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Originally published In Press as doi:10.1074/jbc.M609071200 on November 16, 2006

J. Biol. Chem., Vol. 282, Issue 4, 2156-2162, January 26, 2007
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The Src Homology 3 Domain of the beta-Subunit of Voltage-gated Calcium Channels Promotes Endocytosis via Dynamin Interaction*Formula

Giovanni Gonzalez-Gutierrez{ddagger}1, Erick Miranda-Laferte§1, Alan Neely{ddagger}, and Patricia Hidalgo§2

From the {ddagger}Centro de Neurociencia de Valparaíso, Universidad de Valparaíso 2349400 Chile and the §Abteilung Neurophysiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany

Received for publication, September 25, 2006 , and in revised form, November 14, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
High voltage-gated calcium channels enable calcium entry into cells in response to membrane depolarization. Association of the auxiliary beta-subunit to the {alpha}-interaction-domain in the pore-forming {alpha}1-subunit is required to form functional channels. The beta-subunit belongs to the membrane-associated guanylate kinase class of scaffolding proteins containing a Src homology 3 and a guanylate kinase domain. Although the latter is responsible for the high affinity binding to the {alpha}-interaction domain, the functional significance of the Src homology 3 domain remains elusive. Here, we show that injection of isolated beta-subunit Src homology 3 domain into Xenopus laevis oocytes expressing the {alpha}1-subunit reduces the number of channels in the plasma membrane. This effect is reverted by coexpressing {alpha}1 with a dominant-negative mutant of dynamin, a GTPase involved in receptor-mediated endocytosis. Full-length beta-subunit also down-regulates voltage-gated calcium channels but only when lacking the {alpha}-interaction domain. Moreover, isolated Src homology 3 domain and the full-length beta-subunit were found to interact in vitro with dynamin and to internalize the distantly related Shaker potassium channel. These results demonstrate that the beta-subunit regulates the turnover of voltagegated calcium channels and other proteins in the cell membrane. This effect is mediated by dynamin and depends on the association state of the beta-subunit to the {alpha}1-pore-forming subunit. Our findings define a novel function for the beta-subunit through its Src homology 3 domain and establish a link between voltage-gated calcium channel activity and the cell endocytic machinery.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cellular processes including muscle contraction, endocrine secretion, synaptic transmission, and gene expression (1), depend on the regulated influx of calcium through voltagegated calcium channels (VGCCs).3 VGCCs are multiprotein complexes containing a pore-forming subunit (CaV{alpha}1) and a variable number of auxiliary subunits. Association of the auxiliary beta-subunit (CaVbeta) to a site shared by all CaV{alpha}1, the so-called {alpha}-interaction domain (AID), is mandatory to form a fully functional VGCC. Homology modeling (2) and the recent high resolution crystal structures of three CaVbeta isoforms (3–5) identified CaVbeta as a novel member of the membrane-associated guanylate kinase class of scaffolding proteins containing a Src homology 3 (SH3) and a guanylate kinase (GK) domain (Fig. 1A). As shown by the crystal structure of CaVbeta complexed to AID, the CaVbeta-GK binds to the AID, whereas CaVbeta-SH3 interacts with GK. Although SH3 domains are known to mediate protein-protein interactions by binding to proline-rich motifs in ligand proteins (6), no interactions mediated by the CaVbeta-SH3 have been described yet. Moreover, the functional integrity of CaVbeta-SH3 domain is uncertain since the residues homologous to the ones critical for binding PXXP motifs in canonical SH3 modules are occluded in the crystal structure of CaVbeta. Intriguingly, canine and human cardiac cells express splicing variants encoding short versions of the CaVbeta that only encompass the variable N-terminal region and the SH3 domain (7, 8) (Fig. 1A, V1 and C1, respectively).

