanes 4 and 3, respectively) only from cells expressing WT-Ca_L, not control (untransfected) cells (lanes 7 and 8, respectively), when plated onto FN-coated plates before IP and IB. All blots were repeated at least three times. Prot-G, protein-G.

To confirm that the effect of FN on α_1C-Ca_L association with β₁-integrin was not an artifact of Ca_L overexpression in HEK293-T cells, we examined the same interaction in lysates from arteriolar SM. Arterioles were dissected and then incubated with exogenous FN abluminally (300 μg/ml) for 1 h. When arteriolar SM lysates were immunoprecipitated with anti-α_1C-Ca_L, β₁-integrin was detected only in arterioles incubated with FN (Fig. 3A, lanes 1 and 2), compared with control (lanes 5 and 6). The same blot was also stripped and reprobed for the expression of α_1C-Ca_L. In addition, incubation with FN did not substantially change the level of α_1C-Ca_L expression in arterioles (Supplemental Fig. S1B). These results indicate that the association of α_1C-Ca_L with β₁-integrin in SMC depends on ligation of β₁-integrin by FN.

Fig. 3.

α_1C-Ca_L associates with β₁-integrin in arteriolar smooth muscle incubated with FN. A: representative blot showing co-IP protocol on protein lysed from arteriolar smooth muscle (pulled down with anti-α_1C-Ca_L). β₁-Integrin and α_1C-Ca_L coimmunoprecipitated when arterioles were incubated with FN for 1 h (lanes 1 and 2, respectively). Lanes 1 and 6 were immunoprecipitated with anti-α_1C-Ca_L from Chemicon, whereas lanes 2 and 5 were immunoprecipitated with anti-α_1C-Ca_L from Abcam. Lanes 3 and 4 were lysates alone isolated from control arterioles or arterioles incubated with FN, respectively. The absence of coimmunoprecipitated β₁-integrin by rabbit IgG (lane 7) indicates binding specificity. B: representative blot showing co-IP protocol on protein lysed from arteriolar smooth muscle (pulled down with anti-α_1C-Ca_L). c-Src coimmunoprecipitated with α_1C-Ca_L whether or not arterioles were incubated with FN (lanes 1 and 2 vs. lanes 5 and 6, respectively). IP was performed as described in A. Abluminal incubation of arterioles with exogenous FN did not change the degree of association between α_1C-Ca_L and c-Src (lane 2, 90% and lane 5, 86% of arterioles alone, respectively). The absence of coimmunoprecipitated c-Src by rabbit IgG (lane 7) indicates binding specificity. C: representative blot showing co-IP protocol on protein lysed from arteriolar smooth muscle (pulled down with anti-α_1C-Ca_L). Phosphorylated c-Src was only observed in arterioles incubated with exogenous FN (lanes 1 and 2). IP was performed as described in A. The absence of phosphorylated c-Src by rabbit IgG (lane 7) indicates binding specificity.

c-Src, a tyrosine kinase activated upon integrin ligation by ECM ligands (18), has been shown to modulate Ca_L function in SMC (2, 3, 19, 39). Thus, we also examined whether c-Src associated with α_1C-Ca_L in arteriolar SM and whether FN was important for this association. As shown in Fig. 3B, c-Src was detected in immunoprecipitates using two different anti-α_1C-Ca_L antibodies (Fig. 3B, lanes 1 and 2). However, abluminal incubation with FN did not substantially change the degree of c-Src association with α_1C-Ca_L (Fig. 3B, lanes 1 and 2 vs. lanes 5 and 6). Next, we tested whether incubation with FN altered the phosphorylation status of c-Src in association with α_1C-Ca_L. A basal level of phosphorylated c-Src was observed in untreated control lysate (Fig. 3C, lane 3) and in lysate from arteriolar SM incubated with FN (lane 4). In contrast to Fig. 3B, phosphorylated c-Src coassociated with α_1C-Ca_L only in arteriolar SM incubated with FN, not in control arteriolar SM (Fig. 3C, lanes 1 and 2 vs. lanes 5 and 6, respectively). These results suggest that the association of α_1C-Ca_L with c-Src in arteriolar SM is independent of FN, whereas the association of α_1C-Ca_L with phosphorylated c-Src depends on integrin engagement by FN.

The COOH terminus of α_1C-Ca_L is essential for association with β₁-integrin.

