To examine the role of the intracellular N terminus in the G-protein modulation of the neuronal voltage-dependent calcium channel (VDCC) α1B, we have pursued two routes of investigation. First, we made chimeric channels between α1B and α1C, the latter not being modulated by Gβγ subunits. VDCC α1 subunit constructs were coexpressed with accessory α2δ and β2a subunits inXenopus oocytes and mammalian (COS-7) cells. G-protein modulation of expressed α1 subunits was induced by activation of coexpressed dopamine (D2) receptors with quinpirole in oocytes, or by cotransfection of Gβ1γ2 subunits in COS-7 cells. For the chimeric channels, only those with the N terminus of α1B showed any G-protein modulation; further addition of the first transmembrane domain and I-II intracellular linker of α1B increased the degree of modulation. To determine the amino acids within the α1B N terminus, essential for G-protein modulation, we made mutations of this sequence and identified three amino acids (S48, R52, and R54) within an 11 amino acid sequence as being critical for G-protein modulation, with I49 being involved to a lesser extent. This sequence may comprise an essential part of a complex Gβγ-binding site or be involved in its subsequent action.
The inhibition of N- (α1B) and P/Q-type (α1A) calcium currents by receptors, usually acting through pertussis toxin-sensitive G-proteins, appears to be mediated by Gβγ subunits (Herlitze et al., 1996; Ikeda, 1996). There has been some controversy concerning whether the α1E calcium channel is G-protein-modulated (Page et al., 1998). We have now established that, whereas an N-terminally truncated isoform of rat α1E is not subject to modulation, an isoform with a full-length N terminus is G-protein-modulated, either by coexpression of Gβγ subunits or by activation of a G-protein-coupled receptor (Page et al., 1998), which would agree with results obtained previously for full-length human α1E (Qin et al., 1997).
A number of recent studies have established the importance of the intracellular loop that links transmembrane domains I and II, both in binding Gβγ and in mediating its effects to produce inhibition of the channel (Herlitze et al., 1997; Zamponi et al., 1997). However, this result is controversial, and several studies have suggested either that the I-II loop plays no role in G-protein modulation of α1B (Zhang et al., 1996) or α1E (Qin et al., 1997), or that alone it cannot mediate the effects of the Gβγ subunits (Page et al., 1997,1998; Simen and Miller, 1998). Nevertheless it is not disputed that the I-II loops of α1A, B, and E comprise a major binding site or sites for Gβγ and contain a QxxER amino acid consensus sequence common to many Gβγ-binding sites (De Waard et al., 1997; Herlitze et al., 1997; Zamponi et al., 1997; Dolphin et al., 1999). Secondly, a C-terminal Gβγ-binding site has recently been identified and proposed to be a region responsible for G-protein inhibition of human α1E (Qin et al., 1997). However, it is clear that there are also a number of other sites in the α1 subunit of G-protein-modulated calcium channels that are involved in expression of the inhibition by Gβγ. First, we have found that part of the intracellular N terminus of α1B and α1E is essential for their G-protein modulation (Page et al., 1998). Second, the transmembrane domain I has been found to have an important role (Zhang et al., 1996; Stephens et al., 1998b).
In the present study we have examined the critical nature of the intracellular N terminus of α1B, by making chimeric channels between α1B, which is strongly G-protein-modulated and α1C, which is not G-protein-modulated by this mechanism, and has a completely different N-terminal sequence. We have shown an absolute requirement for the α1B N terminus for observation of G-protein modulation in all the chimeric constructs. Second, we have made specific deletions and point mutations to identify the sequence in the N terminus of α1B that is responsible for conferring G-protein modulation.
MATERIALS AND METHODS
The following cDNAs were used: rat α1C (isoform CII, GenBank accession number M67515), rabbit α1B (D14157), rat β2a (M80545), rat α2δ-1 (neuronal splice variant, M86621), rat D2long receptor (X17458, N5→G), bovine Gβ1 (M13236), bovine Gγ2 (M37183), the C-terminal minigene of β-ARK (M34019), and mut-3 green fluorescent protein (GFP; U73901). All cDNAs were subcloned into the expression vector pMT2 (Swick et al., 1992).