Here, we studied the effect of isolated CaVbeta-SH3 on calcium channel function and expression. The SH3 domain of the rat beta2a isoform of CaVbeta (beta2a-SH3) was expressed in bacteria, purified, and injected into Xenopus oocytes expressing the cardiac CaV{alpha}1 (CaV1.2) subunit isoform. We found that the beta2a-SH3 induces removal of channels from the plasma membrane in a dynamin-dependent fashion. This function is preserved by full-length CaVbeta in the absence of CaV{alpha}1 subunit or when binding to it is disrupted by deleting the AID site. Our results define a novel interaction and outline a new function for the calcium channel beta-subunit.


Figure 1
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  		FIGURE 1.
beta2a-SH3 reduces charge movement in Xenopus oocytes expressing CaV1.2. A, CaVbeta domains. V1, V2, and V3 denote variable regions, and C1 and C2 denote conserved SH3- and GK-like domains, respectively. Numbers refer to the amino acid sequence in the beta2a rat isoform used in this study. B, size exclusion chromatography profile of purified beta2a-SH3 domain and molecular mass calibration curve on Superdex 200 10/30 column (GE Healthcare Life Sciences). 1 denotes void volume, and 2, 3, and 4 denote the elution volume of albumin (67 kDa), ovalbumin (43 kDa), and ribonuclease A (13.7 kDa), respectively. The inset shows a SDS-PAGE performed on purified beta2a-SH3 with numbers corresponding to the molecular mass of standards in kDa. MW, molecular weight. C, average Qon from CaV1.2-expressing oocytes before (132.6 ± 11.6 pC, n = 18) and after beta2a-SH3 (26.0 ± 3.8 pC, n = 25) or buffer injection (123.6 ± 10.5 pC, n = 22) as control. Qon was measured by integrating gating current during a step near IBa reversal potential as shown in the inset. Voltage near IBa reversal potential was determined empirically by stepping to several potentials in 2-mV increments. D, time course of the beta2a-SH3-induced decrease in Qon in CaV1.2-expressing Xenopus oocytes. Each point corresponds to the average of Qon measured as in panel C for several oocytes recorded at different times following the injection of beta2a-SH3. Averages for each time point include measurements up to 30 min before the indicated time, and t = zero corresponds to the average Qon from non-injected oocytes. The data were fitted to Qon = Qo(exp[–t/{tau}]), where Qo is the estimated Qon at time 0 in pC (108 pC), Qmin is the residual Qon (25.7 pC), and {tau} is the time constant in hours (0.90 h). E, average current-voltage plot normalized by Qon from CaV1.2-expressing oocytes before protein injection ({circ}) and after beta2a-SH3 (•) or CaVbeta2a ({diamondsuit}) injection. F, representative gating currents traces and voltage dependence of Qon from CaV1.2-expressing oocytes before ({circ}) and after beta2a-SH3 injection (•) during 20-ms voltage pulses from –80 mV to +40 mV in 5-mV increments from a holding potential of –90 mV recorded in 2 mM external Co2+. G, representative gating currents traces and time dependence of Qon from CaV1.2-expressing oocytes before ({circ}) and after beta2a-SH3 injection (•) during pulses to +40 mV of variable duration (0.5–12.5 ms in 0.5-ms increments) from a holding of –90 mV. Proteins were injected 1–5 h before recordings. See details in supplemental Tables S1 and S2.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Recombinant Proteins—The cDNA encoding the SH3 domain of the rat beta2a isoform (Swiss-Prot entry:Q8VGC3) encompassing residues 24–136 was subcloned between BamHI and EcoRI restriction sites by conventional PCR methods into pRSETB vector (Invitrogen) to introduce a polyhistidine tag at the N-terminal. The molecular mass predicted by the amino acid sequence of the CaVbeta2a-SH3 His-tagged protein is 16.7 kDa. The His-tagged CaVbeta2a-SH3 was expressed in BL-21 (DE-3) E. coli bacteria by a 2-h induction with 0.5 mM isopropyl-beta-D-thiogalactopyranoside at 37 °C. Cells were harvested by centrifugation, flash-frozen, and stored until use at –80 °C. Right before protein purification, the cells were resuspended in phosphate buffer (50 mM sodium phosphate buffer and 300 mM NaCl, pH 7.0) containing EDTA-free protease inhibitor mixture (Roche Applied Science) and disrupted by ultrasonication. After centrifugation, the protein was purified from the cleared cell lysate by using a cobalt-based metal affinity chromatography (Talon, BD Biosciences) according to the manufacturer's instructions followed by size-exclusion chromatography onto a SuperdexTM S-200 column 26/60 (GE Healthcare Life Sciences) pre-equilibrated with buffer containing 20 mM Tris buffer, 300 mM NaCl, 1 mM EDTA, pH 8.0. The fractions containing the protein were pooled, concentrated up to 1–2 mg/ml by centrifugation using Amicon Ultra tubes with 10,000 molecular weight cut-off (Millipore), aliquoted, flash-frozen, and stored at –80 °C until use. The full-length CaVbeta2a was prepared as described (9). The apparent molecular mass of His-tagged CaVbeta2a-SH3 determined from the size exclusion chromatography calibration curve was obtained from the partition coefficient value (Kav) calculated from its elution volume as described (10), where Kav is equal to (Ve – Vo)/(Vt – Vo) and Ve is the elution volume of the protein; Vo is the void volume of the column calibrated with blue dextran, and Vt is the total bed volume. A set of globular protein standards was used as indicated in Fig. 1. Mass spectrometry analysis was performed in the mass spectrometry laboratory, Zentrums Pharmakologie und Toxikologie, Medizinische Hochschule Hannover. The protein was digested by trypsin, and the peptides were analyzed in an Ultraflex matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometer (Bruker Daltonics).


