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NEUROSCIENCE |
1 Laboratory of Cellular and Molecular Neuroscience, Department of Pharmacology, University College London, London, UK
2 School of Crystallography, Birkbeck College, London, UK
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Abstract |
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2
-2/ß1b channels expressed in human embryonic kidney tsA-201 cells, when equivalent proportions of cDNA were used. Furthermore, despite the 24-fold reduced affinity of CaVß1b for the Y388S I–II linker of CaV2.2, all the key features of modulation as well as trafficking by CaVß subunits remained intact. This is in contrast to the much more marked effect of the W391A mutation, which abolished interaction with the CaV2.2 I–II linker, and very markedly affected the trafficking of the channels. However, using the Xenopus oocyte expression system, where expression levels can be accurately titrated, when CaVß1b cDNA was diluted 50-fold, all evidence of interaction with CaV2.2 Y388S was lost, although wild-type CaV2.2 was still normally modulated by the reduced concentration of ß1b. These results indicate that high affinity interaction with the 1 subunit is not necessary for any of the modulatory effects of CaVß subunits, but occupancy of the interaction site is important, and this will occur, despite the reduced affinity, if the CaVß subunit is present in sufficient excess.
(Received 15 March 2006;
accepted after revision 13 April 2006;
first published online 20 April 2006)
Corresponding author A. C. Dolphin: Laboratory of Cellular and Molecular Neuroscience, Department of Pharmacology, Andrew Huxley Building, University College London, Gower Street, London, WC1E 6BT, UK. Email: a.dolphin{at}ucl.ac.uk
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Introduction |
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1 subunit, associated with auxiliary CaVß and 2 subunits (Catterall, 2000). CaVß subunits are crucial for normal HVA channel function (for review see Dolphin, 2003), since they are required for the expression of functional channels at the plasma membrane (Bichet et al. 2000), and modulate their biophysical properties (for review see Dolphin, 2003). The CaV2 family calcium channels are inhibited by Gß
dimers (Herlitze et al. 1996; Ikeda, 1996) which is the main mechanism of presynaptic inhibition by G-protein-coupled receptors (GPCRs). CaVß subunits promote the voltage dependence of modulation of CaV2.2 calcium channels by Gß dimers, although the mechanism involved remains unclear (Meir et al. 2000; Canti et al. 2001; Leroy et al. 2005). We have previously investigated the role of CaVß subunits in the plasma membrane expression and modulation of CaV2.2 calcium channels, by mutating the tryptophan (W391 in CaV2.2) that is conserved in the AID sequence of all CaV1 and 2 channels. This sequence is QQIERELNGYLEWIFKAE in CaV2.2. This tryptophan has been shown both by the original study that identified the AID motif and by the recent structural studies to be key to the interaction between CaVß subunits and the AID (Pragnell et al. 1994; Chen et al. 2004; Opatowsky et al. 2004; Van Petegem et al. 2004). Our results showed that the W391A mutation reduced the binding of ß1b to the I–II linker by at least 1000-fold, prevented the enhancement of functional expression of CaV2.2 by CaVß1b, and also prevented modulation of the biophysical properties of CaV2.2 by this subunit. In addition, although the G-protein modulation of CaV2.2 W391A was present, it was not voltage dependent (Leroy et al. 2005).
We have now investigated the role of the AID tyrosine Y388 in the role of CaVß subunits and in G protein modulation, since the crystal structure showed the W and Y to form a hairpin arrangement with their aromatic rings stacked together (Chen et al. 2004; Opatowsky et al. 2004; Van Petegem et al. 2004). The Y residue has previously been described as essential for CaVß binding to the AID and for functional expression (Pragnell et al. 1994; Witcher et al. 1995). However, a subsequent study disputed the importance of this residue in ß subunit-induced modulation of CaV1.2 currents (Gerster et al. 1999), and its role remains open to question. Since in our previous study, measurement of CaVß binding to the I–II linker by surface plasmon resonance correlated extremely well with the maximum conductance (Gmax) values for CaV2.2 currents and with cell surface biotinylation for the W391A mutation (Leroy et al. 2005), we performed similar studies following mutation of Y388. Our results allow us to conclude that there is no requirement for high affinity binding of CaVß to the AID, since this is reduced 24-fold by the mutation Y388S. However, occupancy of the site is a key factor, since reducing the concentration of ß1b by 50-fold relative to CaV2.2 Y388S removed all influence of ß1b on this channel, whereas the wild-type CaV2.2 was still modulated at this concentration of ß1b.