To determine whether the specific regions in the COOH terminus of α_1C are required for the association of α_1C-Ca_L with α₅β₁-integrin, three mutant α_1C-Ca_L constructs were overexpressed in HEK293-T cells (Fig. 1). If any of the specific regions is required for association between the two proteins, we predicted to observe less α_1C-Ca_L pulled down by anti-β₁-integrin Ab in cells expressing the mutant α_1C-Ca_L. As shown in Fig. 4A, truncated α_1C-Ca_L exhibited a reduced level of association with β₁-integrin (lane 8) and a reduced level of association with c-Src (lane 7), compared with their respective levels of association in WT α_1C-Ca_L (lanes 1 and 2, respectively). Figure 4B shows the same membrane after stripping and reprobing for β₁-integrin as a loading control. The expression of β₁-integrin, c-Src (Supplemental Fig. S3A), and the degrees of association between β₁-integrin and c-Src were not changed in cells expressing truncated α_1C-Ca_L, compared with WT α_1C-Ca_L (quantified data not shown). When normalized to the total amount of WT α_1C-Ca_L, the associations between truncated α_1C-Ca_L and β₁-integrin as well as between truncated α_1C-Ca_L and c-Src were reduced to 40 ± 9% and 55 ± 8% of WT α_1C-Ca_L, respectively (Fig. 4C). These results provide evidence for the requirement of the distal COOH terminus in the associations between α_1C-Ca_L and β₁-integrin and between α_1C-Ca_L and c-Src.

Fig. 4.

Requirement of the α_1C-Ca_L COOH terminus for association of α_1C-Ca_L with c-Src or β₁-integrin. A: representative blot comparing WT- and truncated α_1C-Ca_L immunoprecipitated with either anti-c-Src, anti-β₁-integrin Ab, or mouse IgG, then probed for α_1C. α_1C was only detected in lysates immunoprecipitated with anti-c-Src (lanes 2 and 7) or anti-β₁-integrin Ab (lanes 1 and 8) but not with mouse IgG (lanes 3 and 6). Relatively lower amounts of α_1C-Ca_L were pulled down by anti-c-Src or anti-β₁-integrin Ab in truncated α_1C-Ca_L (lanes 7 and 8, respectively), compared with WT. B: the same blot in A after stripping and reprobing for β₁-integrin as a control for IP and immunoblotting. C: summary graph of the relative levels of immunoprecipitated α_1C in cells expressing WT- or truncated α_1C-Ca_L. Values were obtained as described in methods and are based on the average of at least three experiments. *P < 0.05 vs. WT + anti-β₁-integrin Ab or WT + anti-c-Src Ab.

To confirm that the reduced associations between α_1C-Ca_L and β₁-integrin and between α_1C-Ca_L and c-Src were not due to decreased expression of truncated α_1C-Ca_L construct, we increased the amount of total protein for IP from cells expressing the truncated α_1C-Ca_L construct to twofold that of WT α_1C-Ca_L and reexamined the association of truncated α_1C-Ca_L with β₁-integrin or with c-Src. As shown in Supplemental Fig. S2, A and C, reduced degrees of association between α_1C-Ca_L and β₁-integrin (Fig. S2A, lane 3), α_1C-Ca_L and phosphorylated tyrosine (Tyr-Pi, Fig. S2A, lane 2), and α_1C-Ca_L and c-Src (Fig. S2A, lane 1) were still observed in cells expressing truncated α_1C-Ca_L, when compared with the degrees of their respective associations in cells expressing WT α_1C-Ca_L (70 ± 7%, 51 ± 4%, and 58 ± 11%; % of WT, respectively). However, the expressions of β₁-integrin and c-Src, the degrees of associations between β₁-integrin and c-Src, or between β₁-integrin and proteins containing phosphorylated tyrosine were not changed in cells expressing truncated α_1C-Ca_L, compared with WT α_1C-Ca_L (Supplemental Figs. S2B and S3B; quantified data not shown). These results suggest a requirement for the distal COOH terminus in α_1C-Ca_L association with the β₁-integrin and c-Src.

The collaborative contribution of PKA and c-Src in the association of Ca_L with β₁-integrin.

We previously showed that α₅β₁-integrin engagement leads to potentiation of Ca_L current via phosphorylation of α_1C-Ca_L serine (S¹⁹⁰¹) and tyrosine (Y²¹²²) by PKA and c-Src, respectively (14). Thus, a next logical step was to determine whether phosphorylation of α_1C-Ca_L by PKA and c-Src is required for Ca_L association with β₁-integrin or c-Src. As shown in Fig. 5A, reduced associations between α_1C-Ca_L and β₁-integrin (lane 3), α_1C-Ca_L and Tyr-Pi (lane 2), and α_1C-Ca_L and c-Src (lane 1) were observed in cells expressing S¹⁹⁰¹A/Y²¹²²F-Ca_L, compared with WT α_1C-Ca_L (Fig. 5C, 47 ± 8%, 48 ± 12%, and, 27 ± 7%; % of WT, respectively). However, the expressions of β₁-integrin (Fig. 5B) and c-Src, the degrees of associations between β₁-integrin and c-Src, or between β₁-integrin and proteins containing phosphorylated tyrosine were unchanged in cells expressing S¹⁹⁰¹A/Y²¹²²F-Ca_L, compared with WT α_1C-Ca_L (Supplemental Fig. S4A, quantified data not shown). These results suggest that phosphorylation of α_1C-Ca_L by PKA and/or c-Src is required for the associations between α_1C-Ca_L and β₁-integrin and between α_1C-Ca_L and c-Src.