Figure 2
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  		FIGURE 2.
beta2a-SH3-induced reduction of charge movement is not abolished by bafilomycin. A, chemiluminescence and Qon of oocytes expressing CaV1.2-HA alone or after beta2a-SH3 injection. Chemiluminescence was integrated for 1.0 s and expressed as 105 counts/second (cps). Qon was measured as in Fig. 1C. Non inj., non-injected oocytes. B, average Qon measured from Xenopus oocytes expressing CaV1.2 in control conditions before (184.3 ± 23.3 pC, n = 9) and after beta2a-SH3 injection (54.0 ± 14.6 pC, n = 8). C, average Qon measured from Xenopus oocytes expressing CaV1.2 treated with 500 nM bafilomycin for 24 h before (80.9 ± 9.8 pC, n = 8) and after beta2a-SH3 injection (28.9 ± 7.3 pC, n = 7). In both cases, the reduction in Qon following beta2a-SH3 injection is significant (t test, p < 0.01).

 
The GST-Dyn829–842 peptide was prepared as follows. Two overlapping oligonucleotides were designed according to the dynamin sequence (Swiss-Prot entry: Q05193 [GenBank] ) to encode the peptide sequence from residues 829–842 (829PPQVPSRPNRAPPG842). After annealing, the oligonucleotides were ligated into pGEX vector (GE Healthcare Life Sciences) to fuse a GST at the N-terminal (GST-Dyn829–842 peptide). The GST alone and GST-Dyn829–842 peptide were expressed in bacteria and purified as described (10). Dynamin mutation and CaV1.2 {Delta} AID deletion were done by standard overlapping PCR using complementary oligonucleotides.


Figure 3
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  		FIGURE 3.
beta2a-SH3-induced reduction of charge movement relies on interaction with dynamin. A, Western blot analysis from Xenopus oocytes homogenates. Lane 1, non injected control oocytes; lane 2, oocytes injected with HA-tagged dynamin-WT cRNA; and lane 3, with HA-tagged dynamin-K44A cRNA. Membranes were analyzed with anti-dynamin (anti-Dyn) or anti-HA antibodies as indicated. B, average Qon from oocytes coexpressing CaV1.2 and dynamin-WT before (126.3 ± 13.5 pC, n = 24) and after beta2a-SH3 injection (19.2 ± 3.2 pC, n = 28). C, average Qon from oocytes coexpressing CaV1.2 and dynamin-K44A before (78.3 ± 12.8 pC, n = 19) and after beta2a-SH3 injection (50.6 ± 6.9 pC, n = 19). D, average Qon from CaV1.2-expressing oocytes before (190.8 ± 18.8 pC, n = 25) and after injection of beta2a-SH3 preincubated in an equal weight ratio with either GST (33.3 ± 6.1 pC, n = 37) or GST-Dyn829–842 peptide (98.3 ± 13.1 pC, n = 32) as indicated.

