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Cardiovascular Research 2006 69(2):391-401; doi:10.1016/j.cardiores.2005.11.006
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Copyright © 2005, European Society of Cardiology

Evidence for multiple Src binding sites on the {alpha}1c L-type Ca2+ channel and their roles in activity regulation

Eric Dubuisa, Nichola Rockliffea, Munir Hussainb, Mark Boyettc, Dennis Wrayd and Debra Gawlera,*

aThe Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, United Kingdom
bThe School of Medicine, University of Liverpool, United Kingdom
cThe Department of Medicine, University of Manchester, United Kingdom
dThe School of Biomedical Sciences University of Leeds, United Kingdom

* Corresponding author. Tel.: +44 151 7944786; fax: +44 151 7945322. Email address: dgawler{at}liv.ac.uk

Received 29 July 2005; revised 1 November 2005; accepted 2 November 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Src has been proposed to activate L-type calcium channel activity by binding to the {alpha}1c subunit. In the II–III linker region of this subunit there is a novel consensus sequence for Src binding. We have examined whether this site is a functional Src interaction site and investigated the effect displacing Src from this region has on calcium channel activity.

Methods: In vitro binding assays were performed to map {alpha}1 subunit interaction sites. Cardiac myocytes were isolated enzymatically from rat ventricles. Whole cell patch-clamp technique was used to record Ca2+ channel currents in cells that had been loaded with the Src inhibitor PP1 and/or peptides with amino acid sequence corresponding to the hypothesized Src docking site. Co-immunoprecipitation and pull-down studies were undertaken to identify proteins co-complexing with the {alpha}1 subunit.

Results: Peptides corresponding to the II–III linker region and C-terminal tail of the {alpha}1c subunit, but not scrambled peptide controls, were found to inhibit Src SH3 domain binding to the channel and significantly reduced the channel current amplitude. The II–III linker region peptide shifted the inactivation curve to the left whereas the C-terminal tail region peptide shifted the activation curve to the right when compared to scramble peptide controls. PP1-pre-treatment of myocytes also reduced the current amplitude, decreased the V1/2 for channel inactivation and abolished any further effect on currents by Src binding peptides. The tyrosine kinase PYK2 was found to co-associate with Src and the channel, but PP1 pre-treatment reduced this co-association.

Conclusions: Src binds to both the II–III linker and C-terminal tail regions of the {alpha}1c subunit to differentially modulate channel activity. PYK2 is also able to co-complex with Src when bound to this region of the channel but only when Src is catalytically active. Together the two kinases may synergistically regulate channel activity.

KEYWORDS Ca-channel; e–c coupling; Myocytes; Electrophysiology; Tyrosine protein kinases


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
L-type calcium channels are comprised of {alpha}1, {alpha}2-{delta}, β and {gamma}subunits [1].The {alpha}1 subunit is the pore forming unit and ten {alpha}1 subunit genes have been identified [2]. In cardiac muscle, the predominant {alpha}1 subunit of L-type calcium channels is encoded by the {alpha}1c (also known as the Cav1.2 or CACNA1C) gene [3,4]. L-type calcium channels have already been implicated in heart failure [5,6] atrial fibrillation [7,8] and ischemic heart disease [9]. Indeed, mice lacking Cav1.2 die as embryos due to cardiac dysfunction [10]. Thus, elucidating the mechanisms by which these channels are regulated may lead to a better understanding of normal cardiac function and how alterations can lead to heart disease.

Protein phosphorylation is an important mechanism by which calcium channel activity is regulated [11] and the {alpha}1c subunit is a target for serine/threonine phosphorylation mediated by PKA, PKC and PKG [11–15]. Protein tyrosine kinases (PTKs) are also involved in the regulation of smooth muscle contraction by modulating calcium channel activity [11,16,17]. PTKs can mediate tyrosine phosphorylation of L-type voltage gated calcium channels and enhance channel activity. c-Src has been shown to increase tyrosine phosphorylation of the {alpha}1c subunit [18,20]. Furthermore, proline-rich amino acid sequences resembling predicted Src SH3 domain binding motifs have been identified in the C-terminal tail of the subunit [19,20]. We have identified an amino acid sequence in the {alpha}1c subunit that may be a novel Src SH3 binding site. This stretch of sequence is located within the proposed intracellular II–III linker region. In this study we have examined whether this is a functional Src SH3 domain binding site and investigated the effect that peptides corresponding to this sequence have on channel activity and Src-channel co-association.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Peptides
All peptides used were synthesized at a purity >95% and were HPLC analysed by Sigma-Genosys. Peptide sequences were as follows:


