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J. Biol. Chem., Vol. 281, Issue 20, 14015-14025, May 19, 2006
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4
From the
Department of Medical Pharmacology & Physiology, University of Missouri School of Medicine, Columbia, Missouri 65212, the Department of Systems Biology & Translational Medicine, Texas A&M University College of Medicine, College Station, Texas 77840, and the ¶Smooth Muscle and ||Cellular and Molecular Neurobiology Research Groups, University of Calgary School of Medicine, Calgary, Alberta T2N 4N1, Canada
Received for publication, January 17, 2006 , and in revised form, March 10, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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 51 integrin requires phosphorylation of 1C C-terminal residues Ser1901 and Tyr2122. These sites are known to be phosphorylated by protein kinase A (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 51 integrin activation, native CaL 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 CaL 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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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, Cav1.2c; smooth muscle, Cav1.2b; cardiac, Cav1.2a) 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 CaL channels by serine-threonine 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 (Ser1901 in Cav1.2c; Ser1928 in Cav1.2a), thereby mediating -adrenergic potentiation of the 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).
We recently demonstrated that CaL currents in vascular smooth muscle (VSM) are acutely regulated by the integrin class of cell adhesion molecules. CaL current is inhibited by ligands of v3 integrin but potentiated by ligands of 51 and 41 integrins, including the abundant extracellular matrix (ECM) protein, fibronectin (11). 51-mediated potentiation depends critically on the integrin-associated tyrosine kinases c-Src and focal adhesion kinase (12). This mechanism is consistent with reports that 1C isoforms are potentiated by the growth factors, insulin-like growth factor-1 and platelet-derived growth factor (13–15), and that integrin-CaL channel interactions are required for neurotropin production (16) and acute control of synaptic function in the adult central nervous system (17–22).
Here, we present evidence that activation of 51 integrin acutely potentiates CaL channels in rat brain and smooth muscle. The effect can be reproduced fully in heterologously expressed neuronal and smooth muscle CaL isoforms and depends critically on phosphorylation of two specific C-terminal serine and tyrosine residues, indicating that integrin-ECM interactions are a common mechanism for acute regulation of CaL channels in brain and muscle.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Reagents—The integrin ligands, 5 integrin monoclonal antibody (HM5-1, rat; BD Pharmingen) or 51 integrin polyclonal Ab (human, 10 µg/ml; Chemicon, Temecula, CA), were applied in soluble form or by biotin-streptavidin linking to polystyrene beads (3.2 µm, outer diameter) followed by addition of the beads (typically 5–10) to individual cells using gentle superfusion from a micropipette (11). Soluble reagents (PP2; Calbiochem) were applied using a picospritzer connected to micopipettes positioned 
50 µm from the cell. In some protocols the solution in the recording pipette was exchanged with solutions containing inhibitors (PKA inhibitory peptide (PKI); Sigma) (Src SH2 inhibitory domain peptide; Calbiochem) over a 1–2-min period using a 2PK push-pull pipette exchange system (ALA Scientific Instruments, Westbury, NY).
Heterologous Channel Expression—HEK-293 cells (tsA-201 line) were maintained at 30–37 °C in a 5% CO2 incubator in supplemented Dul-becco's modified Eagle's medium, and transfected with CaL isoforms using CaPO4 (for electrophysiology) or Lipofectamine 2000 (for Western blotting). Rat neuronal 1C, 1b, and 2- DNA, subcloned into pcDNA3.1 vectors, were gifts from T. Snutch; rabbit SM 1C, 2a, and 2-1 DNA were gifts from F. Hofmann and N. Klugbauer; human c-Src subcloned into SR using the EcoRI site was a gift from D. Fujita. Enhanced green fluorescent protein (EGFP) was used as a co-transfectant in all electrophysiological protocols to identify successfully transfected cells. Only single cells were used for electrophysiological protocols, typically 48–72 h after transfection.