Fig. 5.

S¹⁹⁰¹ and Y²¹²² in α_1C-Ca_L are required for α_1C-Ca_L association with β₁-integrin or c-Src. A: representative blot comparing WT α_1C-Ca_L and S¹⁹⁰¹A/Y²¹²²F α_1C-Ca_L immunoprecipitated with either anti-c-Src, anti-phosphorylated tyrosine (Tyr-Pi), or anti-β₁-integrin Ab, and probed for α_1C. Relatively lower amounts of α_1C-Ca_L appear to be pulled down by anti-c-Src, anti-Tyr-Pi, or anti-β₁-integrin Ab (lanes 1, 2, and 3, respectively) in S¹⁹⁰¹A/Y²¹²²F α_1C-Ca_L-expressing cells, compared with WT-α_1C-Ca_L. B: the same blot in A after stripping and reprobing for β₁-integrin. C: summary graph showing the relative levels of immunoprecipitated α_1C in WT- or S¹⁹⁰¹A/Y²¹²²F-Ca_L. Values were obtained as described in methods and are based on the average of at least three experiments. *P < 0.05 vs. WT + anti-β₁-integrin Ab, WT + anti-Tyr-Pi Ab, or WT + anti-c-Src Ab.

The proline-rich domains in the COOH terminus are required for the association of α_1C-Ca_L with β₁-integrin.

Proline-rich domains (PRDs) are binding motifs for Src homology 3 domain (SH3)-containing proteins, such as c-Src (34). Two PRDs have been identified in the COOH terminus of α_1C-Ca_L and have been shown to mediate the association of Ca_L with some proteins containing SH3 domains (12, 13). Thus, we investigated the roles of the COOH-terminal PRDs in determining α_1C-Ca_L association with β₁-integrin or c-Src by expressing a ΔP1/ΔP2-Ca_L mutant with selective deletion of both PRDs in the COOH terminus of α_1C. As shown in Fig. 6A, significantly less association between α_1C-Ca_L and β₁-integrin (lane 6) and between α_1C-Ca_L and c-Src (lane 8), was observed in cells expressing the ΔP1/ΔP2 α_1C-Ca_L mutant, compared with WT α_1C-Ca_L (Fig. 6C, 54 ± 9%, 54 ± 6%; % of WT, respectively). However, the expression of β₁-integrin (Fig. 6B), c-Src, and the degree of association between β₁-integrin and c-Src was unchanged in cells expressing ΔP1/ΔP2 α_1C-Ca_L (Supplemental Fig. S4B, quantified data not shown). These results suggest that the PRDs in the α_1C-Ca_L COOH terminus are partially required for associations between α_1C-Ca_L and β₁-integrin and between α_1C-Ca_L and c-Src.

Fig. 6.

Requirement of proline-rich domains (PRDs) in the α_1C-Ca_L COOH terminus for α_1C-Ca_L association with β₁-integrin or c-Src. A: representative blot comparing WT α_1C-Ca_L and ΔP1/ΔP2 α_1C-Ca_L immunoprecipitated with anti-c-Src, anti-α₅-integrin, or anti-β₁-integrin Ab and probed for α_1C. Relatively lower amounts of α_1C-Ca_L appear to be pulled down by anti-c-Src or anti-β₁-integrin Ab (lanes 8 and 6, respectively) in cells expressing ΔP1/ΔP2 α_1C-Ca_L, compared with WT α_1C-Ca_L. B: the same blot in A after stripping and reprobing for β₁-integrin. C: summary graph showing the relative levels of immunoprecipitated α_1C in WT- or ΔP1/ΔP2-Ca_L. Values were obtained as described in methods and are based on the average of at least three experiments. *P < 0.05 vs. WT + anti-c-Src Ab or WT + anti-β₁-integrin Ab.

Functional modulation of Ca_L current by α₅β₁-integrin is independent of the PRDs in the COOH terminus.

Given the results shown in Figs. 5 and 6, we also investigated the roles of the two PRDs in the modulation of Ca_L current following α₅β₁-integrin engagement. As shown in Fig. 7A, the application of α₅β₁-integrin antibody to the bath solution potentiated whole cell Ca_L current in HEK293-T cells expressing WT-Ca_L, consistent with our previous findings (14). To our surprise, no significant electrophysiological difference was observed between WT-Ca_L and ΔP1/ΔP2-Ca_L (82% of WT) in the degree of current potentiation by α₅β₁-integrin engagement, suggesting that the COOH-terminal PRDs are not required for potentiation of Ca_L current by α₅β₁-integrin engagement. When the degree of Ca_L current potentiation following α₅β₁-integrin ligation in the mutant constructs was normalized to that of WT, both the truncated α_1C-Ca_L and S¹⁹⁰¹A/Y²¹²²F-Ca_L mutants displayed significant impairment in the amount of current potentiation by integrin ligation (Fig. 7B, 9% and 1% potentiation of WT-Ca_L, respectively; see also Ref. 14). The above results confirm that S¹⁹⁰¹ and Y²¹²² residues in the COOH terminus of α_1C-Ca_L are required for Ca²⁺ current potentiation following α₅β₁-integrin ligation. However, selective deletion of the two PRDs in the COOH terminus of α_1C-Ca_L does not impair the current potentiation by α₅β₁-integrin engagement.