Construction of chimeras
Chimeras were created using PCR following the methods described previously (Page et al., 1998; Stephens et al., 1998b). All constructs were subcloned into the pMT2 vector, and the sequences of the PCR products were confirmed using cycle-sequencing. The constructs were assembled as follows: bCCCC, amino acid residues 1–95 of α1B, 125–2143 of α1C; bBcCCC 1–359 α1B, 409–2143 α1C; bBbCCC 1–483 α1B, 525–2143 α1C; cBcCCC 1–124 α1C, 96–359 α1B, 409–2143 α1C; cBbCCC 1–124 α1C, 96–483 α1B, 525–2143 α1C; cCbCCC 1–408 α1C, 360–483 α1B, 525–2143 α1C; and bCbCCC 1–95 α1B, 125–408 α1C, 360–483 α1B, 525–2143 α1C. Chimeric primers were used with the reverse primer CCA CCA GCA GGT CCA GGA TAT TGA (R1). The resulting PCR product was extended against a template using a forward primer (pMT2F2) directed against the vector TCT CCA CAG GTG TCC ACT. The following chimeric primers were used: GTG CTG GGT GTG CTG AGC GGA GAG TTT for bBcCCC; CAG CCA GTA GAA GAC CTG TGC CTT CAC CAT (reverse primer R2) for bBbCCC; CAC CGA GTG GCC TCC ATT TGA AAT AAT T for bCCCC. These chimeras were used as templates to make others. The primers TTT GAG CGG AGA GTT TGC TAA GG and R2 were used to make the first PCR product, which was then extended on bCCCC to give bCbCCC. The chimeras cBbCCC and cBcCCC were made using bBbCCC and bBcCCC as templates. In each case, the PCR product made using the primers TGT TGA ATG GAA ACC GTT CGA GTA CAT G and R1 was extended on α1CpMT2 template to add the N terminus of α1C. For cCbCCC, restriction digestion of an MfeI site in domain I was used to substitute the N terminus of bCbCCC with that of α1C.
Construction of N-terminal deletion and point mutations
The α1B N terminus was truncated at the 5′ end by introducing a start codon before amino acid E7 to make α1B Δ2–6, Y45 (α1B Δ2–44), and Q51 (α1B Δ2–50). The following primers were used; CGC ACT AGT ATG GAG CTG GGC GGC CGC TAT (Δ2–6), CAG ACT AGT ATG TAC AAA CAG TCG ATC GCG (Δ2–44), and CAG ACT AGT ATG CAG CGC GCG CGG ACC AT (Δ2–50). The α1B Δ45–55 construct was made by using the primer GGC CAG CGG GTC CTC ATG GCG CTG TAC AAC to delete the 11 amino acids, YKQSIAQRART. For all of the α1B point mutations, primers were designed so that single residues were mutated to alanines or so that a number of residues were mutated within the same primer. The following primers were used; R52A-R54A, TCG ATC GCG CAG GCC GCG GCG ACC ATG GCG CT; Y45A, CAG CGG GTC CTC GCC AAA CAG TCG ATC; K46A, CGG GTC CTC TAC GCA CAG TCG ATC GCG; Q47A, GTC CTC TAC AAA GCG TCG ATC GCG CAG; S48A, CTC TAC AAA CAG GCG ATC GCG CAG C; I49A, TAC AAA CAG TCG GCC GCG CAG CGC GCG; Q51A, CAG TCG ATC GCG GCG CGC GCG CGG ACC; R52A, TCG ATC GCG CAG GCC GCG CGG ACC ATG; R54A, GCG CAG CGC GCG GCG ACC ATG GCG CTG; 45YKQSIA→AAAAA, GCC GCA GCA GCT GCC GCG CAG CGC GCG CGG (forward) and GGC AGC TGC TGC GGC GAG GAC CCG CTG (reverse); and 45YKQ→AAA, CGG GTC CTC GCC GCA GCG TCG ATC GCG CAG. The reverse primer used in each case was GTC GCT TCT GCT CTT CTT GG. For the PCR extension reactions, the forward primer used was AGC ACT AGT ATG GTC CGC TTC GGG GAC. The sequences of all constructs were verified.
Expression of constructs and electrophysiological recording
Xenopus oocytes. Adult female Xenopus laevis were killed by anesthetic overdose in a 0.25% solution of tricaine, decapitated, and pithed. Oocytes were removed and defolliculated by treatment with 2 mg/ml collagenase type Ia in a Ca2+-free ND96 saline containing (in mm): NaCl, 96; KCl, 2; MgCl2, 1; and HEPES, 5, pH-adjusted to 7.4 with NaOH for 2 hr at 21°C. Plasmid cDNAs for the different α1 subunits, plus accessory β2a and α2δ subunits and rat D2 receptors, were mixed in a ratio of 3:4:1:3 (except where stated), and ∼10 nl was injected into the nuclei of stage V or VI oocytes. Injected oocytes were incubated at 18°C for 3–7 d in ND96 saline (as above plus 1.8 mm CaCl2) supplemented with 100 μg/ml penicillin, 100 IU/ml streptomycin (Life Technologies, Gaithersburg, MD), and 2.5 mm Na pyruvate. Whole-cell recordings from oocytes were made in the two-electrode voltage-clamp configuration with a chloride-free solution containing (in mm): Ba(OH)2, 5; TEA-OH, 80; NaOH, 25; CsOH, 2; and HEPES, 5 (pH 7.4 with methanesulfonic acid). In all experiments, 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) in order to suppress endogenous Ca2+-activated Cl− currents. Electrodes contained 3m KCl and had resistances of 0.3–2 MΩ. The holding potential (V H) was −100 mV, and the test potential (V t) used for time course studies was 0 mV. All illustrated traces are at this potential, and the current amplitude was always measured 20 msec after the start of the test pulse. Membrane currents were recorded every 15 sec, amplified, and low-pass filtered at 1 KHz using a Geneclamp 500 amplifier and digitized through a Digidata 1200 interface (Axon Instruments, Foster City, CA). In all cases currents were leak subtracted on-line by a P/4 protocol.
