Transmitter-mediated inhibition of N-type calcium channels in sensory neurons involves multiple GTP-binding proteins and subunits

Neuron, Vol. 14, 191-200, January,1995, Copyright© 1995 by Cell Press

Transmitter-Mediated Inhibition of N-Type Calcium Channels in Sensory Neurons Involves Multiple GTP-Binding Proteins and Subunits Maria Diversd-Pierluissi,* Paul K. Goldsmith,t and Kathleen Dunlap* *Departments of Physiology and Neuroscience Tufts University School of Medicine Boston, Massachusetts 02111 tMetabolic Diseases Branch National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland 20892

Summary The modulation of voltage-activated Ca 2+ channels by neurotransmitters and peptides is very likely a primary means of regulating Ca2÷-dependent physiological functions such as neurosecretion, muscle contraction, and membrane excitability. In neurons, N-type Ca 2÷ channels (defined as ¢o-conotoxin GVIA-sensitive) are one prominent target for transmitter-mediated inhibition. This inhibition is widely thought to result from a shift in the voltage dependence of channel gating. Recently, however, voltage-independent inhibition has also been described for N channels. As embryonic chick dorsal root ganglion neurons express both of these biophysically distinct modulatory pathways, we have utilized these cells to test the hypothesis that the voltage-dependent and -independent actions of transmitters are mediated by separate biochemical pathways. We have confirmed this hypothesis by demonstrating that the two modulatory mechanisms activated by a single transmitter involve not only different classes of G protein but also different G protein subunits. Introduction Inhibition of high voltage-activated (HVA) neuronal Ca2+ channels has been demonstrated for a variety of transmitters and peptides (Tsien et al., 1988). Because of the potential importance of such modulation to the presynaptic regulation of synaptic transmission, much attention has been placed on characterizing its underlying mechanisms. N-type (e)-conotoxin GVlA-sensitive) channels appear to be one prominent HVA Ca2+channel that serves as a target for transmitter-induced inhibition (Kasai et al., 1987; Plummer et al., 1989; Regan et al., 1991; Cox and Dunlap, 1992). The receptor-mediated inhibition of N-type channels requires the activation of GTP-binding (G) proteins (Holz et al., 1986; Rane and Dunlap, 1990). This fact, coupled with the widespread difficulty in identifying other necessary intermediates (Piummer et al., 1989, 1991; Bley and Tsien, 1989; Bernheim et al., 1991 ; Diverse-Pierluissi and Dunlap, 1993), has led to the notion that N-channel inhibition is produced by a direct G protein-Ca2+channel interaction (Hille, 1992).

Such direct action of G protein subunits has figured prominently in analytical models developed to describe N-channel modulation (Elmslie et al., 1990; Boland and Bean, 1993; Golard and Siegelbaum, 1993). In these models, the activated G protein shifts channels to a closed state from whichthey open slowly (at moderate depolarizations), resulting in the commonly observed slowing of the activation kinetics (Marchetti et al., 1986; Wanke et al., 1987; Bean, 1989; Grassi and Lux, 1989; Kasai and Aosaki, 1989). Depolarizing conditioning pulses reverse the action of the G protein by recruiting modulated channels to the normal control state from which they open rapidly. Although the models accurately describe a portion of the transmitter-mediated effects, they leave a significant fraction of the inhibition unaccounted for. In most cases, the voltage-dependent reversal of transmitter-mediated inhibition is incomplete; typically, 20%-30% (or more) of the inhibition remains even at strongly depolarized potentials (Grassi and Lux, 1989; Elmslie et al., 1990; Beech et al., 1992; Boland and Bean, 1993; Formenti et al., 1993; Zhu and Ikeda, 1993). In addition, a slowing of the activation kinetics has not been uniformly observed in macroscopic (Dunlap and Fischbach, 1981; Bernheim et al., 1991; Beech et al., 1991, 1992; Diverse-Pierluissi and Dunlap, 1993; Formenti et al., 1993) or single-channel (Delcour and Tsien, 1993; but see Elmslie et al., 1994) recordings. Such "steady-state" modulation, unaccompanied by changes in activation kinetics, is voltage independent (Luebke and Dunlap, 1994). The apparent discrepancies among preparations (and among different transmitters within the same preparation) are likely due to the presence of multiple, complex modulatory processes that coexist in cells and that converge at the level of N channels. The existence of multiple modulatory mechanisms raises the issue of their relative importance under variable physiological conditions. A more complete characterization of the biochemical pathways that produce N-channel inhibition is prerequisite to investigating this idea. Embryonic chick sensory neurons have proven to be an excellent experimental preparation in which to dissect the multiple mechanisms underlying N-channel modulation. The HVA Ca2+ current in these neurons is almost entirely N-type, as it is inhibited 100% by e)-conotoxin GVIA in cells tested within 24 hr and >85% in cells tested within 3 days of plating. This current is a specific target for modulation produced by norepinephrine (NE) and 7-aminobutyric acid (GABA; Cox and Dunlap, 1992). Through distinct receptors (Dunlap, 1981; Canfield and Dunlap, 1984), these two transmitters activate at least two biochemical pathways to inhibit N-channel function--one that involves protein kinase C (PKC; Rane et al., 1989) and a second that does not (Divers~-Pierluissi and Dunlap, 1993). Both transmitters also produce at least two biophysically distinct responses in these neurons--a slowing of the activation kinetics that is voltage dependent (Grassi and Lux, 1989; Kasai and Aosaki, 1989) and a steady-state inhibition that is voltage independent (Luebke and Dunlap, 1994). We