peptide P1: SPSTFPRPRPTPPVTPGSRGRPLQPIPTL (residues 1973–2001 of rat {alpha}1c)
peptide P2: EEEPEMPVGPRPRPLSELHLKEKAVPMP (residues 877–905 of rat {alpha}1c)
peptide S1: RLPQRSPTPPSTGLPGVRTPFRPTIPPSP (scrambled peptide P1 sequence)
peptide S2: LEVPEPMEGPASPVRKEPMRLHPKELPE (scrambled peptide P2 sequence)

2.2 Rat ventricular myocytes
Isolations were conducted at 37 °C using the Langendorff technique previously described [21]. Hearts from male Wistar rats (250–300 g) were quickly mounted and perfused with Hepes–Tyrodes buffer comprising of 10 mM Hepes buffer (pH 7.3), 130 mM NaCl, 5.4 mM KCl, 1.4 mM MgCl2, 0.4 mM NaH2PO4, 10 mM creatine, 20 mM taurine, 10 mM glucose and 0.75 mM CaCl2 followed by perfusion with the same buffer containing 0.1 mM EGTA but no CaCl2. Hearts were then perfused with an isolation solution containing 10 mM Hepes (pH 7.3), 130 mM NaCl, 5.4 mM KCl, 1.4 mM MgCl2, 0.4 mM NaH2PO4, 10 mM creatine, 20 mM taurine, 10 mM glucose, 0.75 mM CaCl2 and 1 mg/ml type I collagenase and 0.05 mg/ml type XIV protease. Each ventricle was then removed, chopped and placed in a flask containing the isolation solution supplemented with 10 mg/ml BSA and agitated for 4 min, after which, the solution was filtered and cells collected by centrifugation at 350 rpm for 90 s. Cells were resuspended in low calcium buffer and the process was repeated 5 consecutive times. Cells were finally resuspended in enzyme-free isolation solution containing 0.75 mM CaCl2 and maintained at 4 °C until used within 2 to 6 h. All animals handling in this study conform to the guide for the care and use of laboratory animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.3 Electrophysiological recordings
Cells were loaded for 10 min with 10 µM BAPTA-AM to prevent contraction and then placed into a 200 µl bath chamber. The extracellular solution (140 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 10 mM glucose, 1.8 mM CaCl2 and 10 mM Hepes pH7.3) was superfused at a rate of 1–2 ml/min and experiments performed at 36–37 °C on rod-shaped, non-contracting cardiac ventricle myocytes showing clear striations. Calcium channel currents were recorded using whole-cell patch-clamp technique [22]. Experiments were conducted using an Axoclamp-2B amplifier (Axon Instruments Inc.) and currents were filtered with an 8-pole Bessel filter at 1 kHz and sampled at 10 kHz. Records were digitised with a Digidata-1200 A/D converter (Axon Instruments Inc.) and stored on disk in a PC computer using the Clampex (for voltage-clamp experiments) routine of pCLAMP version 6.02 software (Axon Instruments Inc.). Micropipettes made from borosilicate glass (type GC00F; Clark Electromedical Instruments) were filled with a cesium based solution and pulled to obtain a resistance of 2–2.5 M{Omega}after fire polishing. To assess the effects of the peptides on the calcium current, the cells were clamped at a holding potential of –40 mV and current was generated by consecutive pulses of 40 mV (400 ms duration, 20 s interval) for a period of 20 min. To construct I–V curves, the cells were voltage clamped at a holding potential of –40 mV and calcium currents were generated by stepwise 5 mV depolarizing pulses (400 ms duration, 5 s interval) with a constant holding potential of –40 up to +50 mV. The inactivation of the calcium current was assessed by stepwise 5 mV depolarizing pre-pulses (400 ms duration) with constant holding potentials of –40 up to +50 mV followed by a 2 ms interval at –40 mV and a test pulse (100 ms duration) with an amplitude of 40 mV from a holding potential of –40 mV (6 s intervals between each pre-pulse/test pulse process). At the holding potential of –40 mV, sodium current is fully inactivated and potassium currents were suppressed using CsCl. The membrane potentials were recorded at the same time as the currents and mean values were plotted for I–V and activation or inactivation curves.