 
Binding Assay—Pull-down assays using His-tagged CaVbeta2a-derivatives as baits were performed as described (10). Briefly, His-CaVbeta2a derivatives were coupled to cobalt-based agarose for 1 h at 4°C and incubated for another hour with precleared tsA201 cell extract obtained 24–48 h after transfection with dynamin or with YFP-CaV1.2 expression vector. The beads were pelleted and washed five times, and bound fractions were eluted with SDS-PAGE loading buffer and resolved on SDS-PAGE. In the binding assays to dynamin, the gel was transferred to nitrocellulose membrane and subjected to immunoblot analysis using anti dynamin antibody (BD Biosciences). Binding to YFP-CaV1.2 was visualized by fluorescence scanning using a Typhoon imager (GE Healthcare Life Sciences).


Figure 4
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  		FIGURE 4.
beta2a-SH3 binds in vitro to dynamin but not to CaV1.2. A, anti-dynamin western-blot. His-beta2a-SH3 coupled to Co2+ beads was incubated with lysate from cells expressing dynamin I (lane 2). Control binding was performed with uncoupled Co2+ beads (lane 1). B, asin A, except that either GST (lane 1) or GST-Dyn829–842 peptide (lane 2) was added during incubation with dynamin-containing lysate. C, binding of His-tagged CaVbeta2a derivatives to YFP-CaV1.2. Lane 1, crude lysate from cells expressing YFP-CaV1.2; lane 2, control binding with uncoupled Co2+ beads; lane 3, binding to full-length CaVbeta2a; lane 4, binding to beta2a-core; and lane 5, binding to beta2a-SH3. YFP-CaV1.2 bands were visualized by fluorescence scanning using a Typhoon-9410 imaging system (GE Healthcare Life Sciences). Binding experiments were repeated three, two, and five times in A, B, and C, respectively.

 


Figure 5
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  		FIGURE 5.
beta2a-SH3 and full-length CaVbeta2a reduce ionic currents mediated by Shaker potassium channels expressed in Xenopus oocytes. A, representative ionic currents traces from oocytes expressing Shaker IR (Sh IR) before (top) and after beta2a-SH3 injection either preincubated with GST (middle) or preincubated with GST-Dyn829–842 peptide (bottom). Each panel corresponds to 60 traces recorded once per minute during the pulse protocol depicted on top. B, normalized current amplitudes over time for Sh IR-expressing oocytes before ({blacktriangleup}) (n = 7) and after injection of beta2a-SH3 preincubated with either GST ({diamondsuit}) (n = 11) or GST-Dyn829–842 ({triangledown}) (n = 6). C, time course of current reduction measured as in B from Sh IR-expressing oocytes before ({blacktriangleup})(n = 7) and after injection of CaVbeta2a preincubated with a 6-fold excess (w/w) of either GST ({diamondsuit})(n = 8) or GST-Dyn829–842 ({triangledown})(n = 9). D, anti-dynamin western-blot (as in Fig. 4B). His-CaVbeta2a was coupled to Co2+ beads and incubated with dynamin I cell lysate plus GST (lane 1) or GST-Dyn829–842 peptide (lane 2).