Fig. 7.

Electrophysiological protocols to determine the effects of COOH terminus-specific regions on modulation of Ca_L current by α₅β₁-integrin. A: representative recordings of whole cell Ca_L current from HEK293-T cells expressing WT-Ca_L (left) or ΔP1/ΔP2-Ca_L (right). Top traces in both panels show the amount of basal current in cells expressing WT- or ΔP1/ΔP2-Ca_L, and bottom traces show the current potentiation by α₅β₁-integrin Ab (10 μg/ml). No substantial difference in the relative amount of Ca_L current potentiation was observed in cells expressing ΔP1/ΔP2-Ca_L, compared with WT-Ca_L. B: summary graph showing peak current potentiation by α₅β₁-integrin in WT-Ca_L, truncated α_1C-Ca_L, or ΔP1/ΔP2-Ca_L. Ca_L current potentiated by α₅β₁-integrin Ab was significantly attenuated in truncated α_1C-Ca_L or in S¹⁹⁰¹A/Y²¹²²F-Ca_L, but not in ΔP1/ΔP2-Ca_L. Number of cells recorded is as follows: WT-Ca_L, n = 18; n = 8 in cells expressing ΔP1/ΔP2-Ca_L, S¹⁹⁰¹A/Y²¹²²F-Ca_L, or truncated α_1C-Ca_L. *P < 0.05 vs. WT-Ca_L.

Immunofluorescence imaging analysis demonstrates association of Ca_L with β₁-integrin on the plasma membrane.

The COOH terminus of α_1C-Ca_L has been shown to mediate membrane targeting of the channel (11), so we further investigated the effect of the abovementioned regions in the COOH terminus on membrane targeting of α_1C-Ca_L and its association with β₁-integrin. Immunofluorescence confocal microscopy (IF) was performed to determine the expression, distribution, and relative localization of α_1C-Ca_L and β₁-integrin on the plasma membrane. Images of cells without staining or cells stained with either anti-mouse IgG, anti-rabbit IgG, or the combination of both, exhibited only faint, diffuse, and nonspecific staining (Supplemental Fig. S5). As shown in Fig. 8, neither the membrane expression of α_1C-Ca_L nor β₁-integrin was detectably altered in S¹⁹⁰¹A/Y²¹²²F-Ca_L, truncated α_1C-Ca_L, or ΔP1/ΔP2-Ca_L, compared with WT-Ca_L.

Fig. 8.

Confocal immunofluorescence image analysis to determine the distribution and association of α_1C-Ca_L and β₁-integrin on the plasma membrane. Representative confocal immunofluorescence images from the cells expressing WT-, truncated α_1C-, S¹⁹⁰¹A/Y²¹²²F-, or ΔP1/ΔP2-Ca_L. α_1C and endogenous β₁-integrin expression on the plasma membrane appear as green and red punctate staining in a, e, i, and m and in b, f, j, and n, respectively. The colocalization of α_1C-Ca_L with β₁-integrin on the membrane appears as yellow to orange punctate staining (c, g, k, and o). A noticeably higher degree of α_1C-Ca_L colocalization with β₁-integrin was seen in cells expressing WT-α_1C-Ca_L, compared with mutant α_1C-Ca_L construct-expressing cells. Merged images of the previous three panels with nuclei counterstaining appear in blue (d, h, l, and p). Endogenous paxillin and vinculin staining in HEK293-T cells appears as green and red in q and r, respectively. The degree of colocalization between paxillin and vinculin (yellow to orange staining; s) appears to be much higher than that between α_1C-Ca_L and β₁-integrin in the WT- or mutant α_1C-Ca_L-expressing cells. Merged image with nuclear counterstaining in blue appears in t. Magnification, ×63 oil; scale bar, 10 μm.

When we examined the association between WT α_1C-Ca_L and endogenous β₁-integrin on the plasma membrane, a moderate degree of colocalization was observed. To quantify the degree of colocalization between α_1C-Ca_L and β₁-integrin, we used the overlap of paxillin and vinculin staining as a normalization standard for the following analyses, since these two proteins highly associate at the focal adhesion site (ICA value 0.25) (37). The relatively high degree of colocalization between paxillin and vinculin was set as 100% for subsequent analysis of colocalization between α_1C-Ca_L and β₁-integrin. We used Pearson's coefficient (R), Mander's coefficient (R_r), and ICA to determine the degree of α_1C-Ca_L-β₁-integrin colocalization, as listed in Table 1. The degrees of α_1C-Ca_L colocalization with β₁-integrin in WT-Ca_L, truncated α_1C-Ca_L, S¹⁹⁰¹A/Y²¹²²F-Ca_L, or ΔP1/ΔP2 α_1C-Ca_L were significantly less than the degree of colocalization between paxillin and vinculin (60–88% of paxillin-vinculin colocalization).