For peptide diffusion inside the cell, patch pipettes were filled by dipping the tip of the pipette into the peptide-free filling internal solution (containing 1 mM MgCl2, 0.1 mM CaCl2, 5 mMEGTA, 130 mMCsCl, 4 mM MgATP, 0.19 mM Na2GTP and 10 mM Hepes pH 7.3) before backfilling with the same internal solution this time containing peptides at a final concentration of 5 µM. Analysis of all patch-clamp data was performed using Clampfit (pCLAMP version 6.02) and Origin 4.1 software (Microcal Inc., Northampton, MA, USA).

2.4 Statistical analysis
Results are expressed as mean ± standard error (S.E.M.). Statistical analysis was undertaken using the unpaired Student t test or the Mann–Whitney test when normality test failed (Anderson–Darling test). For comparison between more than two means, analysis of variance followed by Dunnett's test or Mood Median test was used when normality test failed. n=the number of cells and N=number of animals. Differences were considered significant when p<0.05. Statistical analysis was applied using Minitab software (Minitab Inc.).

2.5 Preparation of cell membranes and solubilisation of the channel complex
Crude cell membranes were prepared from isolated cardiac myocytes by resuspending cells in 20 mM Hepes (pH 7.4) followed by homogenization by 15 passages through a 19 g needle and collection of the supernatant by centrifugation for 5 min at 1000 g. The supernatant was centrifuged for 30 min at 20,000 g and the broken membranes collected in the pellet fraction. Membranes were stored at –80 °C until use. Membranes were resuspended in RIPA buffer at a protein concentration of 1 mg/ml and homogenized using a 19 g needle. Samples were incubated with rotation for 30 min and then centrifuged at 20,000 g to pellet and remove non-solubilised tissue. Lysates thus produced were used for immunoprecipitation studies.

2.6 Pull-downs, immunoprecipitations and Western blotting
Pull-downs, immunoprecipitations and Western blotting procedures were performed as previously described [23]. Anti-{alpha}1C antibodies (Sigma), anti-PYK2 antibodies (UBI), anti-phospho-tyrosine antibodies (Sigma) and anti-Src antibodies (Santa Cruz) were used for either immunoprecipitations at 1 µg/ml or for blotting at 1/200 dilution. For Western blotting, the primary antibodies were detected using biotinylated secondary antibodies (goat anti-rabbit or rabbit anti-goat for {alpha}1c and Src detection, respectively) followed by avidin-HRP probing and ECL detection on film. To enhance {alpha}1c subunit detection the "Visualizer Western Blot detection kit" (UBI) was used at the ECL detection stage when probing blots for this proteins.

2.7 Recombinant protein expression and purification
cDNAs encoding for the SH3 domain of rat c-Src (amino acids 85–143) and the C-terminal tail of the {alpha}1C subunit (amino acids 1909–2170) were isolated by PCR amplification from a rat heart cDNA library (Clontech). The cDNA encoding for the linker II–III region (amino acids 793–935) of the rabbit {alpha}1C subunit was amplified using full-length rabbit {alpha}1C cDNA. Each cDNA was subcloned into the pGEX 5X-3 GST fusion protein expression vector and isolated clones were DNA sequenced. All fusion proteins were expressed in E. coli and purified on glutathione agarose beads. The GST–Src SH3 fusion protein was released from beads by incubation in PBS containing 100 mM glutathione pH 7.2. The released protein was dialysed against PBS overnight at 4 °C prior to use in the overlay binding assay. Each of the {alpha}1c subunit fragments were cleaved from their GST tag by incubation with factor Xa at 25 °C for 4–6 h using a 1:50 ratio of factor Xa:GST fusion protein. The liberated {alpha}1C protein fragments were then collected by centrifugation and quantified on SDS-PAGE gels.