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Methods |
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The cDNAs used in this study were CaV2.2 (D14157), CaVß1b (X61394), 2-2 (Barclay et al. 2001) and D2 dopamine receptor (X17458). The green fluorescent protein (GFP-mut3b, U73901) was used to identify transfected tsA-201 cells. All cDNAs were subcloned in pMT2.
Construction, expression and purification of proteins
CaV2.2 Y388S was generated using standard molecular biological methods. The Y388S, Y388F and W391A mutations were introduced into CaV2.2 I–II loop in pGEX2T (Bell et al. 2001) (GST CaV2.2 I–II loop) by site-directed mutagenesis using standard molecular biological methods. The resulting mutated I–II linkers and the wild-type linker were subcloned into pETM6T1 (gift from Dr A. Okorokov, UCL), which encodes an N-terminal NusA-tag and a His-tag, using BamH I and EcoR I, generating NusA Cav2.2 I–II loop fusion proteins.
NusA Cav2.2 I–II loop fusion proteins were expressed in BL21(DE3) codon plus (RIL) E. coli in 1 litre cultures of LB medium containing 30 µg ml–1 kanamycin, 34 µg ml–1 chloramphenicol and 1% (w/v) glucose. Cultures were grown to optical density 0.6 at 560 nm and induced at 20°C for 4 h using 0.1 mM isopropyl-ß-D-thiogalactopyranoside. For protein purification, cells were lysed by sonication in buffer A (20 mM Hepes pH 8.0, 150 mM NaCl, 1 mM DTT, 20 mM imidazole) containing protease inhibitors (Complete EDTA-free, Roche Diagnostics, Lewes, UK). The lysate was cleared by centrifugation at 20 000 g for 20 min and the resulting pellet was washed once with buffer A. The pellet was resuspended in buffer A containing 1.5% CHAPS and incubated for 1 h at 4°C. After centrifugation at 20 000 g for 20 min the supernatant was diluted 1 : 1 with buffer A and applied to 1 ml Ni–NTA resin (Qiagen, Crawley, West Sussex, UK) equilibrated with buffer B (buffer A containing 0.5% CHAPS). The column was washed with 25 volumes buffer B before proteins were eluted with 4 volumes buffer B containing 350 mM imidazole. Eluted proteins were analysed by SDS-PAGE followed by Coomassie blue staining. C-terminally His-tagged Cavß1b (Cavß1b-H6C) was expressed and purified as described by Bell et al. (2001).
Surface plasmon resonance
Assays were performed using a BIAcore 2000 (Biacore AB, Uppsala, Sweden) at 25°C using running buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 0.1% CHAPS). NusA fusion proteins and NusA only were immobilized directly onto the surface of a CM5 sensor chip. Using a 1 : 1 mixture of 400 mM 1-ethyl-3-[3-dimethyl-aminopropyl]carbodiimide hydrochloride and 100 mM N-hydroxysuccinimide to activate the chip surface, 2000 reference units (RU) of NusA I–II loop fusions and the molar equivalent of NusA were immobilized. Cavß1b-H6C was dialysed against running buffer and diluted to the required concentrations in running buffer. These samples were applied for 5 min at a flow rate of 50 µl min–1 over all flow cells and each injection was followed by a 5 min dissociation phase. The sensor chip surface was regenerated between injections by the application of 30 µl of 20 mM glycine pH 2.2 at 10 µl min–1. Sensorgrams were processed using the BIAevaluation 3.0 software (Biacore AB). Sensorgrams recorded from the flow cells containing NusA Cav2.2 I–II loop, either wild-type, Y388S, or Y388F were corrected for passive refractive index changes and for non-specific interactions by subtraction of the corresponding sensorgram recorded from the flow cell containing NusA only. Sensorgrams were analysed using Biacore kinetic analysis software using a model of 1 : 1 interaction. In addition, the maximum responses for the Cav2.2 I–II linker and both mutants after 250 s of sample injection were plotted against Cavß concentration. The resulting curves were analysed by fitting a rectangular hyperbola, using Origin 7 (Microcal Software, Northhampton, MA, USA), and the affinity constant KD was estimated. The dissociation phase of the sensorgrams was also fitted to a single exponential, to determine the dissociation rate, koff.
Cell culture and heterologous expression
The tsA-201 cells were cultured in a medium consisting of Dulbecco's modified Eagle's medium, 10% fetal bovine serum, and 1% non-essential amino acids. The cDNAs (all at 1 µg µl–1) for CaV1 subunits, CaVß, 2-2, D2 dopamine receptor and GFP (when used as a reporter of transfected cells) were mixed in a ratio of 3 : 2 : 2 : 2 : 0.4. The cells were transfected using Fugene6 (Roche Diagnostics, Lewes, UK; DNA/Fugene6 ratio of 2 µg in 3 µl).