COS-7 cells. Cells were cultured and transfected, using the electroporation technique, essentially as described previously (Campbell et al., 1995a). The α1, α2δ, β2a, and GFP cDNAs were used at 15, 5, 5, and 1 μg, respectively. When used, Gβ1 and Gγ2 were included at 2.5 μg each, or β-ARK was included at 5 μg. Blank pMT2 vector was included where necessary to maintain the total cDNA at 31 μg/transfection. Cells were replated using nonenzymatic cell dissociation medium (Sigma, St. Louis, MO), and then maintained at 25°C for between 1 and 16 hr before electrophysiological recording. Maximum GFP fluorescence and voltage-dependent calcium channel (VDCC) expression were observed between 2 and 4 d after transfection (Brice et al., 1997). Ca2+ channel currents were recorded using the whole-cell patch technique. Borosilicate glass 2–5 MΩ electrodes were used. The internal (electrode) and external solutions were similar to those described previously (Campbell et al., 1995b). The patch pipette solution contained in mm: Cs aspartate, 140; EGTA, 5; MgCl2, 2; CaCl2, 0.1; K2ATP, 2; and HEPES, 10; pH 7.2, 310 mOsm with sucrose. GDPβS (2 mm) was included where stated. The external solution contained in mm: tetraethylammonium (TEA) bromide, 160; KCl, 3; NaHCO3, 1.0; MgCl2, 1.0; HEPES, 10; glucose, 4; and BaCl2, 1 or 10, pH 7.4, 320 mOsm with sucrose. Whole-cell currents were elicited fromV H of −100 mV and recorded using an Axopatch 1D amplifier. Data were filtered at 2 kHz and digitized at 5–10 kHz. The junction potential between external and internal solutions was 6 mV, the values given in the figures and text have not been corrected for this. Current records are shown following leak and residual capacitance current subtraction (P/4 or P/8 protocol) and series resistance compensation up to 85%. Current amplitudes were measured 50 msec after the start of depolarization.
All experiments were performed at room temperature (18–20°C). Analysis was performed using pClamp6 and Origin software. Data are expressed as mean ± SEM. Statistical analysis was performed using paired or unpaired Student’s t test, as appropriate.
In a previous study we made chimeras between the rat brain α1E (rbEII) clone, which is not G-protein-modulated, and the strongly modulated α1B. The results of this study showed that rbEII was not modulated because it was N terminally truncated, and a full-length rat α1E isoform showed clear G-protein modulation, although not to such a great extent as α1B. We further showed the importance of the first domain of α1B in increasing the extent of G-protein modulation of α1B/α1E chimeras (Page et al., 1998; Stephens et al., 1998b), as has another recent study (Simen and Miller, 1998). In the present study, we wished to examine the distinct role of the N terminus of α1B in G-protein modulation. To do this we have taken two approaches. First, we have made chimeras between α1B and the α1C channel, which is not modulated by a Gβγ-mediated pathway under any conditions. Second, we have produced selective deletions and mutations of the α1B N-terminal sequence. With such constructs we can determine the domains necessary for the expression of G-protein modulation.
G-protein modulation of α1B/α1C chimeras by activation of the dopamine D2 receptor
In this part of the study, all channels were expressed with the accessory subunits α2δ and β2a (unless stated) inXenopus oocytes, where they were coexpressed with the dopamine D2 receptor. A series of chimeras were made, in which the N terminus, first transmembrane domain, and I-II loop of α1B were systematically substituted for those in α1C, in different permutations. Figure 1 Ashows the chimeras that were made, and the nomenclature employed, which uses capital letters for the transmembrane domains and small letters for the intracellular N-terminal and I-II loop. All chimeras contained the last three domains and C-terminal tail of α1C (denoted CCC), and all showed good expression levels with one exception (Table1). However, because α1B, α1C, and the chimeras between them showed differences in their voltage dependence of activation (Table 1), we could not compare G-protein modulation at a single step potential (Fig.2). Therefore, we have estimated the amount of G-protein modulation in two ways in Xenopusoocytes, first by determining the ability of the D2 agonist quinpirole to cause a depolarizing shift in the voltage dependence of activation, determined from current–voltage plots (Fig. 1 B). Second, we have determined the percentage inhibition by quinpirole of the current activated at all potentials between −20 and +30 mV (Fig.2). In all cases, the modulation by quinpirole occurred within 30–60 sec of its application and was fully reversible.