2.8 Src SH3 overlay binding and in vitro peptide competition studies
Samples of either 100 ng of C-terminal tail region protein or 500 ng of the linker II–III region protein were loaded onto SDS polyacrylamide gels and after electrophoresis were transferred onto nitrocellulose filters. After blocking with 5% (w/v) skimmed milk, filter strips were cut and incubated with GST–Src SH3 fusion proteins (500 ng/ml) in the presence or absence of peptides (20 µg) in 2 ml of blocking solution for either 1 h at 25 °C (C-terminal binding) or overnight at 4 °C (linker binding). Filter strips were then washed twice with PBS and once with PBS containing 0.05% (v/v) Tween 20. GST–Src SH3 protein bound to filters was then probed for using anti-GST antibodies and these antibodies were in turn detected using anti-rabbit-HRP antibodies and ECL signals captured on film.

2.9 PP1 treatment of cells
Pyrazolo pyridine 1 (PP1; Biomol. International) was dissolved in DMSO and then diluted in extracellular solution to give final DMSO and PP1 concentrations of 0.01% and 1 µM, respectively. For electrophysiological recordings, cells were perfused with the PP1 solution for the times indicated in the figures. To pre-treat cells prior to undertaking immunoprecipitation studies, fresh batches of myocytes were isolated. Half of each batch of cells was incubated in enzyme-free isolation buffer and the other half were incubated in the same buffer containing PP1 at a final concentration of 1 µM for 20 min at 37 °C. Crude membranes were then prepared as described above.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Binding of Src to the {alpha}1c subunit
To determine whether the C-terminal tail region and the II–III linker region of the {alpha}1c subunit each contained a bona fide Src SH3 domain binding site, overlay binding assays were performed. To do this, purified synthetic peptides corresponding to rat {alpha}1c subunit amino acid residues 1973–2001 (denoted peptide P1), 877–905 (denoted peptide P2) and scrambled peptide controls (denoted S1 and S2, respectively) were commercially obtained. A GST–Src SH3 domain fusion protein and recombinant proteins representative of either the C-terminal tail of the channel (rat amino acids 1909–2170) or the linker II–III region (rat amino acids 793–935) were constructed and expressed (see Fig. 1). Overlay filters containing each channel protein fragment were probed for Src SH3 binding. Binding to both protein fragments was clearly detected (Fig. 2A and B). However, binding to the C-terminal tail (Fig. 2B) was more strongly detected than that for the linker II–III region binding (Fig. 2A). Competition studies in the presence of each peptide demonstrated that peptide P1 significantly reduced the binding of the Src SH3 domain to both channel protein fragments. However, peptide P2 was only able to inhibit binding to the linker II–III region protein fragment and scrambled control peptides had no significant effect on Src SH3 binding to either channel fragment.


Figure 1
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  		Fig. 1 Schematic diagram showing the position of putative Src SH3 binding sites within the {alpha}1c subunit. Amino acid sequences of consensus Src SH3 binding motifs are indicated and species variations are shown. Peptides P1 and P2 correspond to sequences found within the C-terminal tail and II–III linker regions of the rat {alpha}1c protein, respectively.

 

Figure 2
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  		Fig. 2 Peptides P1 and P2 but not scrambled peptides, S1 and S2, compete for Src binding to the {alpha}1c subunit. A, overlay binding of Src SH3 to the linker II–III region or B, the C-terminal tail region in the presence or absence of peptides as indicated. Src co-immunoprecipitates with {alpha}1c from cardiac myocyte lysates in the presence or absence of peptides. C, the {alpha}1c subunit was immunoprecipitated using anti-{alpha}1c antibodies and samples were probed with either anti-Src or anti-{alpha}1c antibodies as indicated.