Cell surface biotinylation and Western blotting
Cell surface biotinylation experiments were carried out as described in Leroy et al. (2005). For Western blotting, samples (10 µg) from tsA-201 whole-cell lysates from biotinylation experiments (see above) were separated by SDS-PAGE on 4–12% Tris-glycine gels and then transferred to polyvinylidene fluoride membranes. Immunodetection was performed with antibodies to the Cav2.2 II–III linker as previously described (Raghib et al. 2001).
Whole-cell patch clamp in tsA-201 cells
The tsA-201 cells were replated at low density on 35 mm tissue culture dishes on the day of recording. Whole-cell patch-clamp recordings were performed at room temperature (22–24°C). Only fluorescent cells expressing GFP were used for recording. The single cells were voltage clamped using an Axopatch 200B patch-clamp amplifier (Axon Instruments, Inc., Union City, CA, USA). The electrode potential was adjusted to give zero current between pipette and external solution before the cells were attached. The cell capacitance varied from 10 to 40 pF. Patch pipettes were filled with a solution containing (mM): 140 caesium aspartate, 5 EGTA, 2 MgCl2, 0.1 CaCl2, 2 K2ATP, 10 Hepes, titrated to pH 7.2 with CsOH (310 mosmol l–1), with a resistance of 2–3 M
. The external solution contained (mM) 150 tetraethylammonium (TEA) bromide, 3 KCl, 1.0 NaHCO3, 1.0 MgCl2, 10 Hepes, 4 glucose, 10 BaCl2, pH adjusted to 7.4 with Tris-base (320 mosmol l–1). The pipette and cell capacitance, as well as the series resistance, were compensated by 80%. Leak and residual capacitance current were subtracted using a P/4 protocol. Data were filtered at 2 kHz and digitized at 5–10 kHz. The holding potential was –100 mV, and pulses were delivered every 10 s.
Two-electrode voltage clamp in Xenopus oocytes
Xenopus laevis females were killed by non-recovery anaesthesia in accordance with Schedule 1 to the Animals (Scientific Procedures) Act, 1986. Oocytes were then surgically removed and defolliculated by treatment with 1.5 mg ml–1 collagenase type Ia in a Ca2+-free ND96 saline containing (mM): 96 NaCl, 2 KCl, 1 MgCl2, 5 Hepes, pH adjusted to 7.4 with NaOH, for 2 h at 18°C. Ten nanolitres of cDNAs (1 ng nl–1) were injected into the nuclei of stage V and VI oocytes. Injected oocytes were incubated at 18°C for 3–7 days in ND96 saline (as above plus 1 mM CaCl2) supplemented with 100 µg ml–1 penicillin and 100 i.u. ml–1 streptomycin (Life Technologies, Gaithersburg, MD, USA). Further details have been previously described (Canti et al. 2001).
Whole-cell recordings from oocytes were performed in the two-electrode voltage-clamp configuration under continuous superfusion of a chloride-free solution containing (mM): 10 Ba(OH)2, 50 NaOH, 10 TEA-OH, 2 CsOH, 5 Hepes (pH 7.4 with methanesulphonic acid). The oocytes were injected with 30–40 nl of a 100 mM solution of K3-1,2-bis(aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA) to suppress endogenous Ca2+-activated Cl– currents. Electrodes contained 3 M KCl and had resistances of 0.3–1.5 M. The holding potential was –100 mV in all cases. The currents were amplified and low-pass filtered at 1 kHz by means of a Geneclamp 500 amplifier (Axon Instruments, Burlingame, CA, USA), digitized through a Digidata 1200 interface (Axon Instruments). All experiments were performed at 18–20°C.
