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. 1998 May 15;509(Pt 1):15–27. doi: 10.1111/j.1469-7793.1998.015bo.x

Facilitation of rabbit α1B calcium channels: involvement of endogenous Gβγ subunits

Gary J Stephens 1, Nicola L Brice 1, Nicholas S Berrow 1, Annette C Dolphin 1
PMCID: PMC2230940  PMID: 9547377

Abstract

  1. The α1B (N-type) calcium channel shows strong G protein modulation in the presence of G protein activators or Gβγ subunits. Using transient expression in COS-7 cells of α1B together with the accessory subunits α2-δ and β2a, we have examined the role of endogenous Gβγ subunits in the tonic modulation of α1B, and compared this with modulation by exogenously expressed Gβγ subunits.

  2. Prepulse facilitation of control α1B/α2-δ/β2a currents was always observed. This suggests the existence of tonic modulation of α1B subunits. To determine whether endogenous Gβγ is involved in the facilitation observed in control conditions, the βARK1 Gβγ-binding domain (amino acids 495-689) was overexpressed, in order to bind free Gβγ subunits. The extent of control prepulse-induced facilitation was significantly reduced, both in terms of current amplitude and the rate of current activation. In agreement with this, GDPβS also reduced the control facilitation.

  3. Co-expression of the Gβ1γ2 subunit, together with the α1B/α2-δ/β2a calcium channel combination, resulted in a marked degree of depolarizing prepulse-reversible inhibition of the whole-cell ICa or IBa. Both slowing of current activation and inhibition of the maximum current amplitude were observed, accompanied by a depolarizing shift in the mid-point of the voltage dependence of activation. Activation of endogenous Gβγ subunits by dialysis with GTPγS produced a smaller degree of prepulse-reversible inhibition.

  4. The rate of reinhibition of α1B currents by activated G protein, following a depolarizing prepulse, was much faster with Gβ1γ2 than for the decay of facilitation in control cells. Furthermore, βARK1 (495-689) co-expression markedly slowed the control rate of reinhibition, suggesting that the kinetics of reinhibition depend on the concentration of free endogenous or exogenously expressed Gβγ in the cells. In contrast, the rate of loss of inhibition during a depolarizing prepulse did not vary significantly between the different conditions examined.

  5. These findings indicate that, in this system, the voltage-dependent facilitation of α1B that is observed under control conditions occurs as a result of endogenous free Gβγ binding to α1B.

Voltage-dependent G protein-mediated inhibition of neuronal Ca2+ channels is characterized by an inhibition of current amplitude and a slowing of the activation kinetics (for review, see Dolphin, 1996). G protein-mediated inhibition of neuronal N-type (α1B) and P/Q-type (α1A) calcium currents is mediated by Gβγ, and not Gα, subunits (Herlitze, Garcia, Mackie, Hille, Scheuer & Catterall, 1996; Ikeda, 1996). The main binding sites for Gβγ subunits on α1A and α1B have been localized to two domains on the intracellular loop that links transmembrane domains I and II (De Waard, Liu, Walker, Scott, Gurnett & Campbell, 1997; Zamponi, Bourinet, Nelson, Nargeot & Snutch, 1997). Although a binding site for Gβγ has also been found on the C-terminal tail of human α1E (Qin, Platano, Olcese, Stefani & Birnbaumer, 1997), this was not observed for α1B (Zamponi et al. 1997).

G protein modulation is most pronounced at potentials just supra-threshold for activation; this has been interpreted as G protein-modulated channels requiring stronger depolarizations to open (Bean, 1989). Thus, inhibition is weaker at more depolarized potentials. Early models involved binding of activated G protein to both closed and open states of the channel (Elmslie & Jones, 1994; Boland & Bean, 1993). More recently, it has been suggested that these phenomena are due to binding of activated G protein to only the closed state of the channel (Patil, de Leon, Reed, Dubel, Snutch & Yue, 1996). The finding that G protein modulation is voltage dependent provided the basis for the use of large depolarizing prepulses to reverse G protein-mediated inhibition (Elmslie & Jones, 1994). Enhancement of calcium currents in response to prior depolarization is termed facilitation, but it does not necessarily involve reversal of G protein modulation (for review, see Dolphin, 1996). It has also been observed for both cloned and native cardiac calcium channels (Sculptoreanu, Rotman, Takahashi, Scheuer & Catterall, 1993a; Sculptoreanu, Scheuer & Catterall, 1993b; Bourinet, Charnet, Tomlinson, Stea, Snutch & Nargeot, 1994), which do not show direct modulation by inhibitory G proteins.

In the present study, we have examined the G protein modulation of α1B calcium channels in the presence of β2a, a subunit that dramatically slows inactivation, in order to examine changes in the activation properties in isolation. Under these conditions, we observed facilitation of α1B under control conditions, both in terms of an increase in activation kinetics following a depolarizing prepulse and an increase in current amplitude. We have therefore compared the effects of co-expressing exogenous Gβ1γ2 and of activating endogenous G proteins with GTPγS, and conversely of either reducing endogenous G protein activation with GDPβS or reducing free endogenous Gβγ concentrations by exogenous expression of βARK1 (495-689), which contains only the C-terminal Gβγ-binding domain of βARK (Koch, Inglese, Stone & Lefkowitz, 1993; Pitcher, Touhara, Payne & Lefkowitz, 1995). Expression of βARK1 (495-689) has been shown previously to block several Gβγ-mediated signalling pathways in COS-7 cells (Koch, Hawes, Inglese, Luttrell & Lefkowitz, 1994). We now show that the observed control facilitation of α1B is reduced by expression of exogenous βARK1 (495-689), and is thus likely to be due to endogenous free Gβγ subunits.

METHODS

Materials

The following cDNAs were used: rabbit α1B (accession number, D14157; Fujita et al. 1993) was provided by Dr Y. Mori (Seriken, Okazaki, Japan); rat β2a (M80545; Perez-Reyes et al. 1992) was provided by Dr E. Perez-Reyes (Loyola University, Chicago, IL, USA); β1b (X11394) was provided by Dr T. Snutch (UBC, Vancouver, Canada); the full-length rat α2-δ (neuronal splice variant, M86621; Kim, Kim, Lee, King & Chin, 1992) was provided by Dr H. Chin (National Institutes of Health, Bethesda, MA, USA). Bovine Gβ1 (M13236; Fong et al. 1986) and bovine Gγ2 (M37183; Gautam, Baetscher, Aebersold & Simon, 1989) were provided by Dr M. Simon (CalTech, Pasadena, CA, USA). The C-terminal minigene of βARK1 (BTARKB) was provided by Dr R. Lefkowitz (Duke University, Durham, NC, USA). The S65T mutant of green fluorescent protein (GFP) was a gift from Dr S. Moss (University College London, UK) and the mut-3 GFP mutant was a gift from Dr T. Hughes (Yale University, New Haven, CT, USA). All cDNAs were subcloned, using standard techniques, into the pMT2 expression vector (Genetics Institute, Cambridge, MA, USA) for transient expression in COS-7 cells, except that encoding βARK1 (495-689), which was expressed from pRK5 (Koch et al. 1994).