Mutagenesis Methods—A PCR strategy was used to introduce stop codons at various positions in the Cav1.2 1C subunit C terminus. The wild type Cav1.2 clone contained a NotI site at the 3' multicloning site. Thus, primer A was designed to anneal to the Cav1.2 antisense strand immediately upstream of the site intended for the stop codon. The primer included the stop codon and the sequence encoding a NotI restriction site. Primer B was designed to associate with the Cav1.2 residues upstream of both a unique SbfI restriction site and the placement of the intended stop codon. The resulting PCR product that extended from the SbfI site to the stop codon/NotI site was then sequenced, excised via SbfI and NotI, and subcloned into the original Cav1.2 construct via SbfI and NotI. Site-directed mutagenesis to eliminate phosphorylation consensus sites in the C terminus of Cav1.2 was conducted using the QuikChange site-directed mutagenesis kit (Stratagene), using the entire cDNA as a template. Subsequently, a fragment ranging from SbfI to NotI was sequenced to confirm the presence of the mutations, excised, and subcloned into the original WT Cav1.2 construct.
Immunoprecipitation, Immunoblotting, and in Vitro Kinase Assays—TsA-201 cells were lysed 48–72 h after transfection in Tris-buffered saline solution containing 1% Triton X-100 with a mixture of protease inhibitors (4 °C). CaL channels were immunoprecipitated by overnight incubation with 1C Ab (Chemicon) followed by addition of protein A/G beads, pelleting, washing, and resuspension in Laemmli sample buffer. For immunoblotting, lysates or immunoprecipitates (IPs) were separated by SDS-PAGE using 6% acrylamide gels, transferred to nitrocellulose, and probed with appropriate primary and secondary Ab before application of SuperSignal chemiluminescent reagent and exposure to x-ray film. c-Src or cAK-induced phosphorylation was carried out at 30 °C in standard assay solution by incubation of IP complexes with 10 µM [-32P]ATP in the presence of purified recombinant human c-Src or cAK catalytic subunit purified from bovine heart, respectively (24). After separating the phosphorylation mixture by SDS-PAGE, the gels were exposed to x-ray film for 9–36 h at –20 °C; densitometry was performed using Scion software or, in some cases, a PhosphorImager.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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51 Integrin in Brain and VSM Potentiates CaL Current—Neurons (Fig. 1a) were acutely dissociated from the medial septum/diagonal band nucleus (MS/nDB) of the rat. CaL currents were measured using whole cell patch clamp techniques in the presence of 5 µM 
-conotoxin MVIIC and a holding potential of –40 mV to block N and P/Q channels (25). 51 integrins were activated by micropipette application of beads coated with anti-5 integrin Ab (5-Ab, a multivalent 51 ligand). Bead attachment to the neuron whose current is shown in Fig. 1a resulted in 1.6-fold potentiation of CaL current that peaked 3–5 min after bead application; potentiation averaged 1.43 ± 0.11-fold in 4 cells. I-V relationships of whole cell currents before (control) and after attachment of 5 beads are illustrated at the bottom of Fig. 1a. Potentiation did not appear to be associated with a significant shift in voltage sensitivity of the channel.
In rat VSM cells, the application of anti-5 integrin Ab on beads also resulted in time-dependent potentiation of the whole cell CaL current. Peak potentiation of the CaL current was 1.8-fold for the cell shown in Fig. 1b, whereas the average potentiation was 1.64 ± 0.05-fold in 5 cells. This effect was specific for integrin engagement because uncoated beads or beads coated with bovine serum albumin had no significant effect on current. In addition, a non-integrin binding Ab, in soluble form or coated onto beads, was without a significant effect on current (11).
Potentiation of Heterologously Expressed Cav1.2 by Integrin Activation—To explore the mechanism of current potentiation, CaL subunits were subsequently expressed in tsA-201 cells, which constitutively express 51 integrin and demonstrate 51 integrin-dependent adhesion (not shown). Transient transfection with the neuronal 1C channel subunit (Cav1.2c) alone resulted in relatively small currents that were potentiated 1.90 ± 0.13-fold by application of soluble anti-51 integrin Ab (human; Fig. 2a, left). Co-expression of 1b and 2- subunits (Fig. 2a, right) along with Cav1.2c resulted in much larger basal currents that were also potentiated by soluble 51 integrin Ab (2.07 ± 0.15-fold). We also used two additional methods known to cross-link integrins (26): 1) application of microbeads coated with 51 Ab; 2) successive application of soluble 51 Ab (mouse anti-human) and an appropriate secondary Ab (goat anti-mouse IgG). Both methods also led to potentiation of current, 1.85–2.07-fold (Fig. 2b), either with or without the accessory channel subunits. Integrin cross-linking and activation with multivalent ECM ligands, or with the appropriate concentration of monovalent Ab, is known to initiate outside-in signaling in several other cell systems (26, 27).