Electrophysiological data analysis
Current amplitude was measured 10 ms after the onset of the test pulse, and the average over a 2 ms period was calculated and used for subsequent analysis. The current density–voltage (I–V) relationships were fitted with a modified Boltzmann equation as follows: I
=
Gmax(V
–Vrev)/(1 + exp(–(V
–V50,act)/k)) where I is the current density (in pA pF–1), Gmax is the maximum conductance (in nS pF–1), Vrev is the reversal potential, V50,act is the midpoint voltage for current activation, and k is the slope factor. Steady-state inactivation properties were measured by applying 5 s pulses from –120 to +20 mV in 10 mV increments, followed by a 11 ms repolarization to –100 mV before the 100 ms test pulse to +20 mV. Steady-state inactivation and activation data were fitted with a single Boltzmann equation of the form: I/Imax
= (A1
–
A2)/[1 + exp((V
–
V50,inact)/k)]
+
A2, where Imax is the maximal current, V50,inact is the half-maximal voltage for current inactivation. For the steady-state inactivation, A1 and A2 represent the total and non-inactivating current, respectively. Inactivation kinetics of the currents were estimated by fitting the decaying part of the current traces with the equation: I(t) =
C
+
Aexp(–(t
–
t0)/
inact) where t0 is zero time, C the fraction of non-inactivating current, A the relative amplitude of the exponential, and inact, its time constant. Activation kinetics were estimated by fitting the activation phase of the current with either a single or a double exponential. Analysis was performed using pCLAMP6 and Origin 7. Data are expressed as mean ±
S.E.M. of the number of replicates n. Error bars indicate standard errors of multiple determinations. Statistical significance was analysed using Student's paired or unpaired t test (as appropriate).
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Results |
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The amino acid Y388 in CaV2.2 is conserved in the AID sequence of all HVA calcium channels (Dolphin, 2003; Richards et al. 2004) and has been previously described to be crucial for the binding of the CaVß ancillary subunits to HVA calcium channels (Pragnell et al. 1994; Witcher et al. 1995; De Waard et al. 1996). The recent structural analysis of the interaction of CaVß subunits with the CaV1.2 I–II linker showed that the aromatic ring of the Y side chain is stacked with the side chain of the W residue and deeply embedded in the AID binding groove in CaVß (Chen et al. 2004; Opatowsky et al. 2004; Van Petegem et al. 2004).
We first examined by surface plasmon resonance analysis whether mutation of Y388 to either F or S in the AID of CaV2.2 affected the binding of CaVß1b to the I–II linker of CaV2.2. In these experiments, NusA fusion proteins corresponding to the entire I–II linker, including the AID of CaV2.2, CaV2.2 Y388S, CaV2.2 Y388F or NusA alone as control (Fig. 1A), were immobilized chemically onto individual flow cells of a CM5 dextran sensor chip. CaVß subunit solutions (20–1000 nM) were perfused over all flow cells. No concentration-dependent binding of the CaVß subunits to the control NusA fusion protein was detected (data not shown). CaVß1b exhibited specific binding to the full-length I–II linker of CaV2.2 (Fig. 1B). Significant binding of CaVß1b was also observed to both the Y388F and Y388S mutant I–II linkers (Fig. 1C and D). The dissociation constant (KD) for CaVß1b binding to the I–II linker of CaV2.2 was calculated to be 13.7 nM for ß1b binding to the wild-type I–II linker, and 78 and 329 nM for the Y388F and Y388S mutant I–II linkers, respectively, representing a 5.7-fold and a 24-fold reduction compared to the wild-type I–II linker (Fig. 1E). In contrast, negligible binding of the CaVß1b subunit to the CaV2.2 W391A I–II linker was detected, and thus the KD values could not be determined (Fig. 1F), as we have previously shown for a GST fusion protein with a I–II linker construct truncated immediately after the AID sequence (Leroy et al. 2005). These results refine, rather than contradict, the findings of previous studies which indicated that mutation of Y to S in the AID sequence of other CaV channels abrogated CaVß subunit binding (Pragnell et al. 1994; Witcher et al. 1995; De Waard et al. 1996; Berrou et al. 2002), since all previous studies have used non-quantitative overlay or pull-down assays, where low affinity interactions may easily be missed.
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The effect of Y388S mutation on the functional expression of CaV2.2 calcium channels at the plasma membrane
Since the mutation of Y388S reduced the affinity for CaVß1b to a greater extent than the Y388F mutation, we used the Y388S mutation in full length CaV2.2 for further functional studies. We coexpressed full length CaV2.2 Y388S or wild-type CaV2.2, together with accessory CaVß1b and 2-2 subunits and compared the biophysical properties of the wild-type and mutated channels. Surprisingly, and unlike the W391 mutant of CaV2.2 that we studied recently (Leroy et al. 2005), there was no significant difference in Gmax determined from the current–voltage (I–V) relationships between wild-type CaV2.2 and CaV2.2 Y388S (Fig. 2A). The Gmax of CaV2.2 Y388S was 97.5 ± 16% (n
= 17) of that found for the CaV2.2/ß1b/2-2 combination when coexpressed with ß1b. Thus, the auxiliary ß1b subunit was still able to significantly increase the Gmax of CaV2.2 Y388S when compared with either the wild-type CaV2.2 expressed alone (Fig. 2A) or with the CaV2.2 Y388S/2-2 (n
= 4, data not shown). In the transfection system used, the over-expression of CaVß may mean that the AID site is constantly occupied despite the 24-fold lower affinity of the Y388S mutant AID. Thus the high affinity interaction of CaVß, previously suggested to be essential for the trafficking to the plasma membrane may not be necessary, but occupancy of the site may be the most important factor.