Transfection of COS-7 cells

COS-7 cells were cultured and transfected, using electroporation, essentially as described previously (Campbell, Berrow, Brickley, Page, Wade & Dolphin, 1995a). For transfection, 15, 5, 5 and 1 μg of α1, α2-δ, β2a (or β1b), and either S65T or mut-3 GFP constructs were used. When used, 2.5 μg of both Gβ1 and Gγ2 cDNA or 5 μg βARK1 minigene (BTARKB) were included. Cells were maintained at 37°C, then replated using a non-enzymatic cell dissociation medium (Sigma), and kept at 25°C prior to electrophysiological recording. Successfully transfected cells were identified by expression of GFP. Maximum GFP fluorescence and Ca2+ channel expression were observed between 2 and 4 days post-transfection (Brice et al. 1997; Berrow, Brice, Tedder, Page & Dolphin, 1997; Stephens, Page, Burley, Berrow & Dolphin, 1997).

Antibodies

Polyclonal antisera to the calcium channel subunits α1B and β were raised in rabbits using standard methodology. The calcium channel βcommon antiserum was raised against a peptide corresponding to amino acids 65-79 of the rat brain β1b sequence (SRPSDSDVSLEEDRE), as previously described (Berrow, Campbell, Fitzgerald, Brickley & Dolphin, 1995). The α1B antiserum was raised against a peptide corresponding to amino acids 851-867 (RHHRHRDRKDKTSASTPA) of the rat brain α1B sequence (rbB-1; Dubel, Starr, Ahlijanian, Enyeart, Catterall & Snutch, 1992). This epitope forms part of the intracellular loop between transmembrane segments S6 of domain II and S1 of domain III, and is 65 % homologous to the corresponding region of the rabbit brain α1B sequence used for this study (BIII, Fujita et al. 1993). Cross-reactivity of this antiserum with the rabbit α1B was confirmed by expression and immunostaining of the rabbit α1B subunit in COS-7 cells.

Immunocytochemistry

COS-7 cells used for immunocytochemistry were transfected as for electrophysiological experiments, but omitting the GFP. Immunocytochemistry was performed essentially as described previously (Brice et al. 1997). Briefly, the cells were fixed in 4 % paraformaldehyde 2-3 days post-transfection and permeabilized with 0.02 % Triton X-100. The cells were then incubated overnight with α1B antiserum (1 : 500 dilution), calcium channel βcommon antiserum (1 : 500 dilution), or Gβcommon antibody (Santa Cruz, California, USA; 250 μg ml−1, used at 1 : 500 dilution). β1b was used in this study rather than β2a, as the antibody, although raised against a peptide common to all β subunits, recognizes β1b better than β2a (Brice et al. 1997). Bound antibodies were detected using biotin-conjugated goat anti-rabbit IgG antibody and streptavidin-conjugated Texas Red (Molecular Probes). Immunostaining was observed using an MRC 600 confocal microscope (Biorad, Hemel Hempstead, UK). Arbitrary optical density units were calculated using the Kontron KS400 program (Kontron Elektronik, Eshing, Germany).

Electrophysiology

Recordings were made at room temperature (20-22°C) from COS-7 cells which had been replated between 1 and 16 h previously. Cells were viewed briefly using a fluorescein filter block, and only fluorescent cells expressing GFP were used in experiments. The internal (pipette) and external solutions and recording techniques are similar to those described previously (Campbell, Berrow, Fitzgerald, Brickley & Dolphin, 1995b). The patch pipette solution contained (mm): caesium aspartate, 140; EGTA, 5; MgCl2, 2; CaCl2, 0.1; K2ATP, 2; GTP, 0.1 (when used); and Hepes, 10; pH 7.2, 310 mosmol l−1 with sucrose. GTPγS (100 μm) or GDPβS (0.5-2 mm) were included, where stated, in place of GTP. The external solution contained (mm): TEA-Br, 160; KCl, 3; NaHCO3, 1.0; MgCl2, 1.0; Hepes, 10; glucose, 4; and BaCl2 or CaCl2, 1; pH 7.4, 320 mosmol l−1 with sucrose. Pipettes of resistance 2-4 MΩ were used. Whole-cell currents were elicited from holding potentials (Vh) of -100 mV. Cells used had a capacitance of 31.9 ± 4.1 pF (n= 10), 27.1 ± 1.8 pF (n= 15) and 37.3 ± 2.1 pF (n= 10) for control, Gβ1γ2 cDNA-transfected and βARK1 (495-689) cDNA-transfected cells, respectively. Cells were only used where series resistance was compensated to 80 %, and space clamp was adequate as judged by graded activation of ICa or IBa. The voltage errors from the residual uncompensated series resistance were less than 1 mV for the largest currents, and no further correction was made. An Axopatch-1D amplifier (Axon Instruments) was used, and data were filtered at 0.5-5 kHz and digitized at 1-10 kHz. Analysis was performed using pCLAMP 6 (Axon Instruments) and Origin 3.5 (Microcal Software, Northampton, MA, USA). 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). Data are expressed as means ±s.e.m. Statistical analysis was performed using Student's paired or unpaired t tests, as appropriate.

RESULTS

Biophysical properties of α1B subunits co-transfected with β2a and α2-δ

Transient transfection of α1B cDNA together with β2a and α2-δ cDNAs resulted in inward ICa with a similar current density to that which we have previously described for IBa. In most experiments described here, Ca2+ was used as the charge carrier, but a number of the experiments were performed with Ba2+ (where stated), and no major differences were observed. A minor difference was that the mid-point of voltage dependence of activation (V1/2) (see legend to Fig. 1) was -10.5 ± 1.8 mV (n= 7) in 1 mm Ba2+, whereas it was -6.6 ± 2.1 mV (n= 11) in 1 mm Ca2+, as expected for differences in charge screening. The β2a subunit acts to retard entry of channels into the inactivated state (Olcese et al. 1994), and was used here to limit any confounding effects of voltage-dependent inactivation on the observation of G protein modulation (see Jones & Elmslie, 1997, for discussion of this problem). Co-expression of β2a dramatically slowed the voltage-dependent inactivation of α1B, compared with that previously observed when these subunits were co-expressed with β1b subunits (Page, Stephens, Berrow & Dolphin, 1997). The inactivation kinetics of α1B/α2-δ/β2aIBa at -10 mV, measured using 8 s voltage steps, could usually be described by a single exponential, with a time constant of 4980 ± 800 ms (n= 4), together with a plateau phase representing approximately 30 % of the current.

Figure 1. Calcium channel currents in COS-7 cells transfected with α1B, and the effects of Gβ1γ2 and βARK1 (495-689) co-expression.

Figure 1

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A-D, examples of traces of ICa recorded from cells transfected with α1B cDNA together with α2-δ and β2a cDNAs. Steps of 100 ms duration were applied to increasing test potentials (Vt) to maximally activate ICa from a holding potential (Vh) of -100 mV. Vt values are example traces between -40 and +10 mV. A and B, cells recorded with (A) or without (B) 0.1 mm GTP included in the intracellular medium. C, cells were co-transfected with Gβ1 and Gγ2 cDNAs, and ICa was recorded in the absence of added intracellular GTP. D, cells were co-transfected with βARK1 (495-689) cDNA, and ICa was recorded in the absence of added intracellular GTP. E, current density-voltage relationships for cells recorded under conditions A, C and D. Results are given as means ±s.e.m. for 10, 11 and 15 cells, respectively. Individual current density-voltage plots were also fitted with the equation:
graphic file with name tjp0509-0015-mu3.jpg
where Gmax is maximum conductance (nS pF−1), V1/2 is the voltage at which 50 % of the current is activated, Vrev is the null potential and k is the slope factor. From this analysis, V1/2 was -6.6 ± 2.1 mV for control, -4.9 ± 1.6 mV for βARK1 (495-689) and +0.1 ± 0.2 mV for Gβγ co-expression (P < 0.05vs. control); Gmax was 0.6 ± 0.1 nS pF−1 for control, 0.8 ± 0.1 nS pF−1 for βARK1 (495-689) and 0.5 ± 0.1 nS pF−1 for Gβγ co-expression; k was 4.0 ± 0.4 mV for control, 3.1 ± 0.2 mV for βARK1 (495-689) and 4.5 ± 0.3 mV for Gβγ co-expression; and Vrev was 49.5 ± 3.5 mV for control, 53.1 ± 3.7 mV for βARK1 (495-689) and 51.8 ± 2.4 mV for Gβγ co-expression.