Currents were comparably potentiated by activation of 51 integrin in HEK cells expressing either cardiac (Cav1.2a; not shown) or smooth muscle (Cav1.2b) isoforms of CaL. Fig. 3a shows examples of current potentiation by rabbit smooth muscle CaL channels in response to soluble 51 integrin Ab application, when the cells were expressing 1C-b alone or 1C-b together with 2a and 2-1 subunits. The data are summarized in Fig. 3b, where the potentiation averaged 1.99 ± 0.20- and 1.94 ± 0.14-fold, respectively.
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1C Subunit—Compared with smooth muscle and cardiac isoforms, the neuronal channel (Cav1.2c) has 27 fewer amino acid residues on the N terminus (28), including potential PKC phosphorylation sites (6, 29). The comparable potentiation of neuronal and smooth muscle isoforms argues that the N terminus is not the regulatory site for 51 integrinmediated potentiation.
Subsequently, a series of successive, C-terminal truncation mutants of Cav1.2c were created and their responses to 51 integrin activation tested. As shown in Fig. 4, the truncation mutant Stop 5, which terminated proximal to the canonical PKA phosphorylation site (9) at Ser1901 (equivalent to Ser1928 on cardiac Cav1.2a) exhibited a loss of significant potentiation by 51 integrin Ab. In contrast, truncations distal to the PKA phosphorylation site (Stop 6–8), including those missing a proline-rich domain (30), resulted in a significantly reduced (50%), but not completely abolished, response to integrin activation, as compared with wild-type (WT) Cav1.2c. Each of the Stop 6–8 constructs was missing the putative tyrosine phosphorylation site at Tyr2122 previously identified as a regulatory site for insulin-like growth factor-1 (14). These results suggest that two different regulatory sites on the C terminus of Cav1.2 are required for full potentiation following 51 integrin activation, with one of the sites being Ser1901 (Cav1.2c). Consistent with this conclusion is the additional observation that inclusion of PKI in the patch pipette abolished the remaining potentiation associated with integrin activation in the Stop 6–8 constructs (Fig. 4c).
Potentiation of Cav1.2c Depends Partially on 1C Tyr 2122—Our previous work showed that potentiation of CaL in rat smooth muscle was partially but incompletely blocked by tyrosine kinase inhibitors (12). Because only two tyrosine residues in rat Cav1.2c are distal to Ser1901, we constructed single-site mutants in which each of those residues was substituted with phenylalanine (Y2122F-1C and Y2139F-1C). After expression, current through the Y2122F-1C construct continued to be potentiated by the 51 integrin Ab, but potentiation was only 1.39 ± 0.06-fold above basal current, compared with 1.90 ± 0.13-fold for WT-1C (Fig. 5, a and b). In contrast, the magnitude of potentiation of Y2139-1C was statistically indistinguishable from that for WT-1C (1.86 ± 0.09- versus 1.90 ± 0.13-fold, respectively). To further verify that Tyr2122 was critical for full potentiation by 51 integrin, we recorded current through WT-1C while dialyzing the cells with a decoy peptide containing the same amino acid sequence as rat WT-1C-c. This peptide was previously found to interfere with potentiation of native CaL channels in smooth muscle (12), and here it reduced integrin-induced potentiation of WT-1C by 50% (to 1.43 ± 0.05-fold above basal). As controls, neither an identical peptide containing a Y2122F substitution, nor a scrambled peptide, had any effect on integrin-induced potentiation of WT-1C (Fig. 5b). Collectively, these results suggest that Tyr2122 is required for a significant fraction of, but not complete, potentiation of 1C-c following 51 integrin activation.