The modulation of α1B by activation of the dopamine D2 receptor with 100 nm quinpirole was voltage-dependent, as we have shown previously (Page et al., 1998; Stephens et al., 1998b). This is manifested by a depolarizing shift in the voltage for 50% activation of the current (V 50) (Fig.1 B), and also by a reduction in the percentage inhibition at increasing test potentials (Fig. 2 A). Maximum inhibition was usually seen at a test potential ∼10 mV below the peak of the current–voltage relationship (47% at −10 mV for α1B; Fig. 2 A). The transfer of the entire N terminus, first transmembrane domain, and I-II loop sequence of α1B into α1C gave a chimera showing G-protein modulation that was smaller at all potentials than the α1B parent (Figs. 1 B,2 B). The depolarizing shift in theV 50 for α1bBbCCC was less than for α1B (Fig. 1 B), and the maximum modulation was 24% at −20 mV (Fig. 2 B). With respect to both measurements, a similar degree of modulation by quinpirole was seen for α1bBcCCC (Figs. 1 B, 2 C), providing strong evidence that the I-II linker from a modulatable channel such as α1B is not essential for exhibition of G-protein modulation. Modulation by quinpirole was also still present in the chimera α1bCbCCC (18% at −10 mV; Figs. 1 B,2 D). Furthermore, there was still a significant degree of modulation of the minimal chimera α1bCcCCC (13% at −10 mV; Figs. 1 B, 2 E), again indicating that the I-II linker from a modulatable channel is not essential for the observation of G-protein modulation.
In contrast, none of the chimeras containing the N terminus of α1C instead of α1B showed any inhibition by quinpirole at any potential from −30 to +40 mV under these conditions (inhibition by quinpirole at 0 mV: 0.66 ± 1.0% for α1cBbCCC, –0.4 ± 0.3% for α1cBcCCC, and –0.86 ± 0.92% for α1cCbCCC; nvalues given in Table 1) (Fig. 2 F). There was also no quinpirole-induced depolarizing shift in theV 50 for activation (Fig.1 B). This was also the case for α1C (−0.25 ± 0.21% inhibition by quinpirole at 0 mV; Figs. 1 B,2 F). Thus, the N terminus of α1B is essential and sufficient for the expression of any G-protein modulation, whereas the first transmembrane domain and I-II linker of α1B can be substituted by that of α1C, and significant, although reduced, G-protein modulation is still observed.
Antagonism by β2a of G-protein modulation of the α1B/α1C chimeras
It has previously been shown that the G-protein modulation of α1E currents is antagonized by β2a (Qin et al., 1998). To study the interaction between the presence of overexpressed VDCC β2a and the extent of G-protein modulation, we also examined the degree of G-protein modulation by dopamine D2 receptor activation in the absence of exogenously coexpressed VDCC β subunit in Xenopusoocytes. Nevertheless, it should be stressed that Xenopusoocytes contain an endogenous β3-like subunit, and when this was depleted with an antisense construct, no functional currents were seen (Tareilus et al., 1997). The G-protein modulation of α1B and the chimera α1bBbCCC was found to be significantly greater in the absence of coexpressed β2a than in its presence (Fig.2 A,B). In contrast, the extent of quinpirole-induced modulation of the α1bBcCCC and α1bCbCCC chimeras was not significantly increased in the absence of exogenous β2a (Fig.2 C,D). Furthermore, the absence of β2a did not uncover G-protein modulation in any of the chimeras lacking the N terminus of α1B that were not modulated in the presence of β2a (results not shown). We were unable to examine α1bCcCCC currents in the absence of β2a, because no expression was observed (n = 3 experiments). These results suggest that the presence of the I-II linker and first transmembrane domain of α1B, although not being essential for G-protein modulation, are together required for the reduction of G-protein modulation in the presence of the exogenously expressed VDCC β2a subunit, seen under these conditions.
Coexpression of β subunits with α1 subunits in Xenopusoocytes and other systems results in a hyperpolarizing shift in current activation (for review, see Walker and De Waard, 1998), and it is of interest that this is greatest for α1B, α1C and those chimeras in which all the transmembrane domains are identical (α1bCbCCC and α1cCbCCC; Table 1). However, despite the reduced β2a-induced hyperpolarizing shift in the activation of the α1bBbCCC chimera, compared to α1B, there was still a clear β2a-induced reduction in the amount of G-protein inhibition at all potentials (Fig.2 B), indicating that β2a was influencing this channel.