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Biophysical properties of CaV2.2 Y388S expressed with CaVß1b are similar to those of CaV2.2/ß1b
As well as having effects on trafficking, CaVß1b is known to promote the activation of HVA calcium channels at more negative potentials (for review see Dolphin, 2003). In a previous study, we have estimated that the effects of CaVß subunits on the voltage-dependent properties of the channels occurred with an approximately 7-fold lower affinity than the effects on trafficking (Canti et al. 2001). It is therefore possible that CaV2.2 Y388S would be trafficked to the plasma membrane by CaVß1b, despite its reduced affinity, but would lose any interaction following trafficking. However, from the I–V relationships, the CaV2.2 Y388S had a similar V50 for activation (+5.5 ± 1.5 mV; n = 17) to wild-type CaV2.2 in the presence of ß1b (+6.0 ± 0.6 mV; n = 29), which were both significantly less positive compared with V50 for activation for the wild-type CaV2.2 expressed alone (+14.4 ± 0.4 mV; n = 21, P < 0.05) (Fig. 2A). This indicates that the reduction in affinity conferred by the Y388S mutation was insufficient to alter the effect of CaVß1b on the voltage dependence of activation of the channel, and this mutant channel behaved like the wild-type CaV2.2, expressed with a CaVß1b subunit.
Another important feature of CaVß1, ß3 and ß4 subunits is that they hyperpolarize the steady-state inactivation curves of CaV2.2, as well as other HVA calcium channels (Bogdanov et al. 2000). The potential for half-inactivation (V50,inact) was –45.4 ± 0.4 mV (n = 25) for CaV2.2 expressed with CaVß1b (Fig. 3), representing a –15 mV shift compared to CaV2.2 expressed in the absence of a ß subunit. Again, we found no significant effect on the steady-state inactivation for CaV2.2 Y388S/ß1b, as this was superimposed on that of CaV2.2 expressed with CaVß1b (Fig. 3A and B).
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inact) of the CaV2.2 Y388S/ß1b current recorded during 800 ms pulses at +20 mV (550 ± 161 ms; n
= 14) was the same as that of the wild-type CaV2.2/ß1b combination (557 ± 88 ms; n
= 14) (Fig. 3C). Altogether these results indicate that CaV2.2 Y388S is normally regulated by CaVß1b in the plasma membrane. CaV2.2 Y388S channels show normal voltage-dependent modulation by G-protein activation
To investigate the importance of the Y388 residue for the modulation by G-proteins of CaV2.2 calcium channels, the wild-type or mutant CaV2.2, CaVß1b, 2-2 combination was coexpressed with a D2 dopamine receptor, and the receptor was activated with 100 nM quinpirole, which is a maximal concentration of this agonist. Figure 4A shows representative currents obtained before and during application of quinpirole, both before (P1) and immediately after (P2) a 100 ms prepulse to +100 mV. As previously observed (Leroy et al. 2005), the currents measured at +10 mV were inhibited by quinpirole by 64.2 ± 4.0% for the wild-type channel (n
= 29; Fig. 4B). The P2/P1 ratio obtained from traces such as those represented in Fig. 4A reflects the voltage-dependent removal of G-protein inhibition. A value of 2.2 ± 0.1 (n
= 29) was obtained for P2/P1 at +10 mV for the wild-type channel expressed with CaVß1b (Fig. 4C).
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We have shown previously that lowering the concentration of expressed CaVß subunits leads to a slower rate of facilitation of the G-protein-modulated current, with two components of facilitation being present at intermediate CaVß concentrations (Canti et al. 2001). Thus a reduction in affinity of CaVß for the CaV2.2 Y388S channel might be manifested by a reduction in facilitation rate. We therefore determined the time constants of facilitation (facil) by varying the prepulse duration during quinpirole application, and found that the facil was very similar for the wild-type CaV2.2 (10.2 ± 0.7 ms; n
= 11) and CaV2.2 Y388S (11.2 ± 0.8 ms; n
= 12) (Fig. 5).