 
Xenopus Oocytes Preparation, Injection, and Electrophysiological Recordings—Xenopus laevis oocytes were prepared, injected, and maintained as in a previous report (10). All capped cRNAs were synthesized using the MESSAGE-machine (Ambion, Austin TX), resuspended in 10 µl of water and stored in 2-µl aliquots at –80 °C until use. The CaV1.2-subunit used in this study bears a deletion of 60 amino acids at the N-terminal end that increase expression (11). Electrophysiological recordings on CaV1.2-expressing oocytes were performed 2–5 h after protein injection (50 nl of the protein stock solution, 1–2 mg/ml/oocyte) and 5–7 days after cRNA injection using the cut-open oocyte technique with a CA-1B amplifier (Dagan Corp., Minneapolis, MN) as described (9). The external solution contained (in mM)10Ba2+, 96 n-methylglucamine, and 10 HEPES pH 7.0, and the internal solution contained (in mM) 120 n-methylglucamine, 10 EGTA, and 10 HEPES, pH 7.0. Data acquisition and analysis were performed using the pCLAMP system and software (Axon Instruments Inc., Foster City, CA). Currents were filtered at 2 kHz and digitized at 10 kHz. Linear components were eliminated by P/-4 prepulse protocol. The normalized charge movement-voltage plot and the average current-voltage plot were obtained as described (12) using a CA-1B amplifier (Dagan). Currents were filtered at 1 kHz and digitized at 20 kHz. Ionic currents mediated by Shaker potassium channel were recorded 1 day after cRNA injection with two-electrode voltage clamp technique using a Dagan TEV 200A or Warner OC725A and filtered at 10 kHz. For the bafilomycin treatment, oocytes were incubated with 500 nM bafilomycin A1 (Sigma) 24 h prior to protein injection (9). The Shaker channel used, Sh IR (inactivation removed), bears an N-terminal deletion that removes fast inactivation (13).

Surface Expression Measurements in Xenopus Oocytes—Surface expression of CaV1.2 channels bearing the HA epitope (CaV1.2-HA) was measured by immunoassay as described (9). Briefly, 5–7 days after CaV1.2 RNA injection, oocytes were separated in two groups: for electrophysiological recordings and for immunoassay. Oocytes were incubated in blocking buffer containing 1% bovine serum albumin followed by incubation with 1 µg/ml rat monoclonal anti-HA antibody (3F10, Roche Applied Science). After washing, oocytes were incubated with horseradish peroxidase-coupled secondary antibody (goat anti-rat FAB fragments, Jackson ImmunoResearch) and extensively washed, and individual oocytes were placed in 50 µl of SuperSignal enzyme-linked immunosorbent assay femto substrate (Pierce) in 96-well microplates (Optiplate, PerkinElmer Life Sciences) and chemiluminescence-quantified 30 s later with a luminometer (Viktor2, PerkinElmer Life Sciences).


Figure 6
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  		FIGURE 6.
Full-length CaVbeta2a internalizes calcium channels devoid of the AID site but not WT channels. A, representative ionic and gating currents traces from oocytes expressing CaV1.2-{Delta}AID channels during 60-ms voltage pulses to –30, 0, and +30 mV from a holding potential of –80 mV. B, voltage and time dependence (inset) of Qon from CaV1.2-(continuous line) and CaV1.2-{Delta}AID-expressing oocytes before ({square}) and after CaVbeta2a injection ({blacksquare}) measured as in Fig. 1, F and G. See details in supplemental Tables S1 and S2. C, average current-voltage plot normalized by Qon from CaV1.2-{Delta}AID-expressing oocytes before ({square}) and after CaVbeta2a injection ({blacksquare}). For comparison, the data from CaV1.2-WT with CaVbeta2a were included (dotted line). D, average Qon from CaV1.2-expressing oocytes before (135.4 ± 14.4 pC, n = 27) and after (137.3 ± 17.4 pC, n = 31) CaVbeta2a injection. E, average Qon from CaV1.2-{Delta}AID-expressing oocytes before (151.2 ± 12.4 pC, n = 32) and after CaVbeta2a injection (37.4 ± 7.8 pC, n = 39). F, chemiluminescence and Qon on oocytes expressing CaV1.2-{Delta}AID-HA alone (2.1 ± 0.4 x 105 cps, n = 23 and 129.5 ± 9.9 pC, n = 14) or after CaVbeta2a injection (0.5 ± 0.23 x 105 cps, n = 23 and 36.4 ± 11.2 pC, n = 12). Non-inj., non-injected oocytes (0.06 ± 0.02 x 105 cps, n = 35).