G-protein modulation of α1B/α1C chimeras by coexpression of Gβγ subunits
The role of Gβγ in mediating the inhibition observed was confirmed by coexpression of the chimeric α1 channels with α2δ, β2a, and Gβ1γ2 in COS-7 cells. A prepulse protocol was used (Fig.3, left panels), giving steps to potentials between −40 and +40 mV, before (P1) and 10 msec after (P2) a large depolarizing step to +120 mV (Page et al., 1998). The prepulse reverses Gβγ-mediated modulation, and hence P2 acts as an internal control. The Gβγ-mediated modulation was determined from the hyperpolarizing shift in the V 50of the current–voltage relationship in P2 compared to that in P1 (Fig.3, right panels). For α1B, this shift was almost −10 mV (Figs. 3 A, 4), and it was not significantly smaller for the chimeras α1bBbCCC and α1bCbCCC (Figs.3 B, 4). It was reduced, but still significantly different from α1C for the α1bBcCCC and α1bCcCCC chimeras (Figs.3 C, 4). Of the other chimeras, all of which had the N terminus of α1C, none showed any greater shift inV 50 than α1C itself (Figs.3 D, 4). In control experiments recorded in the absence of coexpressed Gβ1γ2 and in the presence of intracellular GDPβS, the shift in V 50 caused by a depolarizing prepulse was approximately −1.8 mV for α1C (n = 10), very similar to the value for α1C coexpressed with Gβ1γ2 (−2.1 mV; Fig. 4). A similar level of control facilitation was observed for α1B (n = 10) (Fig. 4). Similar control results were obtained when the β-ARK1 Gβγ-binding domain was coexpressed, to act as a sink for endogenous Gβγ and prevent tonic modulation (Stephens et al., 1998a,b) (results not shown). This control prepulse potentiation is therefore likely to be caused by a mechanism other than G-protein modulation (Dolphin, 1996). The main discrepancy between the results examining direct Gβγ modulation and those examining receptor-mediated modulation involve the α1bCbCCC chimera, which is strongly modulated by overexpression of Gβ1γ2 (Figs.3 B, 4), and more weakly modulated by receptor-mediated inhibition (Figs. 1 B, 2 D). The reason for this may relate to differences in Gβγ subtype or concentration between the two systems.
Isolation of the amino acid residues of the N terminus essential for G-protein modulation
We have made a number of deletions to determine the amino acid sequences that are essential for G-protein modulation. From our previous study (Page et al., 1998), we found that the truncated Δ1–55 α1B construct was not G-protein modulated, in agreement with the N-terminally truncated α1E (rbEII) isoform, that is also not G-protein-modulated. In the present series of experiments, the effect of quinpirole (100 nm) was determined during steps to 0 mV, because none of the constructs showed major shifts in voltage dependence of current activation, compared to α1B. Inhibition of wild-type α1B was 35.3 ± 2.2% at 0 mV under these conditions (n = 8). We made a number of truncations: α1BΔ2–6 and Δ2–44, in line with regions of homology between the N-terminal sequences of all the G-protein modulated α1 subunits (Fig.5 A). These two constructs were as strongly G-protein-modulated as α1B itself [respectively, 35.8 ± 2.5% (n = 5), and 36.1 ± 5.3% (n = 6) inhibition by quinpirole; Fig.5 B,C]. This identifies the 11 amino acid sequence of α1B 45–55 (YKQSIAQRART) (Fig.5 A) as being required for the G-protein modulation of α1B. To confirm this finding, deletion of only this sequence created a construct, α1B Δ45–55, in which G-protein modulation was completely abolished [−0.7 ± 2.1% inhibition by quinpirole (n = 5); Fig.5 B,C].
Point mutations to alanine (A) were then carried out to identify the specific amino acids in this 11 amino acid sequence that are essential for G-protein modulation. Mutation of both arginines to alanine (R52A, R54A) produced a construct that showed no G-protein modulation (−2.5 ± 2.5% inhibition by quinpirole; n = 8). Point mutations of the individual amino acids in the QRART sequence (Q51A, R52A, and R54A) subsequently identified both arginines as being critical for G-protein modulation, because either mutation produced a construct that showed almost complete loss of inhibition by quinpirole (Fig. 5 B,C).