Control ICa recorded in an intracellular solution without added GTP was normally rapidly activating (Fig. 1A). A few examples of currents with a clearly distinguishable additional slowly activating component were also observed (3/13 cells), and this was also observed when ICa was recorded with GTP in the intracellular medium (2/4 cells) (Fig. 1B). In contrast, in the presence of co-transfected Gβ1γ2 subunits, the inward currents usually consisted of a slowly activating current only (Fig. 1C, representing 12/14 cells, with 2/14 also having a rapidly activating component). To determine whether the slowed activation observed in some control cells was a result of the effect of endogenous free Gβγ subunits, currents were examined in the presence of co-expressed βARK1 (495-689) to bind free Gβγ (Koch et al. 1993), and in the absence of GTP. Under these conditions, currents were always rapidly activating (n= 15, Fig. 1D). From current-voltage relationships, current density was increased by βARK1 (495-689), compared with control currents (Fig. 1E), whereas Gβγ co-expression reduced current density and shifted the mid-point of activation to more depolarized potentials compared with the control (Fig. 1E).

Facilitation of α1B following a large depolarizing prepulse

Current facilitation was examined by comparing current amplitudes and activation rates during a 50 ms test pulse before (P1) and 10 ms after (P2) application of depolarizing prepulses to +120 mV (Fig. 2, see left panel for protocol). The depolarizing prepulse produced a small but significant effect on control α1BICa current density (Fig. 2A, middle) and activation kinetics (Fig. 2A, right). The facilitation ratio of the current amplitudes was also calculated as P2/P1 at 11 ms after the start of the step (Fig. 3). This was designed to measure only rapidly activating current, as an estimate of current that was not modulated at -100 mV. Control facilitation ratios were greater than 1 particularly at the voltages that are just supra-threshold for activation (Fig. 3A, □; e.g. P2/P1 = 2.2 ± 0.4 at -20 mV). Currents were also examined using Ba2+ as the charge carrier (Fig. 3B) to determine whether the facilitation was Ca2+ dependent, and in the presence of GDPβS to limit any tonic activation of endogenous Gαβγ by endogenous GTP. Control facilitation was still present, although to a smaller extent, in the presence of Ba2+ (Fig. 3B, ▪; P2/P1 = 1.5 ± 0.2 at -20 mV), and was reduced, particularly at -30 mV, by the inclusion of GDPβS (Fig. 3B, Inline graphic).

Figure 2. Prepulse potentiation of α1B calcium channel currents.

Figure 2

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ICa was recorded from cells transfected with α1B cDNA, together with α2-δ and β2a cDNAs in the absence of GTP in the intracellular solution, in control conditions (A, n= 9), with Gβ1γ2 co-expression (B, n= 7), or with βARK1 (495-689) co-expression (C, n= 10). Activation of ICa was examined from Vh= -100 mV, immediately before (P1) and 10 ms after (P2) application of a 100 ms depolarizing prepulse to +120 mV, according to the voltage protocol given. Example traces are given in the left panel: Vt values are between -40 and 0 mV. Current-voltage relationships are given in the middle panel from the amplitudes at 50 ms in P1 (▪) and P2 (○). The currents have been normalized for each cell to the maximum current in P1 (normally at 0 mV in A and C, and +10 mV in B). Activation was described by a single exponential fit starting at the beginning of the negative current deflection for the currents in P1 and P2, to obtain the time constant of activation, τact. τact-voltage relationships are given in the right panel, with the same symbols as for the middle panel. All data are given as means ±s.e.m., and statistical significances were examined using Student's t test for paired data; *P < 0.05.

Figure 3. Comparison of facilitation ratios for α1B calcium channel currents.

Figure 3

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The facilitation ratio at the stated potentials is the ratio P2/P1 of the current amplitudes in P1 and P2 measured 11 ms after the start of the step when the rapidly activating current component had saturated. This was to gain a measure of the steady-state inhibition at the holding potential. P2/P1 was determined for each cell under the conditions given. A, Ca2+ as charge carrier: control without GTP (□), βARK1 (495-689) co-expression (Inline graphic) and Gβ1γ2 co-expression (Inline graphic); n= 9, 10 and 6, respectively. B, Ba2+ as charge carrier: control without GTP (▪), GDPβS (Inline graphic), GTPγS (Inline graphic); n= 7, 4 and 12, respectively. All data are given as means ±s.e.m., and statistical significances compared with respective controls were examined using Student's t test; *P < 0.05.

As described above, expression of Gβ1γ2 with α1B caused a prominent slowing of activation kinetics and a reduction in current amplitude (Fig. 2B). Both of these effects were readily reversed by a depolarizing prepulse (Fig. 2B, middle and right), and the facilitated current activated at more hyperpolarized potentials than that before the prepulse (Fig. 2B, middle). The facilitation ratio was 5.9 ± 2.0 (n= 6) at -20 mV (Fig. 3A, Inline graphic). The effect of GTPγS on facilitation was much less marked than that of Gβγ overexpression (Fig. 3B, Inline graphic), the facilitation ratio being 3.0 ± 0.7 at -20 mV. In contrast, co-expression of βARK1 (495-689) almost completely abolished prepulse facilitation (Fig. 2C), both of the current amplitude (Fig. 2C, middle), and of the activation kinetics (Fig. 2C, right). Only a non-significant degree of facilitation remained at submaximal activation potentials (Fig. 3A, Inline graphic; P2/P1 = 1.2 ± 0.4 at -20 mV).

Kinetics of reinhibition of α1B following a depolarizing prepulse

A prominent model of G protein interaction with α1 subunits is one where channels exist in either a normal (‘willing’) or a ‘reluctant’ gating mode; the latter requiring stronger depolarization to open (Bean, 1989; Elmslie, Zhou & Jones, 1990). One possibility is that the transition between these states is determined by binding of activated G protein (βγ) subunits to the channel in a bimolecular reaction. Previous reports have examined the reversal of modulation (possibly representing actual dissociation of Gβγ from the channel), and the reinhibition following reversal (possibly representing re-association of Gβγ and the calcium channel), by using depolarizing prepulse protocols.

In order to characterize further the tonic- and G protein-mediated inhibition of α1B, we examined the kinetics of reinhibition by increasing the delay between the depolarizing prepulse and the subsequent test pulse (P2). This analysis was performed for control facilitation, for that in the presence of GTPγS or co-expressed Gβ1γ2, and for the small degree of facilitation remaining in the presence of βARK1 (495-689).