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
beta2a-SH3 Reduces the Number of Channels Expressed in the Plasma Membrane—The purified SH3 domain of the CaVbeta2a with an expected molecular mass of 16.7 kDa elutes as a mono-disperse peak from a size exclusion chromatography (Fig. 1B). The size exclusion chromatography calibration curve yielded an apparent molecular mass of 20.8 kDa that is compatible with a monomeric conformation of the protein. Mass spectrometry analysis on the purified beta2a-SH3 confirmed its identity (data not shown). beta2a-SH3 was injected in Xenopus oocytes expressing CaV1.2, and gating and ionic currents were measured using the cut-open oocyte voltage clamp technique. Injection of beta2a-SH3 into oocytes causes a dramatic decrease in charge movement (Qon, Fig. 1C) that develops with a time constant of 0.9 h (Fig. 1D). Qon stems from the conformational changes leading to channel opening (14) and, thus, it is proportional to the number of channels. Since decrease in Qon proceeds without changes in the voltage or time dependence (Fig. 1, E–G), it likely reflects a reduction in the number of channels in the cell surface (9). In contrast, injection of full-length CaVbeta, does modify channel gating as expected (15) (Fig. 1E). We corroborated that the drop of Qon upon beta2a-SH3 injection stems from a decrease in the number of channels in the plasma membrane by immunoassay (9). Channel surface expression was measured in oocytes expressing HA-tagged CaV1.2 channels and compared with Qon measurements on the same group of oocytes as shown in Fig. 2A.

Impaired assembly and forward trafficking or enhanced backward trafficking may be responsible for the reduction in the number of channels expressed in the plasma membrane upon beta2a-SH3 injection. To discriminate between these two possibilities, we examined the effect of beta2a-SH3 when incorporation of new proteins into the plasma membrane was inhibited by bafilomycin. We have previously shown that indeed, bafilomycin treatment interrupts incorporation of new CaV1.2 channels in oocytes and causes a net reduction of channel density due to constitutive turnover (9). Injection of beta2a-SH3 in bafilomycin-treated oocytes resulted in 35% reduction in Qon (Fig. 2C) that compares with the 29% observed in control conditions (Fig. 2B). Thus, down-regulation induced by beta2a-SH3 was not prevented by bafilomycin, indicating that this domain interferes with the backward trafficking rather than with the incorporation of newly formed channels.

beta2a-SH3-induced Reduction of Qon Depends on Dynamin—Removal of membrane proteins from the surface implicates endocytosis. Several SH3-containing proteins participate in the regulation of this process by associating with dynamin, a GTPase that excises endocytic vesicles from the plasma membrane (16–19). The proline-rich domain (PRD) of dynamin binds to SH3 domains in the partner protein, and this interaction recruits dynamin to the plasma membrane. Moreover, endocytosis of ion channels and receptors through a dynamin-dependent process has been reported (20–22). Therefore, we investigated the potential role of dynamin in beta2a-SH3 induced channel internalization. We first verified the presence of endogenous dynamin in Xenopus oocytes by Western blot analysis using anti-dynamin antibody and detected a protein of molecular mass similar to a heterologously expressed HA-tagged dynamin I (Fig. 3A). Coexpressing CaV1.2 with dynamin did not have a direct impact on channel expression, and beta2a-SH3 induced reduction of Qon was equivalent (compare Fig. 1C and 3B). We then examined the effect of expressing a dominant-negative mutant of dynamin lacking GTPase activity that inhibits endocytosis (dynamin K44A) (23). Co-expression with dynamin K44A reduced oocyte survival rate and yielded smaller Qon than CaV1.2 alone or with dynamin WT. Although the causes for these changes are unclear, we still observed that beta2a-SH3-induced reduction of Qon was blunted by expression of dynamin K44A (Fig. 3C).