Table 1.

Semiquantitative analysis of Ca_L association with β₁-integrin

	Rr, % of Pax + Vin	R, % of Pax + Vin	ICA, %	ICA, % of Pax + Vin
Paxillin-vinculin	100	100	0.251 ± 0.021	100
WT	60.6 ± 2.7	88.5 ± 1.5	0.22 ± 0.009	85.1 ± 4.2
Truncated-α1C	59.2 ± 4.8	87.9 ± 2.6	0.211 ± 0.01	84.1 ± 5.0
S1901A/Y2122F	56.5 ± 4.5	84.5 ± 2.5	0.21 ± 0.011	84.8 ± 5.3
ΔP1/ΔP2	55.4 ± 5.1	84.1 ± 1.7	0.185 ± 0.013	75.2 ± 7.4

Values are means ± SE, taken from at least eight cells in either wild-type (WT) or mutant L-type voltage-gated Cav1.2 calcium channel (Ca_L) per experiment. The degree of Ca_L association with β₁-integrin was normalized using the association of paxillin (Pax) and vinculin (Vin) as a reference and set as 100% for Pearson's, Mander's, or intensity correlation analysis (ICA). The value of Ca_L association with β₁-integrin in WT, truncated α_1C-, S¹⁹⁰¹A/Y²¹²²F, or ΔP1/ΔP2-Ca_L is presented as the percentage ± SE of paxillin-vinculin association. R_r, Pearson's coefficient; R, Mander's coefficient. Statistical analysis was performed on the basis of the average from at least four individual experiments.

Using ICA analysis (Fig. 9), modest, but not significant, decreases in the degrees of α_1C-Ca_L association with β₁-integrin were observed in the truncated α_1C-Ca_L, S¹⁹⁰¹A/Y²¹²²F α_1C-Ca_L, and ΔP1/ΔP2 α_1C-Ca_L mutants, compared with WT-Ca_L (99 ± 5.9%, 99 ± 6.2%, and 88 ± 5.1% of WT α_1C-Ca_L, respectively). Deconvolution analysis was also used to eliminate the signals collected from out-of-focus emission. However, no substantial differences were noted in the same comparisons when deconvolution analysis was applied (data not shown). Collectively, confocal image analysis demonstrated that the association between α_1C-Ca_L and β₁-integrin on the cell membrane was moderate in cells expressing WT α_1C-Ca_L. No significant differences were observed in the degree of α_1C-Ca_L colocalization with β₁-integrin between the three mutant α_1C-Ca_L constructs, compared with WT α_1C-Ca_L. Taken together, the IF results suggest that α_1C-Ca_L and β₁-integrin colocalize to a moderate degree on the plasma membrane and that the modifications we made in the COOH terminus of α_1C-Ca_L did not significantly alter the membrane targeting of α_1C-Ca_L or β₁-integrin on the plasma membrane. Thus, the reduced associations between the mutant forms of α_1C-Ca_L and β₁-integrin observed in IP protocol are not due to deficient membrane targeting.

Fig. 9.

Bar graph showing the quantified degree of α_1C-Ca_L association with β₁-integrin normalized to the degree of paxillin (Pax)-vinculin (Vin) association using intensity correlation analysis (ICA). The association of paxillin with vinculin was set as 100%. The ICA value of α_1C-Ca_L-β₁-integrin association in WT α_1C-Ca_L, truncated α_1C-Ca_L, S¹⁹⁰¹A/Y²¹²²F-α_1C-Ca_L, or ΔP1/ΔP2 α_1C-Ca_L is presented as the percentage of paxillin-vinculin association. Values were taken from at least 8 cells in either WT- or mutant Ca_L per experiment, and the values in the graph were calculated on the basis of the average of at least four experiments. Statistical analysis was performed on the basis of the average from at least four individual experiments. #P < 0.01 vs. the degree of colocalization between paxillin and vinculin.

DISCUSSION

We present evidence that the pore-forming α_1C-subunit of the L-type calcium channel (α_1C-Ca_L) associates with β₁-integrin and c-Src in arteriolar SM as well as when the channel is heterologously expressed in HEK293-T cells. These associations depend on interactions of the cells with the ECM protein FN, which is known from previous studies to potentiate Ca_L current following α₅β₁-integrin engagement (40). Furthermore, our results show that the associations between α_1C-Ca_L and α₅β₁-integrin and between α_1C-Ca_L and c-Src require specific regions in the COOH terminus of α_1C-Ca_L. Two specific domains that are critical for these interactions are the PKA and c-Src phosphorylation sites that we previously identified as essential for modulation of Ca_L function following α₅β₁-integrin engagement (14). In addition, we found that Ca_L preferentially associates with the phosphorylated, active, form of c-Src in arteriolar SM and that this association depends on FN. The latter result provides evidence for a unique local functional regulation of Ca_L by α₅β₁-integrin through phosphorylation of c-Src. Collectively, our data support the concept of a macromolecular complex composed of multiple focal adhesion molecules including PKA, c-Src, and some fraction of the Ca_L population, which is assembled upon integrin ligation and required for potentiation of Ca_L current following α₅β₁-integrin engagement.