Reinhibition (re-association) kinetics at -100 mV were examined using the protocols shown in Fig. 4. Following the prepulse, the time (Δt) before the subsequent test pulse (P2) was incremented until current was reinhibited to the same extent as during P1, prior to the prepulse. Data were recorded at levels of maximal prepulse potentiation (usually -20 mV). Under control conditions (Fig. 4A), reinhibition occurred with kinetics that were well described by a single exponential (Fig. 4D). The time constant of reinhibition (τreinhibition), determined by exponential fits to data from individual cells, was 350 ± 35 ms (n= 6). This was not affected when Ba2+ was used as the charge carrier (τreinhibition= 361 ± 124 ms, n= 3). For Gβ1γ2 overexpression, the reinhibition kinetics of ICa were much more rapid (Fig. 4B and D), being 41.3 ± 8.9 ms (n= 7; P < 0.01 compared with control). Similarly rapid reinhibition kinetics were obtained using Ba2+ as the charge carrier (τreinhibition= 25 ± 5 ms, n= 5; P < 0.05 compared with control). Reinhibition kinetics of IBa in the presence of GTPγS were slower than for Gβ1γ2 (τreinhibition= 210 ± 30 ms, n= 5; P < 0.05, compared with the τreinhibition in the presence of Gβγ). To determine whether the slow reinhibition kinetics in control conditions were due to a low rate of reassociation with free Gβγ subunits, the time constant of reinhibition was further examined in the presence of expressed βARK1 (495-689) C-terminal polypeptide. Although the amount of facilitation was small, as shown above, reinhibition of ICa was extremely slow (Fig. 4C), τreinhibition being 653 ± 102 ms (n= 9; P < 0.05 compared with control) (Fig. 4D).

Figure 4. Reinhibition of α1B at -100 mV following a depolarizing prepulse to +120 mV.

Figure 4

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ICa was recorded from cells transfected with α1B cDNA together with α2-δ and β2a cDNAs in control conditions (no GTP in the patch pipette, A), and with Gβ1γ2 (B) or βARK1 (495-689) co-expression (C). A-C, facilitating prepulses to +120 mV were applied after a control depolarization (P1), and the time (Δt) between the prepulse and the second test pulse, P2, was incremented on successive recordings until reinhibition was complete. The Δt increments were 150, 20 and 300 ms in A, B and C, respectively, and Vt was to -20 mV where maximal prepulse potentiation was observed. In each case the first two voltage traces are shown above the set of current traces. A dotted line on the current traces indicates the current level at which reinhibition is complete. D, reinhibition of α1B/α2-δ/β2a under control conditions (•, n= 9), with Gβ1γ2 (▪, n= 7) or with βARK1 (495-689) (○, n= 9). Current amplitude was measured at the end of the 50 ms step at P2 for each Δt value and normalized to that at maximum potentiation (i.e. P2-P1 at Δt= 10 ms). The continuous lines represent single exponential fits to the group data.

Kinetics of loss of G protein-mediated inhibition of α1B during a depolarizing prepulse

Dissociation of G protein subunits from α1B at +120 mV was measured using the protocol shown in Fig. 5A. Two test pulses, P1 and P2, were given, with a depolarizing prepulse to +120 mV of 0.25-100 ms duration 10 ms before P2. In the absence of a prepulse, the P1 and P2 currents are identical. The length of the depolarizing prepulse was incremented until the current elicited by the subsequent voltage step had saturated at a maximal level; data were again taken at levels of maximal prepulse potentiation (usually -20 mV). Current amplitude was measured 11 ms after the start of the test pulse, to provide an estimate of only the rapidly activating current component. The time constant of dissociation (τdiss) was then determined by single exponential fits to data from individual cells (see Fig. 5B for examples from three of the conditions: control, Gβ1γ2 and βARK1 (495-689) overexpression). The τdiss did not differ significantly between the different conditions examined here (Fig. 5C), being between 18 and 24 ms, in agreement with the view that relief of block should be independent of free Gβγ subunit concentration at large depolarizations, where rebinding of Gβγ is minimal (Jones & Elmslie, 1997).

Figure 5. Relief of G protein inhibition of α1B at +120 mV.

Figure 5

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Calcium channel currents were recorded from cells transfected with α1B cDNA together with α2-δ and β2a cDNAs under control conditions (ICa), in the presence of 100 μm GTPγS in the patch pipette (IBa) or with Gβ1γ2 (ICa) or βARK1 (495-689) co-expression (ICa). A, two examples of the first (continuous line) and last (dotted line, trace marked with *) currents and the corresponding voltage protocols used are shown for α1B under control conditions. Two test pulses, P1 and P2, were applied before and 10 ms after a depolarizing prepulse to +120 mV, respectively. The prepulse duration (Δt) was incremented from 0.25 ms (thick line) to 120 ms (dotted line). Vt was at levels of maximal prepulse potentiation; for this example, Vt= -20 mV. As the length of the prepulse (Δt) was incremented, current at P2 increased and saturated. B, relief of G protein-mediated block of α1B/α2-δ/β2a in typical examples in control conditions (□), and with βARK1 (495-689) (○) or Gβ1γ2 (▪) overexpression. Current amplitude was measured 11 ms after the start of the P1 and P2 steps for each Δt value, and normalized to that at maximum potentiation (i.e. maximum P2-P1 value). The time constant of dissociation (τdiss) of the active G protein from the α1B channel was determined from a single exponential fit to data from individual experiments (dotted lines). The τdiss values for the examples given are 22.2, 21.2 and 23.3 ms for control, Gβ1γ2 and βARK1 (495-689), respectively. C, mean τdiss±s.e.m. for the different conditions shown, with the number of experiments given in parentheses. The rate of dissociation was similar for all conditions. There was no significant difference between control (▪) and either decreasing free [Gβγ] with βARK1 (495-689) expression (Inline graphic), increasing free [Gβγ] with GTPγS (Inline graphic) or transfection of Gβ1γ2 (□).

Immunolocalization of calcium channel and G protein subunits

The calcium channel β subunit and the Gβγ subunits both have binding sites located on the intracellular I-II loop of the α1B subunit (Pragnell, De Waard, Mori, Tanabe, Snutch & Campbell, 1994; De Waard et al. 1997; Zamponi et al. 1997). As one of the Gβγ-binding sites overlaps directly with that of the calcium channel β subunit, it is important to demonstrate that in the present experiments the results obtained in experiments in which Gβγ is co-expressed are not due to interference with the role of the calcium channel β subunit in localizing the α1 subunits to the plasma membrane (Chien et al. 1995; Brice et al. 1997). In support of the electrophysiological data, Fig. 6 shows that α1B (and also the calcium channel β subunit) was still predominantly membrane localized in the presence of Gβ1γ2; in addition, a pan-specific antibody to Gβ indicated that significant amounts of this subunit also reached the cell membrane. Therefore, Gβγ co-expression does not suppress the calcium channel β subunit function to traffic α1 subunits to the plasma membrane. This is in agreement with electrophysiological results that the maximum conductance (Gmax) obtained from current density-voltage relationships (see legend to Fig. 1) was not significantly reduced in the presence of Gβγ compared with the control. In 1 mm Ca2+, the control Gmax was 0.6 ± 0.1 nS pF−1, whereas for Gβγ overexpression it was 0.5 ± 0.1 nS pF−1 (Fig. 1E). In 1 mm Ba2+, the control Gmax was 0.62 ± 0.11 nS pF−1 (n= 7), whereas for Gβγ overexpression it was 0.65 ± 0.13 nS pF−1 (n= 7).

Figure 6. Immunolocalization of α1B, β and Gβ subunits in COS-7 cells.