The N-terminal part of this 11 amino acid sequence also contains residues that are critical for G-protein modulation. When YKQSI was mutated to AAAAA (Fig. 5 A), the channel was not G-protein-modulated [0.3 ± 2.1% inhibition by quinpirole (n = 4); Fig. 5 C]. To confirm the importance of the amino acids 45–50 (YKQSIA), an intermediate deletion α1B ΔN2–50 was made, to give a construct starting with methionine followed by Q51. This was also found not to be G-protein-modulated (Fig. 5 C). Subsequent point mutations were made of the individual amino acids in the YKQSIA sequence to A (Y45A, K46A, Q47A, S48A, and I49A). This identified only the serine and, to a lesser extent, isoleucine in the sequence as being involved in G-protein modulation. These mutations resulted in reduced quinpirole-induced inhibition of I Ba to 4.5 ± 1.0% (n = 6) for S48A and 17.4 ± 2.1% (n = 11) for I49A, respectively (Fig. 5 C). Although the individual point mutants Y45A, K46A, and Q47A were all strongly G-protein-modulated by quinpirole, the modulation of the construct containing the triple mutation YKQ→AAA was reduced (18.8 ± 3.9% inhibition by quinpirole; n = 5; Fig. 5 C), indicating an influence of these amino acids.
Modulation of N-terminal mutants of α1B by Gβγ
We have confirmed that the identified amino acids are similarly involved in direct Gβγ-induced modulation of α1B by performing experiments with coexpressed Gβ1γ2 in COS-7 cells. Examples of results obtained are shown in Figure 6. For the Q47A mutation, G-protein modulation was still observed, with slowly activating currents in P1 and a clear hyperpolarizing shift in the V 50 for current activation resulting from the depolarizing prepulse (Fig. 6 A). In contrast, for the R52A mutation, no G-protein modulation was observed (Fig. 6 B). The mean results for all the constructs are given in Figure 7, expressed as P2/P1 facilitation ratio at −10 mV (Fig. 7 A). The V 50 for theI Ba current–voltage relationship was also plotted, because this shows a depolarizing shift in G-protein-modulated channels, compared to the control α1B expressed in the absence of Gβγ (Fig. 7 B). These two sets of measurements are strongly correlated (r = 0.76, data not shown), and the depolarizing shift in activationV 50 is also highly correlated to the percentage inhibition by quinpirole observed for the same constructs in the Xenopus oocyte experiments (Fig.7 C), suggesting that direct Gβγ modulation and quinpirole-induced modulation of these constructs are using the same mechanism. The I49A mutation stands out in both these systems as producing a reduction, but not a complete inhibition of modulation (Fig. 7 C).
Basis for the reduction in receptor-mediated modulation of the N-terminal point mutation I49A
G-protein modulation of calcium channels is strongly voltage-dependent, in that more inhibition is observed at low than at high depolarizations (Bean, 1989). To examine the basis for the reduced modulation of the partially modulated mutant (α1B I49A) compared to α1B, we first examined, in Xenopus oocytes, the voltage dependence of the removal of inhibition by quinpirole, during a depolarizing prepulse (see Fig.8 A for voltage protocol). There was no significant effect of the I49A mutation on the voltage dependence of the prepulse-induced facilitation in the presence of quinpirole (Fig. 8 B), or on the time course of removal of quinpirole-induced inhibition during a 100 mV depolarizing prepulse. Single exponential fits gave τ values for removal of inhibition (possibly representing dissociation of Gβγ at this depolarized potential) of ∼20 msec for both constructs (Fig.8 C). The only clear difference between I49A α1B and wild-type α1B was in the more rapid time course of reinstatement of G-protein modulation after its removal by a 100 msec prepulse to +100 mV (Fig. 8 D). This could be fit to a single exponential with τreinhibition of 187 msec for α1B and 85 msec for the I49A mutant (Fig. 8 D, inset).
If we consider G-protein modulation as a simple bimolecular reaction, as has been done previously (Zhang et al., 1996; Stephens et al., 1998a): where C is one of the closed states of the calcium channel α1B subunit, k 1 is the association rate constant, and k −1is the dissociation rate constant for Gβγ. At equilibrium, from the law of mass action: at the holding potential, 1/τreinhibition=k1[Gβγ]+k−1 (1) Equation 2From our previous study (Stephens et al., 1998a), we estimated the Gβγ concentration to reach ∼300 nm, when Gβγ was overexpressed. Taking an approximate value of 100 nm for the concentration of Gβγ resulting from quinpirole-induced receptor activation in the present study [a value of 130 nm can be calculated making the assumptions described in Stephens et al. (1998a)], we can obtain estimates by substitution of steady-state inhibition and τreinhibition values into Equations 1 and 2, of k 1 andk −1. For α1B,k 1 is 25.2 μm −1sec−1, and k −1 is 2.8 sec−1, whereas for the I49A mutantk 1 is 21.0 μm −1sec−1, and k −1 is 9.6 sec−1. Clearly, the major difference is an apparent 3.4-fold increase in the off-rate for Gβγ in the I49A mutant. However, assuming that reassociation of Gβγ is very slow at +100 mV, the dissociation of Gβγ during the prepulse to +100 mV, found from Figure 8 C, is 53.2 sec−1 for α1B and 46.7 sec−1 for α1B I49A, indicating that the apparent off-rate is more rapid for both constructs at this depolarized potential [as previously observed in Stephens et al. (1998a)], and the difference between the parental α1B and the I49A mutant is lost.