 
Next we examined whether our peptides could affect the co-immunoprecipitation of Src with the channel from cardiac myocyte membranes (Fig. 2C). Only one {alpha}1c subunit band with a molecular weight of ~200 kDa was immunoprecipitated from cell membrane preparations (Fig. 2C). Expression levels of the subunit are very low in myocytes and since high levels of tissue were required to detect an {alpha}1c signal, the background on blots was high. However, Src was strongly detected as co-immunoprecipitating with the channel and there appeared to be slight reductions in the amount of Src detected in the presence of peptides P1 and P2. Scanning densitometry provided estimates of co-complexed Src in the presence of peptides to be: S1, 115 ± 10%, S2, 110 ± 15%, P1,17 ± 6% and P2, 80 ± 9% (mean ± S.E.M. values all relative to no peptide control for n=3 blots with each densitometry reading taken twice).

3.2 Effect of peptide diffusion into myocytes on calcium current amplitude over time
Whilst undertaking peptide competition studies, we investigated the effect of peptides P2 and S2 on calcium channel currents using electrophysiological recordings. Peptides were added to cells by patch pipette and the effect of each peptide on the current amplitude was assessed over time (Fig. 3A and B). We observed a rapid and significant decrease in the current amplitude upon application of peptide P2 (33.0 ± 3.2%) when compared to the control peptide, S2, which had no significant effect (6.6 ± 2.2%). In addition, the control current remained steady with time, indicating that no significant run down of currents was occurring.


Figure 3
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  		Fig. 3 Patch pipette application of peptides into rat cardiac myocytes; effect on the current amplitude over time. Left panels: effect of peptide diffusion on current amplitude over time. Right panels: concatenated traces of currents recorded over time. A, peptide P2 application (N=5, n=6) B, peptide S2 application (N=4, n=5). Results represent mean ± S.E.M. values.

 
3.3 Effect of peptides on the I–V relationship, activation and inactivation curves of the calcium channel currents
Analysis of the I–V relationship curves showed that peptide P2 (Fig. 4A) caused a significant decrease in the mean current. Furthermore, whilst having no significant effect upon the activation curve, peptide P2 was found to significantly shift the inactivation curve to the left (corresponding to V1/2 values of –18.4 ± 0.4 mV for control cells and –28.1 ± 0.6 mV for P2 containing cells; Fig. 4C and Table 1). Scrambled peptide S2 had no significant effects on the I–V relationship curves (Fig. 4B) and did not significantly modify the activation or inactivation curves (Fig. 4D and Table 1).


Figure 4
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  		Fig. 4 Effect of peptide application on I–V curves and activation/inactivation curves. Effect of peptides on the I–V relationship curves before ({circ}) or after peptide application (bullet). A, peptide P2 application (N=5, n=6) B, peptide S2 application (N=4, n=5). Results represent mean ± S.E.M. values. Effect of peptide application on activation and inactivation curves. Before ({circ},{square}) or after (bullet,{blacksquare}) peptide application. Activation ({square},{blacksquare}) and inactivation ({circ},bullet) curves for calcium currents. C, peptide P2 application (N=5, n=6) D, peptide S2 application (N=4, n=5). Results represent mean ± S.E.M. values.

 

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  		Table 1 Summary of effects of peptide application on inactivation and activation kinetics

 
3.4 Effect of pre-treating myocytes with the Src kinase inhibitor PP1
Tyrosine phosphorylation within the II–III linker region of the {alpha}1c subunit has previously been looked for, but not detected, by others [20]. So, Src regulation of channel activity within this region may not involve its phosphorylation. Instead, a conformational change within the channel may occur upon Src binding. If this is so, channel activity regulation should be independent of Src enzymatic activity. To test this hypothesis, we pre-treated myocytes with the Src kinase inhibitor PP1 and looked to see if synergistic effects on channel activity were observed upon peptide P2 application (Fig. 5). We found that PP1 treatment inhibited the channel current amplitude to a lesser extent than that observed in response to peptide P2 application (23.2 ± 1.8 and 33.0 ± 3.2% for PP1 and P2 peptide, respectively; Fig. 5A). However, the kinase inhibitor displayed a similar mechanism of channel activity inhibition to that observed for peptide P2 in that, it shifted the inactivation curve V1/2 from –17.48 ± 0.48 to –23.45 ± 0.70 mV (Fig. 5B and Table 1). In addition, when the P2 peptide was applied after PP1 pre-treatment, the ability of the peptide to further inhibit channel activity was abolished (Fig. 5C–F). These data indicate that the enzymatic activity of Src is necessary for its ability to regulate channel activity in this region. However, since application of the P2 peptide alone had a greater inhibitory effect on current amplitude than did PP1 inhibition of Src, this also suggested that Src interaction may further potentiate channel activity regulation when Src's enzymatic activity is functional. One possible mechanism that could account for such an observation would be the (kinase dependent) co-complexing of proteins with Src and the channel.