Figure 6

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Cells were transfected with α1B, α2-δ, β1b and Gβ1γ2 cDNAs, except for A and F where the Gβ1γ2 subunits were omitted, and E which represents transfection of pMT2 vector alone. The cells were labelled with polyclonal antibodies directed against α1B (A, B and E), βcommon (C), Gβ(common) (a pan-specific antibody that recognizes all Gβ isoforms, D) or with α1B pre-immune serum (F). Scale bars are 15 μm. The staining at the plasma membrane (M) and in the intracellular compartment (C) of a random sample of cells from each group were compared using confocal microscopy. The location of membrane and intracellular compartment were determined from an overlaid phase image. For each of the four groups, immunostaining against the underlined subunit (in arbitrary units (a.u.) determined using the Kontron KS400 program) was significantly higher for the plasma membrane than the intracellular compartment: A, -α1B/α2-δ/β1b (M = 117 ± 10 a.u., C = 93 ± 10 a.u.; n= 8, P < 0.005); B, -α1B/α2-δ/β1b, Gβ11γ2 (M = 119 ± 13 a.u., C = 69 ± 9 a.u.; n= 8, P < 0.005); C, α1B/α2-δ/-β1b, Gβ1γ2 (M = 98 ± 8 a.u., C = 62 ± 8 a.u.; n= 17, P < 0.005); D, α1B/α2-δ/β1b, - -Gβ1γ2 (M = 98 ± 16 a.u., C = 64 ± 12 a.u.; n= 8, P < 0.05). Values are means ±s.e.m. No staining was observed in cells transfected with pMT2 vector alone or cells stained using α1B pre-immune serum.

DISCUSSION

Calcium channel facilitation is a phenomenon that has been observed in a number of different systems, for both cloned and native calcium channels (for review, see Dolphin, 1996). In the case of cloned α1C calcium channels, facilitation has been shown to depend on the presence of a β subunit, and possibly to involve phosphorylation (Sculptoreanu et al. 1993a,b; Bourinet et al. 1994). In contrast, for native neuronal calcium channels, it has been suggested that facilitation represents the reversal of tonic G protein modulation (Doupnik & Pun, 1994; Dolphin, 1996; Albillos et al. 1996). The present study has examined the role of endogenous and exogenous Gβγ subunits in the tonic and G protein-mediated modulation of α1B calcium channels, which underlie the neuronal N-type current. In order to separate any effects on voltage-dependent inactivation (Zhang, Ellinor, Aldrich & Tsien, 1994; Herlitze, Hockerman, Scheuer & Catterall, 1997; Page et al. 1997) from voltage-dependent G protein-mediated slowing of activation kinetics, the auxiliary β2a subunit was co-expressed in order to limit inactivation (Olcese et al. 1994). Under control conditions, amplitude facilitation (P2/P1) ratios significantly less than 1 were never seen with β2a, either in the presence or absence of a depolarizing prepulse between the two test pulses. This is in contrast to results obtained with β1b, where the significant degree of inactivation during the prepulse protocol could mask facilitation (G. J. Stephens & A. C. Dolphin, unpublished observations).

Gβγ subunits mediate both G protein-induced and tonic inhibition of the α1B calcium channel

The prepulse facilitation data for the calcium channel α1B subunit under control conditions suggest some tonic facilitation in COS-7 cells (Fig. 3), as previously reported for expression in HEK 293 cells (Toth, Shekter, Ma, Philipson & Miller, 1996). Dialysis with GDPβS reduced control facilitation at -30 mV, indicating that rather than being an intrinsic property of the α1B calcium channel, as may be the case for α1C (Scuptoreanu et al. 1993a), it may be a result of tonic modulation by endogenous free Gβγ subunits, whose level could be influenced by the endogenous GTP concentration, and possibly by tonic activation of endogenous receptors (Koch et al. 1994). It may also reflect the voltage dependence of the influence of the calcium channel β subunit, whose binding may compete with Gβγ (Campbell et al. 1995b; Dolphin, 1996; Qin et al. 1997). To examine further the possibility that α1B is modulated by free Gβγ under control conditions, we examined the effect of co-expression of βARK1 (495-689) (Koch et al. 1994). This is the C-terminal Gβγ-binding domain of βARK1, comprising amino acids 495-689, which has no kinase activity. It has an affinity for Gβγ sufficient to interfere with Gβγ-mediated signalling in other systems (Koch et al. 1994). We observed a marked reduction in the amount of prepulse potentiation in cells co-expressing βARK1 (495-689), presumably because it lowered the amount of free Gβγ able to bind to α1B.

In contrast, dialysis with GTPγS potentiated the observed facilitation of α1B, in agreement with our previous studies in which the auxiliary β subunit was β1b (Page et al. 1997). Co-expression of α1B with Gβ1γ2 caused classical modulation of ICa, in which G protein inhibition is associated with a depolarizing shift in V1/2. This inhibition was readily reversed by a large depolarizing prepulse, both in terms of facilitation of current levels and increase in current activation rate. These findings confirm the involvement of Gβγ in N-type calcium channel inhibition (Herlitze et al. 1996; Ikeda, 1996). It is likely that the action of GTPγS is to recruit endogenous G protein subunits, and in all cases co-expression of Gβ1γ2 led to a greater modulation of activation kinetics than GTPγS. These differences are probably due to a higher effective concentration of the active G protein subunits being present when Gβ1γ2 subunits are co-expressed, as discussed below. In addition, GTPγS produces, in equal proportion, Gα-GTPγS and free Gβγ, whereas when Gβ1γ2 subunits are transfected they are presumably present in excess over Gα subunits.

Kinetics of reinhibition of α1B by Gβγ subunits following a depolarizing prepulse

We further examined the interaction between α1B and activated G protein subunits in terms of reinhibition characteristics for α1B calcium current under control conditions, and compared this with α1B current in the presence of GTPγS (as a measure of maximal modulation of endogenous G proteins), co-expressed with Gβ1γ2 (as a measure of a saturated system), or co-expressed with βARK1 (495-689) to bind endogenous free Gβγ subunits (Koch et al. 1994). Consistent with an increased concentration of active G protein subunits, Gβ1γ2 cDNA transfection produced a much faster reinhibition of α1B current than GTPγS, and this in turn was faster than the reinhibition following facilitation under control conditions. Presumably the action of GTPγS is dependent on the concentration of endogenous Gβγ generated by GTPγS-induced activation of Gα subunits. There is no Gαo in COS-7 cells, but they do contain Gα11 and Gαq (see Lee, Park, Wu, Rhee & Simon, 1992) and also Gαi and Gαs (Daaka, Pitcher, Richardson, Stoffel, Robishaw & Lefkowitz, 1997), all of which could generate endogenous Gβγ subunits in this system. This suggests either that the level of Gβγ attainable from endogenous Gαβγ heterotrimers is much lower than the level reached when Gβγ cDNA is co-transfected into this system, or that the particular endogenous Gβγ subunits generated do not couple well to the calcium channel α1B subunit present. The former explanation seems more likely for our results with α1B, in that N-type calcium channels seem to be fairly promiscuous in the Gβγ isoforms with which they couple (Ikeda, 1996); in agreement, we see similar effects on α1B when Gβ1γ2 is replaced by the Gβ2γ3 combination (G. J. Stephens & A. C. Dolphin, unpublished results). The prolongation of the reinhibition time course in the presence of βARK1 (495-689) suggests that this has lowered the resting level of free Gβγ. Thus, during the depolarizing prepulse, residual Gβγ that was bound to α1B is removed, and rebinding is slow because it occurs from the bulk phase. The fact that rebinding does occur indicates that, at -100 mV, Gβγ binding to the α1B calcium channel is of high affinity. The affinity of Gβγ binding to the interaction domain (AID) on the α1A I-II loop in vitro has been measured to be 63 nm (De Waard et al. 1997). The present results provide evidence that actual dissociation of Gβγ, rather than just a reduction in its effectiveness, occurs during depolarization.