As an additional approach to testing whether the Tyr2122 residue of 1C-c was phosphorylated following integrin activation, we immunoprecipitated the channel after its expression in HEK-293 cells and performed in vitro phosphorylation assays with [-32P]ATP in the presence or absence of purified Src tyrosine kinase. Radiolabeled phosphate incorporation into Y2122F-1C was reduced to 39 ± 6% of the level in WT-1C, whereas [-32P]ATP incorporation into Y2139F-1C averaged 120 ± 26% of that in WT-1C (Fig. 5c, right). Phosphorylation of WT-1C and Y2139F-1C could also be detected (albeit at much lower levels) in the absence of exogenous c-Src (Fig. 5c, left), but it was nearly undetectable in Y2122F-1C under comparable conditions. These results are consistent with the conclusion that phosphorylation of the Tyr2122 residue in 1C-c by c-Src (or other tyrosine kinases) is part of the mechanism for integrin-induced potentiation of the CaL current.
Potentiation of Cav1.2c Is Partly Mediated by c-Src—We further explored the role of c-Src in integrin-induced potentiation of CaL by co-expressing various c-Src constructs along with WT-1C in HEK-293 cells (Fig. 6, a and b). Co-expression of WT-Src (human) with WT-1C resulted in a significantly enhanced potentiation of current following integrin activation (2.34 ± 0.13-fold) as compared with the magnitude of potentiation when WT-1C was expressed alone (1.90 ± 0.13-fold). In contrast, co-expression of a kinase-dead (Kd) form of c-Src (24), along with WT-1C, resulted in 50% reduction of potentiation of current after integrin activation (to 1.43 ± 0.05-fold). Although we attempted various additional methods of enhancing the amount of Kd-Src expression, we were unable to completely abolish the 5 integrin-induced potentiation of current (not shown). As an alternative approach to Kd-Src co-expression, we tested the effects of PP2 (100 nM), a membrane permeable inhibitor of Src family kinases, which reduced potentiation of WT-1C by 40% following integrin activation (1.58 ± 0.06- versus 1.90 ± 0.13-fold). Collectively, these results are consistent with the conclusion that phosphorylation of the channel by c-Src accounts for some, but not all, of the potentiation following 51 integrin activation.
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1C significantly enhanced tyrosine phosphorylation of 1C (by 5-fold; Fig. 6c). However, when Kd-Src was co-expressed instead, tyrosine phosphorylation of 1C was undetectable under comparable conditions. Although tyrosine phosphorylation of 1C by endogenous Src (Fig. 6c, lane 1) is not apparent in Fig. 6, longer film exposure times revealed detectable phosphorylation of the channel in the absence of c-Src co-transfection (not shown).
Full Potentiation of Cav1.2c Depends on Dual Phosphorylation of 1C Residues Ser 1901 and Tyr 2122—The results shown in Figs. 4 and 5 collectively suggest that full potentiation of 1C-c by 51 integrin activation requires phosphorylation of both 1C residues, Ser1901 and Tyr2122. To directly test this hypothesis, we constructed an 1C mutant in which the PKA site was altered to alanine (S1901A-1C) and a double mutant in which both the PKA and Src phosphorylation sites were altered (S1901A/Y2122F-1C). We initially checked the mutants to verify that regulation by PKA was impaired in response to the membrane-permeable cAMP analog, 8-Br-cAMP (Fig. 7, a and b, top). 8-Br-cAMP (1 mM) potentiated the WT-1C current by 1.64 ± 0.12-fold and this effect was completely abolished in both S1901A-1C and S1901A/Y2122F-1C constructs. No significant potentiation by 8-Br-cAMP was observed in cells transfected with WT-1C if PKI was included in the recording pipette.
We also tested whether the S1901A-1C mutant could be phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase (cAK). Compared with phosphorylation of WT-1C, phosphorylation of immunoprecipitated S1901A-1C by cAK was reduced to 65 ± 8% (Fig. 7d). In contrast, the phosphorylation of neither Y2122F-1C nor Y2139F-1C by cAK was significantly different from that for WT-1C (88 ± 14 and 108 ± 11%, respectively; Fig. 7d). The remaining, background [-32P]ATP incorporation into S1901A-1C likely represents phosphorylation of the channel at additional sites containing cAK/cGK consensus sequences (3, 9) that may become modified under the in vitro assay conditions.