Figure 5
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  		Fig. 5 Effect of PP1 treatment on calcium currents with or without peptide P2 application. To apply PP1, cells were perfused with the extracellular solution containing PP1 (1 µM). Peptide P2 was applied by patch pipette as described in Materials and methods section at the times indicated. A, effect of PP1 application on current amplitude over time, B, effect of PP1 application on activation and inactivation curves. Before ({circ},{square}) or after (bullet,{blacksquare}) PP1 application. Activation ({square},{blacksquare}) and inactivation ({circ},bullet) curves for calcium currents, C, effect of peptide P2 application after PP1 application on current amplitude over time, D, effect of PP1 and peptide P2 combined application on activation and inactivation curves. Before ({circ},{square}) or after (bullet,{blacksquare}) PP1 and peptide P2 application. Activation ({square},{blacksquare}) and inactivation ({circ},bullet) curves for calcium currents, E, effect of PP1 and peptide P2 application on I–V curves before ({square}) PP1 application or after ({blacksquare}) PP1 application or after both PP1 and peptide P2 application (bullet), F, histogram showing the effect of PP1 and peptide applications on mean current amplitudes. Results represent mean ± S.E.M. values (N=4, n=6).

 
We therefore looked for tyrosine phosphorylated proteins co-complexing with the {alpha}1c subunit before and after PP1 treatment of cells. Our rationale was that candidate co-complexing proteins could be Src-dependent phospho-tyrosine containing proteins whose association with the channel would be inhibited when Src enzyme activity was inhibited with PP1. So, these proteins would be expected to have a reduced phospho-tyrosine content and reduced channel co-association after PP1 treatment. We immunoprecipitated {alpha}1c from myocytes (+/– PP1 treatment) and Western blot probed for co-associated phospho-tyrosine proteins (Fig. 6). Two proteins with molecular weights of ~200 and 116 kDa were clearly detected in this way (Fig. 6B). We reasoned that the 200 kDa protein is probably the {alpha}1c subunit that we detect with the {alpha}1c antibody in myocyte membranes (Fig. 6A). Because Src is known to bind to the tyrosine kinase PYK2, which has a molecular weight of ~115 kDa [33], we probed blots with anti-PYK2 antibodies to see if this was our 116 kDa channel co-complexing protein (Fig. 6C). We successfully detected PYK2 in our samples and found, upon PP1 treatment of cells, the amount of PYK2 co-immunoprecipitating with the {alpha}1c subunit was indeed reduced (Fig. 6C). Furthermore, when we probed for Src in these samples we found that the amount of Src co-immunoprecipitating with the channel was not significantly altered by PP1 treatment (Fig. 6C). In order to determine whether PYK2 was co-complexing with Src in the II–III linker region of the channel we employed pull-down assays using either GST or GST-II–III linker region fusion proteins (Fig. 6D). We found that PYK2 was indeed detected as co-complexing with the II–III linker region but not with the GST control protein.


Figure 6
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  		Fig. 6 Effect of PP1 pre-treatment on proteins co-complexing with the {alpha}1c subunit in rat myocytes. A, the anti-{alpha}1c subunit antibody specifically detects only one protein band of apparent molecular weight ~200 kDa in rat cardiac myocytes membranes. Panels B and C; the {alpha}1c subunit was immunoprecipitated from cells with or without PP1 pre-treatment as indicated using anti-{alpha}1c antibodies. Samples were then probed with either B, anti-phosphotyrosine antibodies or C, (upper panel) anti-{alpha}1c antibodies or (middle panel) anti-PYK2 antibodies or (lower panel) anti-Src anti-{alpha}1c antibodies. D, PYK2 co-complexes with the {alpha}1C subunit in pull-down assays; GST (lane 1), GST-II–III linker region (lane 2).