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From the foregoing, it is clear that a 24-fold reduction in affinity of CaVß1b for the CaV2.2 AID containing the Y388S mutation is insufficient to have any effect on the ability of ß1b to modulate the channel, by all the parameters we have studied, although we know from the W391A mutation that binding to the AID region is essential for these effects of ß1b to occur (Leroy et al. 2005). We also know from our previous study in Xenopus oocytes that the amount of ß1b expressed when CaV2.2 and ß1b cDNAs are injected in an equivalent ratio is at least 30-fold in excess of that required to hyperpolarize the voltage dependence of steady-state inactivation of the entire channel population (Canti et al. 2001). We therefore examined the properties of wild-type CaV2.2 and CaV2.2 Y388S coexpressed with ß1b either at a normal cDNA ratio or using 50-fold diluted ß1b cDNA in Xenopus oocytes. For wild-type CaV2.2, the effects of both concentrations of ß1b were identical, both in terms of peak current amplitude at +10 mV (Fig. 6A), and in terms of hyperpolarization of the steady-state inactivation (Fig. 6B). For the steady-state inactivation, the V50,inact was –51.0 ± 1.1 mV for CaV2.2/ß1b injected in a standard ratio (n
= 10) and –46.8 ± 1.3 mV for CaV2.2/ß1b using 50-fold-diluted ß1b cDNA (n
= 7). This result is in agreement with our previous findings (Canti et al. 2001). It can be attributed to the fact that CaVß subunits, being low molecular weight cytoplasmic proteins, are transcribed and translated more rapidly and are therefore likely to be present in the cytoplasm at much higher concentrations than the concentration of functional transmembrane CaV2.2 channels at the plasma membrane. However, for CaV2.2 Y388S there was a clear difference between the effects of the two concentrations of CaVß1b, in that the currents in the presence of the 50-fold diluted ß1b were significantly reduced by 74% (P < 0.01) compared to those in the presence of the standard concentration of ß1b (Fig. 6A), and the steady-state inactivation became more positive, to the same extent as in the absence of any ß subunit (Fig. 6B). The V50,inact was –44.9 ± 2.0 mV for CaV2.2 Y388S/ß1b injected in the standard ratio (n
= 7) and –16.5 ± 0.45 mV for CaV2.2 Y388S/ß1b injected in the diluted ratio (n
= 10, P < 0.001 compared to the standard ratio). This is very similar to the V50,inact obtained for steady-state inactivation curves of CaV2.2/2-2 currents, which was –18.0 ± 1.5 mV (n
= 10; Fig. 6B). Thus a reduction in the concentration of expressed CaVß subunits reveals the functional effect of the lower affinity of the CaV2.2 Y388S I–II linker for CaVß subunits.
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Discussion |
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1 subunits (Fig. 1A) and a Src homology 3 domain (SH3) (Hanlon et al. 1999). The 18 amino acid AID motif contains a conserved W that is crucial for binding CaVß (Pragnell et al. 1994; Berrou et al. 2002; Leroy et al. 2005), and also a conserved Y three residues proximal to the W. Recent structural data from three groups has provided detailed information about the interaction between the AID–CaVß complex and confirmed that both W and Y are deeply embedded in the binding groove within the GK of CaVß (Van Petegem et al. 2004; Opatowsky et al. 2004; Chen et al. 2004). The importance of the Y in CaVß binding and functional effects is controversial. It was first found that mutation of this Y to S in the AID of CaV2.1 completely abolished binding to ß3, and almost completely abolished binding to ß2a, whereas the mutation of Y to F appeared to be slightly less effective (Witcher et al. 1995). This Y residue was also originally described as being essential for functional expression (Pragnell et al. 1994; Witcher et al. 1995). However, Berrou et al. (2002) found that although no direct binding of the 35S-labelled Cavß3 subunit to a GST Cav2.3 AID containing the Y to S mutation could be detected, Cav2.3 Y383S was still in part modulated by the Cavß3 subunit when coexpressed in Xenopus oocytes. Berrou et al. (2005) subsequently found that both conserved and non-conserved mutations in Y383 of CaV2.3 had little effect on CaVß modulation of whole-cell currents in Xenopus oocytes but the same mutations in an AID peptide almost abolished 35S-labelled Cavß3 binding, using a non-quantitative assay. In addition, Neuhuber et al. (1998) found that whilst Y366S mutation in CaV1.1 appeared to prevent co-localization of CaV1.1 with CaVß1a in transfected tsA-201 cells, expression of CaV1.1 currents was not affected. Similar results were found by the same group for the same Y to S mutation in CaV1.2, which prevented co-localization of CaV1.2 with ß subunits at the plasma membrane, as determined by immunocytochemistry, but did not affect calcium current expression (Gerster et al. 1999). This group concluded that increased incorporation of channels into the plasma membrane is not required for the CaVß-mediated enhancement of functional calcium currents. Our evidence that ß subunits increase the number of CaV2.2 channels incorporated into the plasma membrane, as determined by cell surface biotinylation (Leroy et al. 2005), and that this is reduced by the W391A mutation (Leroy et al. 2005) but not by the Y388S mutation (as shown here using the same transfection conditions), are in agreement with the effect of these two mutations on CaV2.2 current density, although the effect on cell surface incorporation was always less than the overall influence on current density. Our results strongly support the view that both the CaVß-mediated increase in channel number in the plasma membrane, as well as the undisputed effect of CaVß subunits on channel properties (Neely et al. 1993; Gerster et al. 1999; Meir et al. 2000), both normally contribute to the increase in whole-cell current that is observed. It is likely that previous immunocytochemical results, using intracellular epitopes that require cell permeabilization (Neuhuber et al. 1998; Gerster et al. 1999), do not allow the distinction between sub- plasma membrane channels, and those that are actually in the membrane, whereas cell surface biotinylation is a more accurate reflection of proteins that are incorporated into the membrane.