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51 integrin activation on whole cell current recorded from cells expressing channels mutated at the PKA regulatory site. Potentiation of the S1901A-1C construct following integrin activation was reduced by 45% (1.90 ± 0.13- versus 1.47 ± 0.13-fold) compared with the potentiation of WT-1C; however, currents were still significantly higher than basal. In contrast, in cells expressing S1901A/Y2122F-1C, integrin activation did not result in any significant potentiation of current above basal levels (Fig. 7b, bottom, and c). To further test this idea, we tested five different methods of interfering with phosphorylation of 1C at Ser1901 and Tyr2122: S1901A-1C plus PP2 added to the bath; S1901A-1C plus co-expression of Kd-Src; WT-1C co-expressed with Kd-Src plus PKI in the recording pipette; Y2122F-1C plus PKI in the recording pipette; and the S1901A/Y2122F-1C double mutant. In all five cases, no significant potentiation of current was observed following integrin activation (Fig. 7c). These results strongly support the conclusion that potentiation of the Cav1.2c current by 51 integrin activation requires dual phosphorylation of sites Ser1901 and Tyr2122 on 1C-c.
Potentiation of Native CaL Channels Requires Both PKA and c-Src—The evidence for dual phosphorylation of 1C-c by PKA and c-Src underlying integrin-dependent potentiation of the CaL current prompted a more careful examination of the magnitude and time course of native CaL current potentiation in rat cells during inhibition of Src and PKA. Fig. 8a shows the time course of current potentiation following application of beads coated with 5-integrin Ab onto single vascular myocytes. Consistent with previous findings, potentiation peaked 3–4 min after bead application and then spontaneously declined (11) toward control levels. When Src was inhibited just before peak potentiation by PP2 (100 nM in the bath) or by a Src SH2 inhibitory peptide (2.7 µM in the recording pipette), 75 and 60% less current potentiation was observed, respectively, compared with that recorded in the absence of inhibitors (Fig. 8b). PKI alone, applied by exchange of the recording pipette solution, resulted in only 17% reduction of integrin-potentiated current. PP3 (100 nM), the inactive analog of PP2, was without significant effect (not shown) on either basal or integrin-potentiated current (12). However, the combination of PP2 and PKI peptide completely blocked integrin-induced potentiation of current (Fig. 8b). These results are consistent with those from heterologously expressed channels (Figs. 4, 5, 6, 7) and suggest that the same dual-phosphorylation mechanisms are operating to regulate native CaL channels.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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51 integrin through an intracellular signaling pathway involving phosphorylation of the 1C channel subunit by PKA and c-Src. Integrin activation produces up to 2-fold potentiation of Ca2+ current over a relatively short time frame (1–5 min). Given the central role of CaL channels in nerve and smooth muscle excitability, the ECM-integrin-CaL signaling pathway is likely to have major effects on both short- and long-term control of [Ca2+]i and Ca2+-dependent processes in these respective cell types.
Ion Channel Regulation by Integrins—A growing body of literature supports our conclusion that ion channels are acutely regulated by integrin-ECM interactions (31). 1 integrins are known to modulate excitatory synaptic transmission through an as yet defined role of c-Src (16, 22, 32), observations that are consistent with the well known regulation of ligand-gated channels by tyrosine kinases (33). Recently, 1 integrins have also been implicated in the regulation of several different K+ channels, with evidence for both physical (34, 35) and functional (36–38) associations between the channels and integrins.
Several independent lines of evidence support the idea that integrins also regulate CaL channels. For example, bidirectional regulation of native rat smooth muscle CaL channels is observed following engagement of 51, 41, and v3 integrins with ECM proteins or integrin antibodies (11, 39). In the case of 51 integrin, selective activation by multivalent integrin ligands results in 70% potentiation of CaL current above basal levels and this is substantially reduced by inhibition of focal adhesion kinase and c-Src tyrosine kinases (12). In Lymnaea, soluble fibronectin potentiates neuronal HVA current, enhances the [Ca2+]i response to KCl, and leads to an increase in neuronal excitability (40). The specific Lymnaea integrins responsible have not yet been identified but cRGD peptide, which interacts with multiple integrins, produces dose-dependent, biphasic changes in HVA current (composed of both N- and L-type channels), suggesting that the responses are mediated by multiple integrins and/or multiple Ca2+ channel types. The regulation of mammalian cardiac CaL channels by muscarinic and adrenergic receptors is substantially influenced by the adhesion of cardiac myocytes to their substratum. Specifically, CaL responses to 2 adrenergic agonists are potentiated in cells adhered to laminin or 1 integrin Ab (compared with adhesion on glass), whereas responses to acetylcholine are attenuated (41, 42). However, another study suggests that adhesion-dependent potentiation of cardiac CaL is not specific to integrins (43). In cardiomyocytes derived from embryonic stem cells, muscarinic inhibition of CaL current is absent in 1-integrin–/– cells (44), although the effect is at least partly due to defective coupling of Gi subsequent to 1 integrin knockout. In this context it is interesting that G1 co-localizes extensively with the focal adhesion proteins talin and vinculin (45) known to be critically involved in integrin-mediated signaling (46). In E63 skeletal muscle cells, engagement of 71 integrin by laminin or 7-integrin Ab triggers both Ca2+ release and influx (27). Interestingly, the extracellular domain of 71 integrin associates with the dihydropyridine receptor/channel (1S), which shares substantial sequence homology with Cav1.2. Collectively, these findings support our contention that the pathways elucidated in the present study may be common mechanisms for regulation of Ca2+ entry in many nerve and muscle cells.