In this study, three different approaches, IP, IF, and patch-clamp recording of Ca_L current, were used to assess structural and functional interactions between α_1C-Ca_L and α₅β₁-integrin. In some respects, the data obtained using these methods support each other, while in other respects the data conflict. How can we reconcile the conflicting results? What do the collective data tell us about the direct or indirect association of the channel with α₅β₁-integrin? What signaling components are required to mediate α_1C-Ca_L association with α₅β₁-integrin upon adhesion to FN? How does the spatial interaction between α_1C-Ca_L and α₅β₁-integrin contribute to the functional regulation of Ca_L? What is the physiological significance of α_1C-Ca_L association with α₅β₁-integrin? These issues will be addressed in the following sections.

Direct or indirect association of Ca_L with α₅β₁-integrin.

After demonstrating an association between α_1C-Ca_L and α₅β₁-integrin, using IP and IB methods, we sought to determine the nature of the interaction and the regions in α_1C-Ca_L that were required. Because the crystal structure of α_1C-Ca_L has not been resolved, interactions between these proteins are difficult to predict. If α_1C-Ca_L directly interacts with α₅β₁-integrin, a potential binding region would be the canonical Arg-Gly-Asp (RGD)-integrin-binding motif. In fact, a previous study by McPhee et al. (31) identified the functional significance of an RGD sequence located in the extracellular loop, between the first membrane-spanning region and the pore, of the G protein-activated inward rectifier K⁺ channel (GIRK). This RGD sequence mediates the direct interaction between GIRK and the β₁-integrin. The functional significance of the interaction appears to involve targeting of GIRK to the plasma membrane and/or regulation of its stability rather than acute regulation of channel function (31). However, in Cav1.2, the only conserved RGD sequence (aa 448–450) is found in a conserved transmembrane region of all three Ca_L isoforms, so that direct binding of α_1C-Ca_L to RGD-binding integrins is unlikely due to potential steric hindrance. Thus, an indirect association between α_1C-Ca_L and α₅β₁-integrin through cytoplasmic or cytoskeletal proteins seems more probable.

To determine regions in α_1C-Ca_L that provide binding motifs for other focal adhesion proteins that are known to associate with α₅β₁-integrin, we used a site-directed mutagenesis strategy. On the basis of differences in the amount of coimmunoprecipitated α_1C-Ca_L with β₁-integrin in WT versus mutated α_1C-Ca_L, we identified several regions in the α_1C-Ca_L COOH terminus that are involved in the interaction of α_1C-Ca_L with α₅β₁-integrin: two phosphorylation sites for PKA and c-Src, along with the PRDs. These findings therefore implicate PKA, c-Src, and potentially other SH3 domain-containing proteins in mediating the interaction of α_1C-Ca_L with α₅β₁-integrin.

The roles of PKA in the functional regulation of Ca_L and its association with α₅β₁-integrin.

PKA is known to regulate all three Ca_L isoforms. In neurons, PKA colocalizes with adenylate cyclase, β₂-adrenergic receptor (β₂-AR), A-kinase anchoring protein (AKAP), and protein phosphatase (PP2A) in a macromolecular signaling complex; PKA mediates the increase in neuronal Ca_L current following stimulation of β₂-AR (6). Our results suggest that PKA is also a component of another macromolecular complex containing various focal adhesion proteins recruited following ligation of α₅β₁-integrin and that signaling between proteins in this complex also can exert a significant degree of control over Ca_L function. Evidence to support this conclusion derives from two aspects of our results. First, reduced association between α_1C-Ca_L and β₁-integrin was observed in an α_1C-Ca_L mutant with altered PKA and c-Src phosphorylation sites. Second, our previous electrophysiological studies demonstrated a substantial reduction in the amount of Ca_L current that was potentiated following ligation of α₅β₁-integrin when α_1C-Ca_L mutants with altered PKA and/or c-Src phosphorylation were used (14). When S¹⁹⁰¹ in α_1C is mutated, it not only leads to reduction in the association between α_1C-Ca_L and β₁-integrin, but also to reduction in the phosphorylation of Ca_L by PKA (14). Findings from other laboratories also support the notion that S¹⁹⁰¹ in the COOH terminus of α_1C-Ca_L is a focal assembly point for PKA, AKAP, PP2A, and WAVE/WASP, which collectively modulate Ca_L function (5). In support of this idea, the macromolecular complex composed of PKA, AKAP, PP2A, and WAVE/WASP has been shown to provide a spatial link to the integrin-cytoskeleton network in modulation of other cellular functions (15) and engagement of β₁-integrin is known to facilitate PKA targeting to specific cellular locations (26, 32). On the basis of all the above, faulty trafficking/targeting of PKA in the cytoplasm could contribute to reduced interaction of PKA with other focal adhesion components in the macromolecular complex, thereby disrupting the spatial link to the integrin-cytoskeleton network and reducing the extent to which Ca_L function is influenced by α₅β₁-integrin signaling.