Neuron 192

Table 1. Concentration Dependencies of GABA- and NE-Mediated Inhibition of N Current in Chick Sensory Neurons and the Effects of GTP~,S

Treatment

# Cells Tested

# Cells with Kinetic Slowing

% Inhibition of Total Charge Entry

% Inhibition Associated with Kinetic-Slowing Componenta

Time Constant of Slow Componenta (ms)

500 nM GABA Control 100 ~.M GTPyS

'5 5

0 5

10 ± 2 40 _ 5

0 24 _+ 8

16 _+ 4

1 ~M GABA Control 100 ~.M GTPTS

5 5

3 5

19 _ 3 50 -- 7

5 _+ 3 23 ± 5

10 _ 1 19 ± 5

100 ~M GABA Control 100 p.M GTP~,S

6 6

5 6

35 ± 5 47 ± 5

10 ± 5 23 ± 7

10 ± 2 16 ± 3

Control 100 p.M GTP~,S

4 8

0 5

7 ± 1 25 _ 3

0 11 _ 6

10 _ 1

10 ~M NE Control 100 ~M GTP~,S

10 10

3 10

25 _ 7 37 _+ 6

12 _ 5 11 _ 4

10 _ 2 16 + 4

100 ~M NE Control 100 ~M GTP'yS

8 4

4 4

30 - 8 42 _.+ 7

10 _ 6 25 _ 8

10 ± 3 15 ± 4

1 ~M NE

a Averages calculated only from those cells demonstrating kinetic slowing.

have employed affinity-purified antibodies selective for the (~subunits of particular G protein subtypes and I~y subunits purified from G proteins in turkey erythrocytes to test the hypothesis that slowing of the N-current activation kinetics involves a species of G protein or G protein subunit distinct from that involved in steady-state inhibition. Receptor coupling to multiple G protein subtypes would be one means of bringing about the variety of transmitter effects on ion channels thus far described.

Results Identification of Two Components to Transmitter-Induced Ca2+-Current Inhibition HVA Ca 2+ currents were recorded from cell bodies of embryonic chick dorsal root ganglion neurons within 1-3 days in vitro. Previous results have demonstrated that these currents are blocked 8 5 % - 1 0 0 % by 1 ~M co-conotoxin GVIA (Cox and Dunlap, 1992), thus defining them as N-type. Step depolarizations to 0 mV from a holding potential of - 8 0 mV produced inward Ca 2+ currents that activated rapidly in control cells with a time course approximated (after the first 0.5 ms) by a single exponential function ('~ = 1.5 -+ 0.6 ms; n = 47). Total charge entry during 100 ms pulses averaged 0.13 _ 0.04 nC in these cells. Application of 100 ~.M NE produced an average 37.5% -4- 8% reduction in charge entry in 30 neurons studied; similarly, 100 I~M GABA inhibited N currents by an average of 45% _ 6% (n = 30). As significant interplating variability in the magnitude of transmitter-mediated inhibition was observed, measurements in a given experimental group were always compared with those in untreated control cells from the same plating on the same day. These reductions in charge entry were most often, but

not always, accompanied by a slowing of the activation kinetics, manifest (as previously described) by the appearance of a second, slower exponential component of the current activation (Marchetti et al., 1986; Wanke et al., 1987; Bean, 1989; Grassi and Lux, 1989; Kasai and Aosaki, 1989). The time constant of this slow component ranged from 10 to 20 ms among the 45 cells tested (average 16 _+ 5 ms). The presence of such kinetic slowing (and its contribution to the overall percentage inhibition) could also be detected with the current subtraction method outlined in Experimental Procedures. Since the kineticslowing component subsides with time during a depolarizing pulse, its contribution to the total inhibition declines with increases in pulse duration. With the long pulses used in our experiments (100 ms), the kinetic-slowing component comprised approximately 10% of the total inhibition of charge entry produced by 100 I~M of either transmitter.