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51 Integrin—Clues to the regulation of CaL by integrins may lie in the structure of the 1C C terminus. Our finding of comparable integrin-mediated potentiation of both smooth muscle and neuron channels suggest that the N terminus, which is 27 amino acids shorter in the neuronal isoform (6, 28), is not critical for this regulation. The 1C C terminus is composed of 550 amino acid residues known to regulate channel function in a highly complex manner. The C terminus contains domains that mediate Ca2+-induced inactivation, calmodulin binding, and interactions with the subunit (47–50). The terminal 150 amino acids (2021–2171 in Cav1.2a) contain an inhibitory domain that interacts internally with more proximal (1733–1900) residues (51). 1C-a constructs missing substantial portions of the C terminus exhibit up to a 10-fold increase in basal current (30, 51, 52), which we observed to a lesser extent with our 1C-c truncation mutants. Thus, it is possible that potentiation of CaL following phosphorylation of the C terminus is mediated by partial relief of tonic inhibition.
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2-adrenergic receptor and at least one heterodimeric G-protein in what appears to be a signaling complex that might facilitate rapid and specific [Ca2+]i signaling (53). This complex is assembled on a scaffold of membrane-bound proteins including AKAP 79 (54, 55), AKAP 15 (56), MAP2B (57), and possibly cytoskeletal proteins such as -actinin (58) and actin (59). The Cav1.2a II-II linker region and C terminus both contain proline-rich, class I and class II, Src homology 3 (SH3) domains that mediate binding of c-Src and Src family kinases to the cardiac CaL channel (60). Residues containing the C-terminal SH3 domain (1974–2000) are important for tethering 1C-a to the plasma membrane (30) and the 2 channel subunit (48). Importantly, both cardiac and smooth muscle channel 1C isoforms have been shown to physically associate with and be phosphorylated by c-Src (13, 30, 51, 60, 61), observations that are consistent with our present in vitro phosphorylation studies (Figs. 5 and 6). Likewise, the growth factor PDGF is also known to potentiate native rat smooth muscle channels through an unknown mechanism involving c-Src (13, 62).
Collectively, this evidence points to the possibility that CaL channels co-localize with integrins and/or focal adhesion proteins in excitable cells and that CaL-integrin-focal adhesion signaling complexes potentially overlap with CaL-PKA-AKAP complexes. The activation of PKA and subsequent phosphorylation of Cav1.2 channels following integrin activation observed in our study are consistent with activation of the cAMP signaling cascade in endothelial cells following application of mechanical stress through RGD-containing integrins (63); indeed, the activation of the cAMP cascade required both integrin receptor occupation and intact G signaling. Thus, it is possible that scaffolding proteins such as WAVE1, which associate with both PKA and integrin-linked tyrosine kinases (64), will be found to play critical roles in the regulation of CaL channels. The possible crossover between these signaling pathways will be an important issue to resolve with respect to the regulation of CaL in excitable cells.