The molecular determinants for the inhibition of neuronal VDCC α1 subunits by Gβγ have been the subject of several studies. However, there remains no consensus of opinion concerning the functional importance of biochemically identified Gβγ-binding sites on the I-II loop and C terminus (De Waard et al., 1997; Page et al., 1997; Qin et al., 1997; Zamponi et al., 1997) (for review, see Dolphin, 1998). Furthermore, there has been little agreement on the extent of modulation of the E-type VDCCs (Bourinet et al., 1996; Toth et al., 1996; Yassin et al., 1996; Mehrke et al., 1997; Page et al., 1997; Qin et al., 1997). However, following our recent study (Page et al., 1998), it now seems clear that all α1E orthologues are G-protein-modulated when the long N terminus is present.
Requirement for the N terminus of α1B for G-protein modulation
The present study was performed to further our understanding of the involvement of the N terminus of the VDCC α1B in G-protein modulation, first identified by Page et al. (1998). We therefore made a series of chimeras between α1B, which is strongly G-protein-modulated, and α1C, which is not modulated by Gβγ, in the systems studied. Our conclusions are that the N terminus of α1B is absolutely essential for its G-protein modulation. No modulation was observed of any channel that contained the N terminus from α1C. The sequences of the intracellular N termini of α1B and α1C show little homology, and it is thus clear that the N terminus of α1B plays a role in G-protein modulation that cannot be substituted by that of α1C.
Role of the I-II linker and first transmembrane domain of α1B in G-protein modulation
In contrast to the results concerning the N terminus, the I-II linker of α1B was not completely essential; significant G-protein modulation was observed in the chimeras α1bBcCCC and α1bCcCCC, although the extent of modulation was less than for the control α1B. These results are of mechanistic interest because of the inability of the α1C I-II linker to bind Gβγ (De Waard et al., 1997; Qin et al., 1997; Dolphin et al., 1999), presumably because of the lack of the QxxER-binding motif. It is possible that Gβγ binding to the I-II linker of α1B increases its concentration close to its site of action, but is not directly involved in its functional effects.
Both the I-II linker and the first transmembrane domain of α1B are, however, essential for the observation of a reduction of G-protein modulation by overexpression of exogenous β2a subunit in theXenopus oocyte system. Whereas α1B itself and α1bBbCCC showed significantly greater modulation by quinpirole in the absence of coexpressed VDCC β2a subunit, the α1bBcCCC and α1bCbCCC chimeras exhibited a similar degree of inhibition by quinpirole in the presence and absence of coexpressed β2a. The mechanism of this partial antagonism by β2a remains unclear, but is not completely shared by other β subunits such as β1b (C. Canti and A. C. Dolphin, unpublished results).
The first transmembrane domain of α1B clearly has a role in G-protein modulation, as suggested previously (Zhang et al., 1996; Stephens et al., 1998b). We have found that, although it can be substituted by that of α1C, the α1bCbCCC chimera is less modulated than α1bBbCCC by quinpirole in the Xenopus oocyte system. It is possible that the first transmembrane domain mediates the effects of Gβγ subunits to slow current activation, via interference with the function of its voltage sensor. Evidence suggests that only one Gβγ subunit binds per α1 subunit, in a voltage-dependent manner (Stephens et al., 1998a; Zamponi and Snutch, 1998). We previously estimated the off-rate (k −1) of Gβγ subunits to be ∼1.3 sec−1 at −100 mV and 50 sec−1 at +120 mV (Stephens et al., 1998a). Thus, the binding of Gβγ is probably of higher affinity to the channel with the voltage sensors in their resting state. The action of Gβγ subunits is to delay channel opening and to produce a depolarizing shift in the voltage dependence of activation (Patil et al., 1996). Presumably, this is achieved either by slowing the movement of the voltage sensors (and the IS4 sensor in particular), in response to a change in transmembrane voltage, or reducing the efficiency of coupling of the voltage sensor to channel opening (Jones et al., 1997).
Some of our chimera results and conclusions do not agree with those of a previous work (Furukawa et al., 1998), which also made a chimera with the I-II linker of α1B in α1C and showed it to be G-protein-modulated, thus indicating that the I-II linker alone could mediate G-protein modulation. However, their chimera, together with other chimeras described in their paper, involved substitution of more than just the I-II linker of α1B. In the chimera in question, a region of α1B from part of IS5 through to IIS2 was substituted into α1C, with in addition several amino acid substitutions and deletions, and the results are thus not directly comparable. Furthermore, in their study the reciprocal chimera, made up of α1B with a region including the I-II linker of α1C, was also G-protein-modulated.