Low affinity interactions of different CaVß subunits with the N- and C-termini of various calcium channels have also been reported (for reviews see Walker & De Waard, 1998; Dolphin, 2003), although in a yeast two-hybrid screen we did not observe any interaction of CaVß1b with the N- or C-terminus of CaV2.2, under conditions where the interaction of CaVß1b with the I–II linker was robust (Raghib et al. 2001). Furthermore, it is unlikely that such interactions could be responsible for the effects of CaVß subunits in the absence of an anchor to the AID region of the I–II linker, since all the effects of ß1b were abrogated by the W391A mutation (Leroy et al. 2005). However, palmitoylated ß2a was still able to modulate the biophysical properties of CaV2.2 W391A, indicating that the plasma membrane anchoring afforded by its palmitoylation can substitute for high affinity interaction with the I–II linker (Leroy et al. 2005). Thus it seems likely that several other regions of the calcium channel 1 subunits are involved in mediating the effects of CaVß subunits.
Lack of evidence for the binding of CaVß subunits to additional regions on the I–II linker, apart from the AID
In this study we obtained a similar affinity of CaVß1b for the full-length CaV2.2 I–II linker to that which we found previously for a I–II linker construct truncated immediately after the AID (13.8 nM; Leroy et al. 2005). If there were an additional binding site, for example for the ß subunit SH3 domain, to a site on the I–II linker distal to the AID, as suggested previously (Maltez et al. 2005), the combination of two binding sites would lead to the measurement of a higher overall affinity of CaVß for the full-length I–II linker. Our results, combined with the complete lack of binding of ß1b to the full-length CaV2.2 W391A I–II linker, do not provide evidence that there is an additional binding site for other domains of ß1b on the distal I–II linker of CaV2.2, in contrast to the previous conclusion (Maltez et al. 2005).
The Y388S mutation in the AID of CaV2.2 appears not to influence the functional effects of ß1b, despite producing a 24-fold reduction in affinity for ß1b binding to the AID
One of the main effects of CaVß subunits on HVA calcium channels is to increase current density. Our studies have shown that there are fewer channels present at the cell surface when no CaVß subunits were coexpressed or when mutated CaV2.2 W391A channels were cotransfected with a CaVß (Leroy et al. 2005). It has been suggested that a CaVß bound to the I–II linker may mask an endoplasmic reticulum retention signal present in the I–II linker of HVA calcium channels and favour the trafficking of the channel to the cell surface (Bichet et al. 2000). Our previous data suggested that the endogenous CaVß3 that we have identified in tsA-201 cells was responsible for trafficking some wild-type CaV2.2 to the plasma membrane in the absence of a coexpressed ß subunit, and that the markedly reduced affinity of the W391A mutated channel for CaVß subunits abolished interaction with the endogenous CaVß3 subunits, and thus prevented any trafficking to the plasma membrane (Leroy et al. 2005). Our results therefore provided very strong evidence that the binding of a CaVß subunit to the channel is an essential requirement for the functional expression of CaV2.2 at the plasma membrane.