 
3.5 Effect of peptide P1 diffusion into myocytes on amplitude, the I–V relationship and activation and inactivation curves of the calcium channel currents
Application of peptide P1 to cells resulted in a significant decrease in the current amplitude (28.8 ± 2.2%) when compared to the control peptide, S1 (8.1 ± 1.9%; Fig. 7). The inhibitory effect of peptide P1 was maximal within 15 min (Fig. 7A). Analysis of the I–V relationship curves showed that peptide P1 (Fig. 8A) caused a significant decrease in the mean current. However, whilst having no significant effect upon the inactivation curve, peptide P1 was found to shift the activation curve to the right (corresponding to V1/2 values of –9.7 ± 0.6 mV for P1 containing cells and –19.2 ± 0.4 mV for control cells; Fig. 8C and Table 1). Peptide S1 had no effects on the I–V relationship curves (Fig. 8B) and did not modify the activation or inactivation curves (Fig. 8D and Table 1).


Figure 7
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  		Fig. 7 Patch pipette application of peptides into rat cardiac myocytes; effect on the current amplitude over time. Left panels: effect of peptide diffusion on current amplitude over time. Right panels: concatenated traces of currents recorded over time. A, peptide P1 application (N=4, n=6) B, Peptide S1 application (N=5, n=5). Results represent mean ± S.E.M. values.

 

Figure 8
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  		Fig. 8 Effect of peptide application on I–V curves and activation/inactivation curves. Effect of peptides on the I–V relationship curves before ({circ}) or after peptide application (bullet). A, peptide P1 application (N=4, n=6). B, peptide S1 application (N=5, n=5). Effect of peptide application on activation and inactivation curves. Before ({circ},{square}) or after (bullet,{blacksquare}) peptide application. Activation ({square},{blacksquare}) and inactivation ({circ},bullet) curves for calcium currents. C, Peptide P1 application (N=4, n=6). D, Peptide S1 application (N=5, n=5). Results represent mean ± S.E.M. values.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
4.1 Src SH3 domain binding to the {alpha}1c subunit
Protein sequences that selectively bind SH3 domains have been identified [24–26]. Src SH3 domains bind to proline rich sequences containing a consensus sequence of either: RXXPXXP [20] or RPXPXXP [24] (denoted class I sites) or XPXXP or XPXXPXR [25,26] (denoted class II sites). There may be two Src SH3 binding sites in the C-terminal tail region of the rat {alpha}1c subunit; a class I site located between amino acids 1979–1985: RPRPTPP and a class II binding site located between amino acids 1993–1997: RPLQP. Although the class II site has been confirmed in vitro [19], the class I site is still only a proposal [20]. However, enhanced Src SH3 binding is observed when both sites are present in binding assays [19]. Here we have identified a novel docking site for Src on the {alpha}1c subunit within the II–III linker region of the channel. Amino acids 883–887 in the rat {alpha}1c subunit contain a class II Src SH3 consensus sequence: MPVGP and a sequence very similar to a class I sequence between amino acids 888–894: RPRPLSE. The peptides we have used span both potential sites in the C-terminal tail region (peptide P1) and both potential sites in the linker region (peptide P2).

The rationale of our study was that by using sequence specific peptides to displace Src from the channel we could evaluate their effect on channel calcium currents. Peptide P1 was intended as a positive control in that it contains a previously shown Src SH3 binding site. Here we have shown that the Src SH3 domain does indeed bind directly to the II–III linker region of the {alpha}1c subunit in vitro. This binding was blocked by both peptide P2 and peptide P1. Moreover, these interactions were specific because scrambled control peptides had no effect upon Src SH3 domain binding. Furthermore, since peptide P2 did not prevent the Src SH3 domain from binding to the C-terminal tail region, but peptide P1 did, we conclude that there is some degree of selectivity for Src binding to each of these regions. The affinity for Src binding to the II–III linker region may be weaker than that for the C-terminal tail; possibly reflective of the classification of the SH3 binding site. Peptide P1 (and the corresponding rat C-terminal protein sequence) contains sequences that are 100% identical to both a class I and a class II SH3 binding site. However, peptide P2 (and the corresponding II–III linker region) only contains a class II binding site, and a weaker (85% identical) class I binding site. We consistently detected more Src SH3 domain binding to the C-terminal tail protein fragment than to the II–III linker region even though we used ~5 times more linker protein fragment than C-terminal tail fragment and overnight binding conditions for the linker fragment assay in order to optimize the detection of Src binding to this region.