The roles of PRDs and c-Src in functional regulation of Ca_L and its association with α₅β₁-integrin.

c-Src is another kinase that is implicated in the signaling pathway between α₅β₁-integrin and Ca_L. The involvement of c-Src is supported by several findings in the present study: 1) co-IP of α_1C-Ca_L with c-Src in arteriolar SM appears to be independent of FN; 2) co-IP of α_1C-Ca_L and phosphorylated c-Src is only observed in arteriolar SM incubated with FN; 3) co-IP of α_1C-Ca_L and c-Src is only observed upon adhesion of HEK293-T cells to FN; and 4) mutation of the phosphorylation site for c-Src in the COOH terminus in α_1C-Ca_L (Y²¹²²) leads to reduced co-IP of α_1C-Ca_L with β₁-integrin and of α_1C-Ca_L with c-Src. These results are consistent with previous findings that purified c-Src associates with the COOH terminus of α_1C-Ca_L (19, 20) and that a basal level of Ca_L current depends on c-Src activity in colonic smooth muscle (16). Our co-IP findings are also consistent with a previous functional study from our laboratory showing that the potentiation of Ca_L current following α₅β₁-integrin ligation is significantly reduced in Y²¹²²F α_1C-Ca_L constructs (14). Thus, preventing this tyrosine residue from being phosphorylated not only reduces the overall phosphorylation of Ca_L by exogenous c-Src, but also reduces the spatial interaction between α_1C-Ca_L and β₁-integrin and between α_1C-Ca_L and endogenous c-Src, indicating that the interactions between α_1C-Ca_L and β₁-integrin and between α_1C-Ca_L and c-Src are required for current potentiation following α₅β₁-integrin ligation.

Our IP studies also identified a region in the α_1C-Ca_L COOH terminus containing two PRDs (P1/P2) as another potential binding domain for c-Src. Reduced associations between α_1C-Ca_L and β₁-integrin and between α_1C-Ca_L and c-Src were observed in the ΔP1/ΔP2 α_1C-Ca_L mutant. Our results agree with a previous study demonstrating that fusion proteins containing α_1C PRDs mediate interactions with c-Src in SY5Y cells expressing the neuronal Ca_L isoform upon IGF stimulation (1). Taken together, the results suggest multiple roles for PRDs in association with the macromolecular complex containing β₁-integrin, c-Src, and α_1C-Ca_L.

FAK is another proline-rich tyrosine kinase known to provide a binding site for SH2 and SH3 domain-containing proteins such as paxillin, vinculin, talin, p130Cas, and Crk (42). Although we did not specifically examine whether α_1C-Ca_L associates with FAK, our previous studies in vascular SMC revealed that a significant amount of the Ca_L current potentiation following α₅β₁-integrin engagement was attenuated by dialysis of the cells with an antibody against FAK (40). A precedent for an association between FAK and another ion channel has been established by studies of Cherubini et al. (4), who demonstrated the association between FAK and the human ether-a-go-go-related gene (hERG) channel. In addition, PYK2, a family member of the FAK tyrosine kinases, was demonstrated to associate with cardiac α_1C-Ca_L via binding to PRDs (9). On the basis of the above, the PRDs in the α_1C-Ca_L appear to play significant roles in the assembly of a macromolecular complex containing Ca_L, α₅β₁-integrin, c-Src, and potentially other protein tyrosine kinases.

It should be noted that the roles of PRDs in the physical and functional association of α_1C-Ca_L with α₅β₁-integrin may be more complicated than at first suggested by the preceding discussion. When the ΔP1/ΔP2 α_1C-Ca_L mutant is expressed, the IP results (Fig. 6, A and C) reveal a reduced association between α_1C-Ca_L and β₁-integrin and between α_1C-Ca_L and c-Src, compared with WT α_1C-Ca_L, whereas the electrophysiological data (Fig. 7, A and B) reveal no substantial impairment in the degree of Ca_L current potentiation following α₅β₁-integrin ligation. The discrepancy may arise from two issues. First, the PRD regions that we deleted from α_1C-Ca_L lie within the distal COOH terminus inhibitory region (DCT). Two studies have shown that deletion of the homologous PRDs (aa 1966–2004) in the cardiac Ca_L isoform leads to enhancement of basal Ca_L current (13, 38). Deletion of the PRDs removes part of the DCT, which may partially relieve constitutive channel inhibition by the DCT segment, though this has not been specifically tested. However, the major differences between the neuronal, cardiac, and smooth muscle isoforms of the Ca_L channel reside in the more distal regions of the respective COOH termini, and whether or not the DCT segment exerts strong inhibitory control of the neuronal Ca_L has not been determined.