The rate of calcium current reinhibition has been shown previously to be dependent on the concentration of agonist (Elmslie & Jones, 1994; Zhou, Shapiro & Hille, 1997). In oocytes, reinhibition of α1B following a depolarizing prepulse during modulation by a receptor agonist had a time constant of about 77 ms (Zhang, Ellinor, Aldrich & Tsien, 1996). These studies were performed in the presence of both the agonist somatostatin and exogenously expressed Gβγ, although Gβγ expression may not be maximal as the ligand was still able to inhibit currents. In general, reinhibition rates in native neurons following inhibition of the calcium current by agonist application have time constants of between 50 and 140 ms (see Dolphin, 1996; Zhou et al. 1997). It seems likely that transient overexpression of Gβ1γ2 in the present study causes maximal inhibition of the α1B current.

Estimation of free Gβγ concentrations

For the reaction:

graphic file with name tjp0509-0015-mu1.jpg

C is one of the closed states of the calcium channel α1B subunit, k1 is the association rate constant and k-1 is the dissociation rate constant, both of which may be (indirectly or directly) voltage dependent. At equilibrium, from the law of Mass Action:

graphic file with name tjp0509-0015-mu2.jpg

(where [Gβγ] and [CGβγ] are the concentrations of free and channel-bound Gβγ, respectively), at -100 mV

graphic file with name tjp0509-0015-m1.jpg (1)

and

graphic file with name tjp0509-0015-m2.jpg (2)

therefore,

graphic file with name tjp0509-0015-m3.jpg (3)

Substituting into eqn (1) the τreinhibition values for control, Gβγ or βARK1 (495-689) overexpression obtained from Fig. 4, and substituting into eqn (3) the facilitation ratios P2/P1 determined in Fig. 3A for a step to -20 mV, k-1 at -100 mV can be calculated to be 1.25 s−1 for control, 4.26 s−1 for Gβ1γ2 overexpression, and 1.28 s−1 for βARK1 (495-689) overexpression. It is of interest that the k-1 value is similar in the two cases involving endogenous Gβγ, but is larger for co-expressed Gβ1γ2, suggesting that the dissociation rate from the α1B channel for different Gβγ subunits may not be identical. However, if we assume the affinity of Gβγ binding to α1B at -100 mV to be similar to that calculated for Gβγ binding to the α1A I-II intracellular loop (63 nm; De Waard et al. 1997), for all the conditions described here, we can estimate k1 to be 19.8, 67.6 and 20.3 μm−1 s−1 for control, Gβ1γ2 and βARK1 (495-689) overexpression, respectively, at -100 mV. Taking these values, the free [Gβγ] under these three conditions is 50, 295 and 11.8 nm, respectively.

Kinetics of dissociation of Gβγ from α1B during a depolarizing prepulse

The time constants for relief of G protein-mediated inhibition of α1B currents by varying the depolarizing prepulse are very similar under control conditions, or in the presence of GTPγS, or with Gβ1γ2 or βARK1 (495-689) co-expression (τdiss= 18-24 ms). The similarity of these dissociation rates under conditions of varying free Gβγ concentration indicates that there is little reassociation of Gβγ during the depolarizing prepulse. Thus, there is a strong voltage dependence of k1, and dissociation will be favoured at this potential (see Patil et al. 1996). Assuming k1 to be negligible at +120 mV, then k-1 under control conditions is approximately 50 s−1 at this potential, about 50-fold more rapid than at -100 mV.

Dissociation time constants, measured by varying the depolarizing prepulse, of between 5 and 12 ms have been reported for neuronal N-type calcium channels in the presence of GTPγS (Currie & Fox, 1997; Jones & Elmslie, 1997), and 3 ms for somatostatin inhibition of α1B currents in oocytes (Zhang et al. 1996), which are somewhat faster than those measured in the present study. One major difference between this and the study of cloned α1B expressed in oocytes (Zhang et al. 1996) is that α1B was co-expressed with the β2a subunit here. The role of this subunit in the kinetics of G protein modulation requires further comparative investigation; however, it may be speculated that the kinetics of interaction between Gβ1γ2 and α1B are dependent on the β subunit present, since there are binding sites for both on the I-II loop. Some preliminary data support this hypothesis, in that transmitter-induced slowing of calcium channel kinetics was prominent with β2a, but not seen with β1b (Patil, Brody, Snutch & Yue, 1997). The molecular composition of neuronal calcium channels, and in particular β subunit diversity, may thus have important consequences not only for voltage-dependent inactivation, but also in the fine-tuning of modulation by Gβγ subunits.

We also show that Gβγ co-expression does not suppress calcium channel β subunit function to traffic α1 subunits to the plasma membrane. Conversely, current amplitude and activation rates were clearly inhibited by Gβ1γ2 in the presence of β subunits; and the reversal of inhibition by depolarizing prepulses suggests that the nature of the interaction of Gβγ with the channel is highly dynamic. In contrast, the α1-β interaction might be expected to be much more stable with very high affinity binding reported in vitro (De Waard, Witcher, Pragnell, Liu & Campbell, 1995). Nevertheless, it is likely that this binding is also voltage dependent, and it will be of interest to determine the effects of the β subunit on Gβγ binding to the I-II loop, to examine more fully the hypothesis that β subunits and G proteins may interact competitively with calcium channel α1 subunits (Campbell et al. 1995b; Bourinet, Soong, Stea & Snutch, 1996; Qin et al. 1997).

In conclusion, the data shown here indicate that under control conditions there is sufficient free Gβγ present in the vicinity of the α1B calcium channel to produce a significant tonic block of α1B current. If this also occurs in native neurons and neurosecretory cells, it would lead to frequency- or depolarization-dependent facilitation of calcium entry through N-type channels, as has been observed in several cell types (e.g. Doupnik & Pun, 1994; Albillos et al. 1996).

Acknowledgments

We thank the following for generous gifts of cDNAs and reagents: Dr Y. Mori (Seiriken, Okazaki, Japan), α1b; Dr H. Chin (NIH, USA), α2-δ; Dr E. Perez-Reyes (Loyola University, USA), β2a; Dr T. Snutch (Vancouver, Canada), β1b; Dr M. Simon (CalTech, CA, USA), Gβ1 and Gγ2; Dr S. Moss (UCL, London, UK), S65T GFP; Dr T. Hughes (Yale University, USA) mut-3 GFP; Dr R. Lefkowitz (Duke University, USA), βARK minigene; Genetics Institute (CA, USA), pMT2. We also gratefully acknowledge financial support from The Wellcome Trust, and thank Ms A. Odunlami, Mr I. Tedder and Ms M. Li for technical assistance. This work benefitted from the use of the Seqnet facility (Daresbury, UK).