Co-immunoprecipitation experiments also agreed with the in vitro binding assays in that both peptides did appear to reduce the amount of Src co-associating with the {alpha}1c subunit. However, peptide P1 reduced the co-association more than peptide P2. From our blotting experiments, the only form of the {alpha}1c subunit we were reproducibly able to detect in our immunoprecipitates appeared to be the smaller form with a molecular weight of ~200 kDa. This may reflect either the truncated form of the protein previously reported [12,27] or a proteolytically degraded form of the full-length channel due to insufficient inhibition of proteases in our preparations. This truncated form of the protein has previously been reported as the predominant form in these cells when blotting [27], but others report that the full length protein is also found in these cells [28]. The antibody we have used for these studies was raised to the rabbit amino acid residues 818–835 and so would be expected to precipitate both truncated and full-length {alpha}1c forms. It would similarly be expected to co-immunoprecipitate any proteolytically cleaved C-terminal tail regions that may remain co-associated with the channel [29].

4.2 Effect of peptides on calcium channel activity
Since peptide P2 showed selective displacement of Src at only one site on the {alpha}1c subunit, we assume that peptide P2 is highly specific. Our results indicate that, when Src is prevented from binding to this region of the {alpha}1c subunit, channel inactivation kinetics are altered and channel activity is subsequently inhibited. These data are consistent with Src playing a role in the augmentation of channel activity [30]. However, our observations are intriguing because, whilst the C-terminal tail is known to be important in calcium dependent channel inactivation [31] and when removed leads to an increase in the open probability of the channel [32], the II–III linker region has not been previously implicated in channel inactivation. Also peptide P1 surprisingly had no effect on the inactivation kinetics of the channel. Given that the in vitro binding data indicate that this peptide causes displacement of Src at both the C-terminal tail and II–III linker regions, it is possible that by shifting the activation curve, the detection of a further shift in the inactivation curve could be masked. However, if combined effects on activation and inactivation were simultaneously occurring we would expect a synergistic reduction of current amplitude. However, this was not observed (Fig. 7A). An alternative explanation could be Src dependent intra-molecular interactions between the C-terminal tail and the II–III linker region. That is, when peptide P1 displaces Src from the C-terminal tail, an interaction between the tail and the II–III linker region could be prevented. Furthermore, docking of Src at one or both sites may result in selective phosphorylation of the channel and/or co-complexed proteins.

4.3 Role of Src kinase catalytic activity in the regulation of calcium channel function
We propose that Src can bind to the {alpha}1c subunit via its SH3 domain at two distinct sites and, when catalytically active, brings with it other regulatory co-complexing proteins. One of these proteins is PYK2. This protein can bind to the SH2 domain of Src when Src is catalytically active [33]. Intriguingly, the kinase activity of Src is not required for Src to interact with the {alpha}1c subunit, but it is required for co-complexing of PYK2 with the channel to occur. Also, the Src:{alpha}1c subunit molecular ratio appears to be far greater than 1:1; indicating either that Src may be multimerizing and/or there are multiple Src molecules simultaneously docking to the channel. However, because peptide P1 is not selective, we cannot investigate specific protein complex formations at each site at this time. Finally, we realize that whilst such in vitro site mapping studies provide mechanistic clues they do not definitively prove these interactions occur in vivo. However, such studies may facilitate advances in our understanding of channel function and heart disease.


   Acknowledgements
 
This work was funded by grants from the British Heart Foundation (to M.B., D.W. and D.G) and The Wellcome Trust (to D.G.).


   Notes
 
Time for primary review 21 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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