Figure 7
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  		FIGURE 7.
Model for CaVbeta and dynamin interaction. The schematic depicts that association of CaVbeta to CaV1.2 masks the SH3-module. Upon dissociation from CaV{alpha}1, the SH3 domain becomes available to interact with dynamin, leading to down-regulation of calcium currents through endocytosis. How the interaction with CaVbeta-SH3 recruits dynamin to its anchor site remains to be investigated.

 
To further test the role of dynamin, we fused a 14-amino-acid residue peptide spanning the proline-rich region of dynamin I to GST protein to produce GST-Dyn829–842 peptide. This peptide is known to disrupt the interaction between dynamin and SH-3 domains and to inhibit endocytosis in synaptic vesicles (24, 25). Fig. 3D shows that preincubation of beta2a-SH3 with GST-Dyn829–842 peptide, but not with GST alone, inhibits its potency to reduce Qon. Furthermore, beta2a-SH3 binds in vitro to dynamin (Fig. 4A) and, consistently with the electrophysiological data, this binding is partially blocked by GST-Dyn829–842 peptide but not by GST (Fig. 4B). We tested the ability of beta2a-SH3 to bind to the full-length channel. Using a similar pull-down assay, we did not observe binding of beta2a-SH3 to CaV1.2 fused to the yellow fluorescent protein (YFP-CaV1.2; Fig. 4C). In contrast and as expected, CaVbeta and the functional core of CaVbeta (26) encompassing the SH3 and GK domains (Fig. 1A, C1–C2) bound to the channel.

beta2a-SH3 and Full-length CaVbeta2a Down-regulate the Distantly Related Shaker Potassium Channel Expressed in Xenopus Oocytes—Because beta2a-SH3 promotes channel internalization without binding to the channel protein, we examined the effect of beta2a-SH3 onto the distantly related Shaker potassium channel that lacks binding activity to the CaVbeta-subunit (27). Injection of beta2a-SH3 to oocytes expressing the Shaker channel resulted in no changes in channel gating (supplemental Fig. S1), but ionic currents were reduced by ~60% 1 h after protein injection (Fig. 5A). This current reduction was also partially blocked by preincubation of beta2a-SH3 with GST-Dyn829–842 peptide but not with GST (Fig. 5B). Full-length CaVbeta2a preserves the ability of beta2a-SH3 to down-regulate Shaker channels expressed in oocytes. CaVbeta2a reduced ionic currents to a similar degree as beta2a-SH3 (Fig. 5C) without changes in the voltage dependence (supplemental Fig. S2). This current decrease was also antagonized by GST-Dyn829–842 peptide. Moreover, CaVbeta2a bound in vitro to dynamin, and this interaction was inhibited by GST-Dyn829–842 peptide (Fig. 5D). These results indicate that CaVbeta still acts through the dynamin-dependent endocytic pathway.

Full-length CaVbeta2a Reduces the Number of Plasma Membrane CaV1.2 Channels Lacking the AID but Not WT Channels—A corollary from the above results is that free CaVbeta may also be able to reduce surface expression of CaV1.2 channels when the CaV{alpha}1-CaVbeta primary interaction site is disrupted. To test this possibility, we deleted the AID site of CaV1.2 (residues 459–475) to obtain CaV1.2-{Delta}AID channels. This mutated channel yields gating currents that, with respect to their voltage and time dependence, are indistinguishable from wild type CaV1.2 (Fig. 6, A and B), but as expected, CaVbeta2a loses its ability to potentiate ionic currents (Fig. 6C). As recently corroborated by chemiluminescent enzyme immunoassay (9), surface expression of CaV1.2 channels in oocytes is not altered by injection of CaVbeta2a protein (Fig. 6D). In contrast, in oocytes expressing CaV1.2-{Delta}AID, injection of CaVbeta2a reduces Qon to the same extent as did beta2a-SH3 in oocytes expressing wild type CaV1.2 (Fig. 6E). To further prove that channels lacking the AID are indeed expressed in the plasma membrane and that CaVbeta decreases the number of channels at the cell surface, we performed the surface expression assay with CaV1.2-{Delta}AID HA-tagged channels. CaVbeta2a injection decreased Qon and chemiluminescence signal in CaV1.2-{Delta}AID-expressing oocytes (Fig. 6F).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We here show that the SH3 domain of the beta-subunit of the voltage-gated calcium channels promotes internalization of membrane proteins in a dynamin-dependent manner. In addition, we found that CaVbeta-SH3 binds in vitro to dynamin and, since this association is inhibited by GST-Dyn829–842 peptide, we propose that this interaction is mediated by the PRD of dynamin. Nevertheless, simulated docking predictions indicate that CaVbeta-SH3 is unlikely to interact with PXXP motifs unless a considerable structural rearrangement occurs (4). The dynamin PRD-CaVbeta-SH3 interaction may be mediated by non-canonical PXXP binding residues in CaVbeta-SH3 or, alternatively, exposition of canonical residues may be tunable by a yet unknown regulatory protein or event. The interaction between recombinant beta2a-SH3 and dynamin may reflect an in vivo phenomenon given that a SH3-only form of the CaVbeta protein is expressed in cardiac cells (8).