References

  1. Albillos A, Gandía L, Michelena P, Gilabert JA, Del Valle M, Carbone E, García AG. The mechanism of calcium channel facilitation in bovine chromaffin cells. Journal of Physiology. 1996;494:687–695. doi: 10.1113/jphysiol.1996.sp021524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bean BP. Neurotransmitter inhibition of neuronal calcium channels by changes in channel voltage dependence. Nature. 1989;340:153–156. doi: 10.1038/340153a0. [DOI] [PubMed] [Google Scholar]
  3. Berrow NS, Brice NL, Tedder I, Page K, Dolphin AC. Properties of cloned rat α1A calcium channels, transiently expressed in the COS-7 cell line. European Journal of Neuroscience. 1997;9:739–748. doi: 10.1111/j.1460-9568.1997.tb01422.x. [DOI] [PubMed] [Google Scholar]
  4. Berrow N, Campbell V, Fitzgerald EM, Brickley K, Dolphin AC. Antisense depletion of β-subunits modulates the biophysical and pharmacological properties of neuronal calcium channels. Journal of Physiology. 1995;482:481–491. doi: 10.1113/jphysiol.1995.sp020534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boland LM, Bean BP. Modulation of N type calcium channels in bullfrog sympathetic neurons by luteinising hormone releasing hormone: kinetics and voltage-dependence. Journal of Neuroscience. 1993;13:516–533. doi: 10.1523/JNEUROSCI.13-02-00516.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bourinet E, Charnet P, Tomlinson WJ, Stea A, Snutch TP, Nargeot J. Voltage-dependent facilitation of a neuronal α1C L-type calcium channel. EMBO Journal. 1994;13:5032–5039. doi: 10.1002/j.1460-2075.1994.tb06832.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bourinet E, Soong TW, Stea A, Snutch TP. Determinants of the G protein-dependent opioid modulation of neuronal calcium channels. Proceedings of the National Academy of Sciences of the USA. 1996;93:1486–1491. doi: 10.1073/pnas.93.4.1486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brice NL, Berrow NS, Campbell V, Page KM, Brickley K, Tedder I, Dolphin AC. Importance of the different β subunits in the membrane expression of the α1A and α2 calcium channel subunits: studies using a depolarisation-sensitive α1A antibody. European Journal of Neuroscience. 1997;9:749–759. doi: 10.1111/j.1460-9568.1997.tb01423.x. [DOI] [PubMed] [Google Scholar]
  9. Campbell V, Berrow N, Brickley K, Page K, Wade R, Dolphin AC. Voltage-dependent calcium channel β-subunits in combination with alpha1 subunits have a GTPase activating effect to promote hydrolysis of GTP by G alphao in rat frontal cortex. FEBS Letters. 1995a;370:135–140. doi: 10.1016/0014-5793(95)00813-o. [DOI] [PubMed] [Google Scholar]
  10. Campbell V, Berrow NS, Fitzgerald EM, Brickley K, Dolphin AC. Inhibition of the interaction of G protein Go with calcium channels by the calcium channel β-subunit in rat neurones. Journal of Physiology. 1995b;485:365–372. doi: 10.1113/jphysiol.1995.sp020735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chien AJ, Zhoa X, Shirokov RE, Puri TS, Chang CF, Sun D, Rios E, Hosey MM. Roles of a membrane-localised β subunit in the formation and targeting of functional L-type Ca2+ channels. Journal of Biological Chemistry. 1995;270:30036–30044. doi: 10.1074/jbc.270.50.30036. 10.1074/jbc.270.50.30036. [DOI] [PubMed] [Google Scholar]
  12. Currie KPM, Fox AP. Comparison of N- and P/Q-type voltage-gated calcium channel current inhibition. Journal of Neuroscience. 1997;17:4570–4579. doi: 10.1523/JNEUROSCI.17-12-04570.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Daaka Y, Pitcher JA, Richardson M, Stoffel RH, Robishaw JD, Lefkowitz RJ. Receptor and Gβγ isoform-specific interactions with G protein-coupled receptor kinases. Proceedings of the National Academy of Sciences of the USA. 1997;94:2180–2185. doi: 10.1073/pnas.94.6.2180. 10.1073/pnas.94.6.2180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. De Waard M, Liu H, Walker D, Scott VE, Gurnett CA, Campbell KP. Direct binding of G-protein βγ complex to voltage-dependent calcium channels. Nature. 1997;385:446–450. doi: 10.1038/385446a0. 10.1038/385446a0. [DOI] [PubMed] [Google Scholar]
  15. De Waard M, Witcher DR, Pragnell M, Liu H, Campbell KP. Properties of the α1-β anchoring site in voltage-dependent Ca2+ channels. Journal of Biological Chemistry. 1995;270:12055–12064. doi: 10.1074/jbc.270.20.12056. [DOI] [PubMed] [Google Scholar]
  16. Dolphin AC. Facilitation of Ca2+ current in excitable cells. Trends in Neurosciences. 1996;19:35–43. doi: 10.1016/0166-2236(96)81865-0. 10.1016/0166-2236(96)81865-0. [DOI] [PubMed] [Google Scholar]
  17. Doupnik CA, Pun RYK. G-protein activation mediates prepulse facilitation of Ca2+ channel currents in bovine chromaffin cells. Journal of Membrane Biology. 1994;140:47–56. doi: 10.1007/BF00234485. [DOI] [PubMed] [Google Scholar]
  18. Dubel SJ, Starr TVB, Ahlijanian MK, Enyeart JJ, Catterall WA, Snutch TP. Molecular cloning of the α1 subunit of an ω-conotoxin-sensitive calcium channel. Proceedings of the National Academy of Sciences of the USA. 1992;89:5058–5062. doi: 10.1073/pnas.89.11.5058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Elmslie KS, Jones SW. Concentration dependence of neurotransmitter effects on calcium channel kinetics in frog sympathetic neurones. Journal of Physiology. 1994;481:35–46. doi: 10.1113/jphysiol.1994.sp020417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Elmslie KS, Zhou W, Jones SW. LHRH and GTP-γ-S modify calcium current activation in bullfrog sympathetic neurons. Neuron. 1990;5:75–90. doi: 10.1016/0896-6273(90)90035-e. 10.1016/0896-6273(90)90035-E. [DOI] [PubMed] [Google Scholar]
  21. Fong HK, Hurley JB, Hopkins RS, Miake-Lye R, Johnson MS, Doolittle RF, Simon MI. Repetitive segmental structure of the transducin beta subunit: homology with the CDC4 gene and identification of related mRNAs. Proceedings of the National Academy of Sciences of the USA. 1986;83:2162–2166. doi: 10.1073/pnas.83.7.2162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Fujita Y, Mynlieff M, Dirksen RT, Kim M-S, Niidome T, Nakai J, Friedrich T, Iwabe N, Miyata T, Furuichi T, Furutama D, Mikoshiba K, Mori Y, Beam KG. Primary structure and functional expression of the ω-conotoxin-sensitive N-type calcium channel from rabbit brain. Neuron. 1993;10:585–598. doi: 10.1016/0896-6273(93)90162-k. 10.1016/0896-6273(93)90162-K. [DOI] [PubMed] [Google Scholar]
  23. Gautam N, Baetscher M, Aebersold R, Simon MI. A G protein γ subunit shares homology with ras proteins. Science. 1989;244:971–974. doi: 10.1126/science.2499046. [DOI] [PubMed] [Google Scholar]