In contrast, the markedly reduced affinity of the Y388S AID (from 13.7 to 329 nM) for ß1b does not translate into a reduced expression of the channels at the plasma membrane, or any effect on the voltage dependence of activation or inactivation or voltage dependence of G-protein modulation. We have determined previously, from experiments in which varying concentrations of ß subunits were expressed together with a constant amount of CaV2.2 in Xenopus oocytes, that there appeared to be two different affinities of ß subunits for trafficking the channels (approximately 17 nM for ß3) and for hyperpolarizing the steady-state inactivation (about 7-fold lower affinity, approximately 120 nM). However, in Xenopus oocytes the concentration of CaVß subunits obtained following the standard conditions of heterologous expression used in this study was estimated to be far in excess of this, at 2–3 µM (Canti et al. 2001). If similar amounts are expressed in the mammalian expression system then it is not surprising that little effect was observed of a 24-fold reduction in the affinity of ß1b for the AID. Occupancy would remain very high because of the excess of free CaVß subunits.
Support is given to this conclusion by our experiments in Xenopus oocytes in which dilution of ß1b by 50-fold abolished the influence of this CaVß subunit on the steady-state inactivation of CaV2.2 Y388S but had no effect on that of wild-type CaV2.2.
These experiments demonstrate the limitation of coexpression studies in that the concentration of the expressed proteins may be very different, particularly when coexpressing membrane proteins with cytoplasmic proteins, despite the use of similar cDNA concentrations, and in this way they may not mimic the ratios of subunits present in vivo. However, the stoichiometry and occupancy of the AID site on endogenous calcium channels by endogenous CaVß subunits remains an open question to be addressed in the future.
The Y388S mutation in the AID of CaV2.2 has no effect on G-protein modulation of CaV2.2 channels
It has been proposed that G-protein modulation of CaV2 channels involves competition between Gß and CaVß subunits, with displacement of ß subunits being a key step (Bourinet et al. 1996; Sandoz et al. 2004). Our results do not support this view as, despite the 24-fold reduction in affinity of CaV2.2 Y388S for ß1b, there was no enhancement of G-protein modulation. If there were simple competition between Gß and CaVß, then a 24-fold reduction in affinity of the I–II linker for CaVß1b should result in an increased occupancy by Gß at the peak of the response to the agonist quinpirole. The present result concurs with our previous results that did not provide evidence for simple competition between CaVß and Gß (Meir et al. 2000).
All parameters of G-protein modulation were identical, including the rate of facilitation, which has been interpreted as resulting from the dissociation of Gß (Ikeda, 1996; Zamponi & Snutch, 1998). In our previous study we found that the facilitation rate during a +100 mV prepulse was a sensitive measure of changes in CaVß subunit concentration (Canti et al. 2001). It was 20-fold slower in the absence than in the presence of coexpressed CaVß subunits, and could be resolved into different proportions of fast and slow components in the presence of intermediate concentrations of CaVß subunits (Canti et al. 2001). Our interpretation of these two components was that the fast component represented Gß dissociation from channels to which CaVß was already bound, and the slow rate represented increased CaVß binding at +100 mV, followed by Gß dissociation, since the slow rate depended on CaVß subunit concentration (Canti et al. 2001). In agreement with our previous results, this suggests that CaVß subunit displacement by Gß is not involved in G-protein modulation (Meir et al. 2000; Canti et al. 2000; Leroy et al. 2005), but in contrast the presence of bound ß subunits is essential to promote the loss of Gß at positive potentials (prepulse facilitation) (Meir et al. 2000; Leroy et al. 2005).
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Acknowledgements |
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2-2; Dr E. Perez-Reyes (Loyola University, Chicago, IL, USA) for rat ß2a; Professor P. G. Strange (Reading, UK) for rat D2 dopamine receptor; Dr T. Hughes (Yale, New Haven, CT, USA) for mut-3 GFP and Genetics Institute (Cambridge, MA, USA) for pMT2. We thank Dr K. Page for critical reading of this manuscript. We also thank K. Chaggar and L. Douglas for technical assistance.
Author's present address
J. Leroy: Inserm U-446, Laboratoire de Cardiologie Cellulaire et Moleculaire, Faculté de Pharmacie, Université Paris-Sud, 5, Rue J.-B. Clement, F-92296 Chatenay-Malabry cedex, France.
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), CaV2.2 Y388F I–II linker (•) and CaV2.2 Y388S I–II linker (
) and CaVß1b concentration. Curves were fitted using a rectangular hyperbola function, and the KD values determined to be 13.7, 78.0 and 329 nM, respectively. F, representative Biacore sensorgrams showing negligible interaction between NusA CaV2.2 W391A I–II linker and CaVß1b at concentrations up to 1000 nM.
, n
= 7), or CaV2.2 Y388S/
, n
= 10), and compared to data obtained without any ß subunit coexpressed (
, n
= 10). The normalized data are plotted against the conditioning potential. The mean data are fitted with a Boltzmann function, whose V50,inact values are given in the text.