Binding of CaVbeta-SH3 to the protein being sequestered is not required since no interaction between the beta2a-SH3 and the whole CaV1.2 channel was observed, and certainly, no association occurs with the Shaker channel. CaVbeta-SH3 has been reported to associate only with isolated regions or truncated CaV{alpha}1 channels (28, 29). Thus, it is conceivable that other cytoplasmic regions within the whole channel hinder this association.

In the presence of the full-length CaVbeta-subunit, calcium channels lacking the AID site, but not WT channels, are down-regulated, as though binding to CaVbeta prevents the channel complex to be internalized. Since association of the CaVbeta to VGCCs ensures normal channel activity, this would constitute an efficient quality control mechanism in which the same protein ensures functional fitness and survival of the channel in the plasma membrane (Fig. 7). Our recent finding that the CaV{alpha}1-CaVbeta interaction is reversible at the level of the plasma membrane (9) supports this mechanism. The ability of this auxiliary subunit to influence internalization of other membrane proteins anticipates that replacement of complete signal transduction assemblies may be triggered by the presence of free CaVbeta. Although the whole picture is certainly still incomplete, our findings outline a novel signaling pathway for the regulation of intracellular calcium concentration.


   FOOTNOTES
 
* This work was supported by grants from the Deutsche Forschung Gemeinschaft (Grant FOR 450, TP1) (to P. H.) and the Fondo para el Desarrollo de Ciencia y Tecnologia (Grant FONDECYT-1020899) and Anillo de Ciencia y Tecnologia (Grant ACT-46) (to A. N.). The authors declare no competing financial interests. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental tables and figures and equations. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Medizinische Hochschule Hannover, Abteilung Neurophysiologie, Carl-Neuberg-Str. 1, 30625 Hannover. Tel.: 49-511-532-2883; Fax: 49-511-532-2776; E-mail: hidalgo.patricia{at}mh-hannover.de.

3 The abbreviations used are: VGCC, voltage-gated calcium channel; AID, {alpha}-interaction domain; CaVbeta, beta-subunit of VGCCs; CaV{alpha}1, pore-forming {alpha}-subunit of VGCCs; CaV1.2, cardiac isoform of the {alpha}-subunit of VGCCs; SH3, Src-homology 3 domain; GK, guanylate kinase domain; beta2a-SH3, Srchomology 3 domain from beta2a isoform; GST, glutathione S-transferase; GST-Dyn829–842, dynamin peptide encompassing residues 829–842; Qon, charge movement; PRD, proline-rich domain; WT, wild type; HA, hemagglutinin; pC, picocoulomb; cps, counts/second. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Christoph Fahlke and Dr. David Naranjo for insightful discussion, Ute Scholl for kindly providing us with the dynamin constructs, Dr. Matthias Gaestel for kindly sharing the luminometer Victor2, and Dr. Andreas Pich for the mass spectrometry.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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