Our finding is that substitution into α1C of the region from the N terminus to the end of the I-II linker of α1B (α1bBbCCC) does not produce a channel that is as strongly modulated as α1B in theXenopus oocyte assay, although in the Gβγ overexpression assay, there was no significant difference between α1bBbCCC and α1B. Thus, it is likely that other regions in the rest of α1B may also contribute to the extent of G-protein modulation of α1B, possibly including the C terminus (Qin et al., 1997; Hamid et al., 1999), which may form part of a complex Gβγ-binding pocket.
Amino acids in the N terminus of α1B that are critical for G-protein modulation
From our mutational study of the N terminus, we identified the sequence between amino acids 45 and 55 (YKQSIAQRART) as being essential for G-protein modulation, because a deletion to amino acid 55 produced a construct that showed no G-protein modulation (Page et al., 1998), whereas a channel truncated to amino acid 44 was fully modulated, and a deletion of these 11 amino acids (45–55) resulted in a nonmodulated construct. Subsequently, we have identified three amino acids within this sequence, S48, R52, and R54, that when mutated to alanine, markedly reduce G-protein modulation of α1B, and a fourth amino acid (I49) that also shows an involvement. The substitution of just two amino acids (R52A, R54A) completely abolished G-protein modulation, whereas constructs containing the individual mutations still showed a small degree of modulation (4 and 9% inhibition by quinpirole, respectively). The R52A and R54A constructs also individually showed some slowing of current activation when coexpressed with Gβγ, whereas the double mutant did not (Fig.6 B; results not shown). The RAR motif is reminiscent of the RAK motif found in one of the Gβγ-binding sites on GIRK4 (Krapivinsky et al., 1998).
The I49A mutation stands out as producing a reduction in G-protein modulation in both systems (Fig. 6 C). It is of interest that the 11 amino acid motif we have identified is identical in rat α1E and α1A, except for I49, whose equivalent is lysine in α1E, and methionine in α1A. Furthermore, both α1A and α1Elong show less G-protein modulation than α1B in a number of systems (Zamponi et al., 1997; Page et al., 1998), possibly involving this amino acid substitution. In the present study we have observed that the τreinhibition after a depolarizing prepulse is more than twice as fast for α1B I49A (85 msec) than for α1B (187 msec; Fig. 8 D). However, in our previous study we observed that the τreinhibition for both α1B and α1Elong was ∼95 msec (Page et al., 1998). We are currently re-examining the comparison between α1B and α1Elong under the present conditions (5 mm Ba2+, BAPTA-injected oocytes), to investigate whether our previous lack of observation of any difference in τreinhibitionbetween α1B and α1Elong was caused by an influence of niflumic acid, which we have subsequently found to affect G-protein modulation of α1B currents.
It is possible that the N terminus forms a Gβγ or VDCC β-binding site, or it may be involved in the downstream effects of Gβγ binding. We have observed that inactivation is increased in a number of the α1B mutants, suggesting an impairment of interaction with β2a (G. J. Stephens and A. C. Dolphin, unpublished results). However, one can consider that these mutations in the N terminus of α1B may alter the binding affinity for Gβγ (see Results for I49A). From this analysis the major difference is an apparent 3.4-fold increase in the off-rate for Gβγ in the I49A mutant. Thus, YKQSIAQRART may form part of a Gβγ-binding site, with I49 playing a modulatory role in binding affinity, or it may be involved in the interaction between Gβγ and VDCC β subunits.
This work was supported by The Wellcome Trust and the European Community (Marie Curie Fellowship to C.C.). We thank the following for generous gifts of cDNAs: T. Snutch (University of British Colombia, Vancouver, Canada), rat α1C; H. Chin (National Institutes of Health, Bethesda, MD), rat α2δ-1; Y. Mori (Seriken, Okazaki, Japan), rabbit α1B; E. Perez-Reyes (Loyola University, Chicago, IL), rat β2a; P. G. Strange (Reading, UK), rat D2 receptor; M. Simon (CalTech, Pasadena, CA), bovine Gβ1 and Gγ2; R. Lefkowitz (Duke University, Durham, NC), β-ARK1; T. Hughes (Yale, New Haven, CT), mut-3 GFP; Genetics Institute (Cambridge, MA), pMT2. We also thank M. Li and J. Richards for technical assistance.
Correspondence should be addressed to Prof. A. C. Dolphin, Department of Pharmacology (Medawar Building), University College London, Gower Street, London WC1E 6BT, UK.