It is interesting that the heterologously expressed rat neuronal isoform (Cav1.2c) showed a similar degree of potentiation as native rat neuronal and VSM CaL channels following 51 integrin activation (compare Fig. 1 with Figs. 2 and 3). Similar physiological responses might be predicted based on the fact that there is >99% sequence homology between the rat neuronal (Cav1.2c) and rat smooth muscle (Cav1.2b) isoforms. For other species, there is less (80%) homology in the regulatory portion (distal 2/3) of the C terminus. Notably, rabbit Cav1.2b lacks the critical tyrosine residue corresponding to Tyr2122 on Cav1.2c that mediates 50% of integrin-induced potentiation and that has also been implicated in regulation of rat Cav1.2c by the growth factor IGF-1 (14). However, rabbit Cav1.2b is also potentiated to a comparable degree by 51 integrin activation (Fig. 3). Whether potentiation of rabbit Cav1.2b depends both on phosphorylation by PKA at Ser1928 and c-Src at an alternate tyrosine residue are important questions that need to be answered. However, it is also known that rabbit smooth muscle CaL channels may be regulated by c-Src through more indirect mechanisms (4, 13, 65).
ECM-Integrin-Cav1.2 Signaling Axis in Cellular Responses to Injury—The regulation of CaL channels by integrin-ECM interactions can potentially play a role in a number of important physiological processes in both the central nervous system and the cardiovascular system. In the brain, integrins are concentrated at sites of synaptic contact (66) and are critical for the formation, maturation, and maintenance of synaptic structure (67). For example, laminin, along with its receptor, 71 integrin, plays a role in the clustering of acetylcholine receptors at the neuromuscular junction (68). Integrin engagement is required under some conditions for neurotrophic signaling (16) and integrin clustering can initiate receptor tyrosine kinase signaling in the absence of neurotrophins, as has been documented for other growth factors in other cell systems (69, 70). In this context, integrin-CaL channel interactions appear to be critical for the up-regulation of brain-derived neurotrophic factor mRNA in hippocampal neurons (16). Finally, a number of studies have now confirmed that integrins are instrumental in modulating long term potentiation and therefore in controlling synaptic function in the adult central nervous system (17–22). However, whether their role involves changes in cellular adhesion or modulation of intracellular signaling has not yet been defined.
Integrin-CaL channel interactions could also play a major role in the responses of neurons and blood vessels to injury and repair. Both neuronal activity and vascular reactivity are modulated by integrin signaling through the generation or exposure of new integrin ligands from limited degradation of extracellular matrix and/or turnover of new integrins (71–73). For example, successful axonal regeneration is highly correlated with the induction of integrins on the surface of peripheral neurons; therefore, peptides derived from ECM proteins have the potential to act as therapeutic agents for neuronal regeneration (74). In blood vessels, it is well established that VSM sensitivity to agonists and mechanical forces is altered following ischemic injury and during hypertrophic and eutrophic remodeling (75, 76). Many factors contribute to these processes, but among them are changes in the number and composition of VSM integrins, changes in ECM components, and changes in the density of CaL channels. Thus the interactions between CaL channels and integrins are likely to play a major role in the ability of neuronal and vascular cells to recover from tissue injury.
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FOOTNOTES |
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The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) P22002
[GenBank]
(1C-c) and P15381
[GenBank]
(1C-b). 
1 Recipient of studentship awards from the Alberta Heritage Foundation for Medical Research and the Canadian Institutes of Health Research.
2 Alberta Heritage Foundation for Medical Research Senior Scholar.
3 Senior Scholar of the Alberta Heritage Foundation for Medical Research and a Canada Research Chair in Molecular Neurobiology.
4 To whom correspondence should be addressed: 1 Hospital Drive, M451, Columbia, MO 65212. Tel.: 573-884-5181; Fax: 573-884-4276; E-mail: davismj{at}health.missouri.edu.
5 The abbreviations used are: CaL, L-type voltage-gated Ca2+ channel; PKG, protein kinase G; PKA, protein kinase A; VSM, vascular smooth muscle; ECM, extracellular matrix; Ab, antibody; MS, medial septum; nDB, diagonal band nucleus; PKI, PKA inhibitory peptide; 8-Br-cAMP, 8-bromo-cAMP; cAK, cyclic A kinase; IP, immunoprecipitation; WT, wild type; SH, Src homology domain.
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ACKNOWLEDGMENTS |
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1C-b, 2a, and 2-1 cDNA, Dr. D. Fujita (University of Calgary) provided the human c-Src cDNA, and Dr. T. Snutch provided 1C-c, 1b, and 2- cDNA. |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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