  24. Herlitze S, Garcia DE, Mackie K, Hille B, Scheuer T, Catterall WA. Modulation of Ca2+ channels by G-protein βγ subunits. Nature. 1996;380:258–262. doi: 10.1038/380258a0. 10.1038/380258a0. [DOI] [PubMed] [Google Scholar]
  25. Herlitze S, Hockerman GH, Scheuer T, Catterall WA. Molecular determinants of inactivation and G protein modulation in the intracellular loop connecting domains I and II of the calcium channel α1A subunit. Proceedings of the National Academy of Sciences of the USA. 1997;94:1512–1516. doi: 10.1073/pnas.94.4.1512. 10.1073/pnas.94.4.1512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ikeda SR. Voltage-dependent modulation of N-type calcium channels by G protein βγ subunits. Nature. 1996;380:255–258. doi: 10.1038/380255a0. 10.1038/380255a0. [DOI] [PubMed] [Google Scholar]
  27. Jones SW, Elmslie KS. Transmitter modulation of neuronal calcium channels. Journal of Membrane Biology. 1997;155:1–10. doi: 10.1007/s002329900153. 10.1007/s002329900153. [DOI] [PubMed] [Google Scholar]
  28. Kim HL, Kim H, Lee P, King RG, Chin H. Rat brain expresses an alternatively spliced form of the dihydropyridine-sensitive L-type calcium channel α2 subunit. Proceedings of the National Academy of Sciences of the USA. 1992;89:3251–3255. doi: 10.1073/pnas.89.8.3251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Koch WJ, Hawes BE, Inglese J, Luttrell LM, Lefkowitz RJ. Cellular expression of the carboxyl terminus of a G protein-coupled receptor kinase attenuates Gβγ-mediated signalling. Journal of Biological Chemistry. 1994;269:6193–6197. [PubMed] [Google Scholar]
  30. Koch WJ, Inglese J, Stone WC, Lefkowitz RJ. The binding site for the βgamma subunits of heterotrimeric G proteins on the β-adrenergic receptor kinase. Journal of Biological Chemistry. 1993;268:8256–8260. [PubMed] [Google Scholar]
  31. Lee CH, Park D, Wu D, Rhee SG, Simon MI. Members of the Gq alpha subunit gene family activate phospholipase C beta isozymes. Journal of Biological Chemistry. 1992;267:16044–16047. [PubMed] [Google Scholar]
  32. Olcese R, Qin N, Schneider T, Neely A, Wei X, Stefani E, Birnbaumer L. The amino terminus of a calcium channel β subunit sets rates of channel inactivation independently of the subunit's effect on activation. Neuron. 1994;13:1433–1438. doi: 10.1016/0896-6273(94)90428-6. 10.1016/0896-6273(94)90428-6. [DOI] [PubMed] [Google Scholar]
  33. Page KM, Stephens GJ, Berrow NS, Dolphin AC. The intracellular loop between domains I and II of the B-type calcium channel confers aspects of G protein sensitivity to the E-type calcium channel. Journal of Neuroscience. 1997;17:1330–1338. doi: 10.1523/JNEUROSCI.17-04-01330.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Patil PG, Brody DL, Snutch TP, Yue DT. β-subunit modulation of G-protein inhibition and voltage-dependent inactivation of P/Q type (A-class) neuronal calcium channels. Biophysical Journal. 1997;72:A22. [Google Scholar]
  35. Patil PG, De Leon M, Reed RR, Dubel S, Snutch TP, Yue DT. Elemental events underlying voltage-dependent G-protein inhibition of N-type calcium channels. Biophysical Journal. 1996;71:2509–2521. doi: 10.1016/S0006-3495(96)79444-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Perez-Reyes E, Castellano A, Kim HS, Bertrand P, Baggstrom E, Lacerda A, Wei X, Birnbaumer L. Cloning and expression of a cardiac/brain β subunit of the L-type calcium channel. Journal of Biological Chemistry. 1992;267:1792–1797. [PubMed] [Google Scholar]
  37. Pitcher JA, Touhara K, Payne ES, Lefkowitz RJ. Pleckstrin homology domain-mediated membrane association and activation of the beta-adrenergic receptor kinase requires co-ordinated interaction with G βγ subunits and lipid. Journal of Biological Chemistry. 1995;270:11707–11710. doi: 10.1074/jbc.270.20.11707. 10.1074/jbc.270.20.11707. [DOI] [PubMed] [Google Scholar]
  38. Pragnell M, De Waard M, Mori Y, Tanabe T, Snutch TP, Campbell KP. Calcium channel β-subunit binds to a conserved motif in the I-II cytoplasmic linker of the α1-subunit. Nature. 1994;368:67–70. doi: 10.1038/368067a0. 10.1038/368067a0. [DOI] [PubMed] [Google Scholar]
  39. Qin N, Platano D, Olcese R, Stefani E, Birnbaumer L. Direct interaction of Gβγ with a C terminal Gβγ binding domain of the calcium channel α1 subunit is responsible for channel inhibition by G protein coupled receptors. Proceedings of the National Academy of Sciences of the USA. 1997;94:8866–8871. doi: 10.1073/pnas.94.16.8866. 10.1073/pnas.94.16.8866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Scuptoreanu A, Rotman E, Takahashi M, Scheuer T, Catterall W. Voltage-dependent potentiation of the activity of cardiac L-type calcium channel α1 subunits due to phosphorylation by cAMP-dependent protein kinase. Proceedings of the National Academy of Sciences of the USA. 1993a;90:10135–10139. doi: 10.1073/pnas.90.21.10135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sculptoreanu A, Scheuer T, Catterall WA. Voltage-dependent potentiation of L-type Ca2+ channels due to phosphorylation by cAMP-dependent protein kinase. Nature. 1993b;364:240–243. doi: 10.1038/364240a0. 10.1038/364240a0. [DOI] [PubMed] [Google Scholar]
  42. Stephens GJ, Page K, Burley JR, Berrow NS, Dolphin AC. Functional expression of rat brain cloned α1E calcium channels in COS-7 cells. Pflügers Archiv. 1997;433:523–532. doi: 10.1007/s004240050308. [DOI] [PubMed] [Google Scholar]
  43. Toth PT, Shekter LR, Ma GH, Philipson LH, Miller RJ. Selective G-protein regulation of neuronal calcium channels. Journal of Neuroscience. 1996;16:4617–4624. doi: 10.1523/JNEUROSCI.16-15-04617.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zamponi GW, Bourinet E, Nelson D, Nargeot J, Snutch TP. Crosstalk between G proteins and protein kinase C is mediated by the calcium channel α1 subunit I-II linker. Nature. 1997;385:442–446. doi: 10.1038/385442a0. [DOI] [PubMed] [Google Scholar]
  45. Zhang J-F, Ellinor PT, Aldrich RW, Tsien RW. Molecular determinants of voltage-dependent inactivation in calcium channels. Nature. 1994;372:97–100. doi: 10.1038/372097a0. [DOI] [PubMed] [Google Scholar]
  46. Zhang J-F, Ellinor PT, Aldrich RW, Tsien RW. Multiple structural elements in voltage-dependent Ca2+ channels support their inhibition by G proteins. Neuron. 1996;17:991–1003. doi: 10.1016/s0896-6273(00)80229-9. [DOI] [PubMed] [Google Scholar]
  47. Zhou J, Shapiro MS, Hille B. Speed of Ca2+ channel modulation by neurotransmitters in rat sympathetic neurons. Journal of Neurophysiology. 1997;77:2040–2048. doi: 10.1152/jn.1997.77.4.2040. [DOI] [PubMed] [Google Scholar]

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