A second explanation for the discrepancy between the structural and functional PRD results is the possible contribution of another PRD that has been identified in the II–III linker region of α_1C-Ca_L (aa 857–861). A study by Dubuis et al. (9) suggests that the II-III linker region appears to play a role in extending the activation range of cardiac Ca_L, which consequently alters Ca_L function. Our IP results revealed that deletion of the COOH-terminal PRDs only reduced c-Src coassociation with α_1C-Ca_L by 42%, even when the amount of truncated COOH-terminal α_1C-Ca_L mutants for IP was increased by twofold (Supplemental Fig. S2, A and C). Thus, deletion of only the PRDs in the COOH terminus of α_1C-Ca_L may not be sufficient to fully abolish the potentiation in Ca_L current following α₅β₁-integrin engagement. Overall, our electrophysiological and IP results agree with the findings of Dubuis et al. and suggest the possibility of another important c-Src binding region in α_1C-Ca_L.

Reduced association between α_1C-Ca_L and β₁-integrin is not due to faulty membrane targeting of Ca_L.

The amino acid residues 1623–1733 in the COOH terminus of cardiac α_1C-Ca_L have been shown to mediate the membrane targeting of the channel (11). Using IF, we detected no significant difference in membrane targeting of α_1C-Ca_L or β₁-integrin in S¹⁹⁰¹A/Y²¹²²F-Ca_L, truncated α_1C-Ca_L at amino acid residue 1862, or ΔP1/ΔP2-Ca_L, compared with WT-Ca_L. Our results suggest that the reduced association between α_1C-Ca_L and β₁-integrin observed in the above mentioned α_1C-Ca_L mutants is not due to unsuccessful membrane targeting of α_1C-Ca_L. Our findings agree with those of Gao et al. (11) that the regions in the COOH terminus required for successful membrane targeting of cardiac Ca_L are amino acid residues 1623–1733. Because those specific regions are not altered in our mutant α_1C-Ca_L constructs, the reduced association between α_1C-Ca_L and β₁-integrin in our mutant constructs are unlikely attributable to incorrect membrane targeting of Ca_L. In addition, our IF results also suggest that the association between α_1C-Ca_L and β₁-integrin on the plasma membrane is independent of the PKA/c-Src phosphorylation sites, the PRD domains, or the distal COOH terminus after amino acid residue 1862 in α_1C-Ca_L. These results suggest that α₅β₁-integrin modulates Ca_L function independent of the interactions of the two proteins on the plasma membrane. The IF results agree with the IP and patch-clamp findings in that the formation of a macromolecular complex including PKA, c-Src, α₅β₁-integrin, and Ca_L in the cytoplasm following α₅β₁-integrin engagement appears to play an important role in Ca_L current potentiation by α₅β₁-integrin.

Physiological significance of Ca_L association with integrin.

Our findings of an association between Ca_L and β₁-integrin and between Ca_L and c-Src provide evidence for a macromolecular signaling complex composed of α₅β₁-integrin, Ca_L, c-Src, PKA, and possibly other focal adhesion components. Formation of this complex appears to be important for regulation of Ca_L function following integrin-ECM interaction. Our IP results are consistent with previous findings that c-Src is involved in the increase of Ca_L current potentiation in response to PDGF (17) or IGF (1) and with the finding that a c-Src-FAK complex forms in colonic smooth muscle in response to PDGF stimulation (16). Interestingly, cross talk between integrins and growth factors has been demonstrated in several studies, with the interaction of FAK and c-Src as a point of convergence (10, 28, 35). Although it is not known whether these two signaling pathways converge to regulate an ion channel, an intriguing possibility is that PDGF or IGF act in conjunction with α₅β₁-integrin to produce additive or synergistic potentiation of Ca_L function by integrin.

In summary, this is the first study to provide evidence for the association of α_1C-Ca_L with α₅β₁-integrin or α_1C-Ca_L with c-Src in arteriolar smooth muscle. This association depends on engagement of α₅β₁-integrin by FN and specific regions in the α_1C-Ca_L COOH terminus, including two phosphorylation sites for PKA and c-Src and proline-rich domains that provide a binding motif for SH3 domain-containing proteins, such as c-Src. Identification of these regions, along with our electrophysiological findings, suggests that a macromolecular complex is assembled upon engagement of α₅β₁-integrin and that the interactions of proteins within this complex contribute to modulation of Ca_L function. Our study provides new insights into a Ca_L macromolecular regulatory complex that is required for functional regulation of the channel following α₅β₁-integrin engagement.

GRANTS

This project was funded by National Institutes of Health Grants HL-071796, HL-072989, and P01 HL-095486 (to M. J. Davis). G. W. Zamponi holds funding from the Heart and Stroke Foundation and is a Canada Research Chair and Alberta Heritage Foundation for Medical Research Scientist.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).