Concentration Dependence of the Steady-State and Kinetic-Slowing Components With standard intracellular solutions, GABA produced both kinetic slowing and steady-state inhibition, but the two components showed different concentration dependencies (Table 1). At low concentrations (<1 p.M), only steady-state inhibition was observed. Above 1 ~M GABA, kinetic slowing could also be detected, with near maximal effects evoked at 100 I~M. Testing with 1 and 100 I~M GABA, 60% and 83% of the neurons, respectively, responded with a slowing in the activation kinetics (Table 1). NE produced concentration-dependent inhibition as well. As with GABA, lower concentrations of NE (<10 I~M) evoked only steady-state inhibition; kinetic slowing developed at higher concentrations (t>10 pM) in a subset of cells. The higher the NE concentration, the higher the per-

N-Channel Inhibition 193

PKC-Dependent Inhibition Is Confined to the Steady-State Component

A

•J75

pA 25 pA 10 ms

B

NE

Figure 1. Staurosporine Blocks NE-Mediated, Steady-State Inhibition, but Not Its Slowing of the Activation Kinetics Ca2÷current was measured using tight-seal, whole-cell recording before and after application of 100 ~M NE (as marked). The holding potential was -80 mV, and the cells were depolarized every 10 s to 0 mV for 100 ms. Traces were recorded from cells in normal external solution with (B) or without (A) 10 I~M staurosporine (added to bath 10 min prior to recording).

centage of neurons displaying kinetic slowing (30% of the cells tested with 10 I~M and 50% of the cells tested with 100 t~M). These results demonstrate that GABA and NE are capable of activating both steady-state and kineticslowing components of inhibition; however, GABA receptors appear to be more consistently coupled to the pathway producing a change in activation rate. The efficacy of the transmitters, as well as their efficiency in activating kinetic slowing, could be enhanced by intracellular application of GTPTS (100 I~M). As summarized in Table 1, all concentrations of NE and GABA tested in the presence of the nucleotide produced kinetic slowing. This suggests that the absence of kinetic slowing produced under normal conditions by low concentrations of transmitter results from the short lifetime and/or low levels of active G protein intermediates.

Previous results from our laboratory have shown that NEinduced inhibition of Ca 2+current is mimicked by PKC activators (Rane and Dunlap, 1986) and diminished by intracellular application of specific inhibitors of PKC (Rane et al., 1989). To test for a differential involvement of PKC in the kinetic slowing and steady-state inhibition of Ca 2+ currents produced by NE, the effects of 100 I~M transmitter were measured in dorsal root ganglion neurons bathed in extracellular solution with or without 10 I~M staurosporine (an inhibitor of PKC). Cells in normal extracellular solution displayed a mean Ca 2÷ current of 0.3 _+ 0.04 nA at peak and 0.21 _+ 0.03 nA at 100 ms (n = 7). NE produced robust inhibition of these currents (average 45% _+ 6%; n = 7; Figure 1A), of which approximately 15% was associated with kinetic slowing (~ = 12 _+ 0.5 ms). Cells from the same plating bathed in staurosporine (Figure 1B) exhibited Ca 2* currents of similar amplitude (0.28 _+ 0.06 nA at peak; 0.20 _+ 0.07 at 100 ms), but the average percentage inhibition produced by NE was reduced to 21% _+ 3% (n = 5). The kinetic-slowing component (~ = 12.5 _+ 1.0 ms) now comprised approximately 80% of the total modulation. This differential inhibition by staurosporine suggests that the two kinetically distinct components of the attenuation produced by NE are mediated by separate biochemical pathways. In contrast, it has been demonstrated previously that Ca2÷-current in hibition mediated by GABA is entirely PKC independent (DiversePierluissi and Dunlap, 1993).

Steady-State Inhibition and Kinetic Slowing Are Mediated by Separate G Proteins To test for the differential involvement of G protein types in kinetic slowing and steady-state inhibition, we used two antibodies, each of which blocks receptor-mediated G protein activation of a single class of G protein a subu n i t - ~ o or o.i (Cerione et al., 1988; McKenzie et al., 1988; Spiegel, 1990). The affinity-purified antibodies (1:100 dilution) were injected into dorsal root ganglion neuron cell bodies in a buffer solution containing 0.1% fluoresceinconjugated dextran. Approximately 1 hr following injection, the neurons were identified using fluorescence optics, whole-cell Ca 2+ currents were recorded, and the effects of NE or GABA were measured. Control experiments demonstrated no effects after injecting vehicle alone, in the absence of antibodies (n -- 20; data not shown). In addition, we found no effects of the antibodies

Table 2. Effect of Antibodies on Sensory Neuron Ca2+Currents Injected Ca2÷-Current Characteristics

Uninjected (n = 15)

Anti-m (n = 13)

Anti-no (n = 19)

Peak amplitude (nA) Amplitude at 100 ms (nA) Activation rate (ms)

-1.23 - 0.3 -0.8 ± 0.03 1.5 ± 0.4

-1.03 -+ 0.4 -0.67 -+ 0.02 1.57 ± 0.7

-1.08 --_ 0.5 -0.76 - 0.05 1.4 ___0.13

a Significantly smaller than control (.05 < p < .1). This indicates a slight alteration in the rate of inactivation between control and anti-m-injected cells. This difference cannot explain the effects of anti-m on the NE-induced steady-state inhibition.

Neuron 194

NE

GABA

GTPyS

charge entry in control cells (n = 10) and a 21.5% ± 9% inhibition in cells injected with AS/7 (n = 8). When the steady-state and kinetic-slowing components of charge entry were analyzed separately, AS/7 was found to inhibit the steady-state component selectively, leaving kinetic slowing unaffected (Figure 2; Figures 3B and 3C). This was confirmed by measurements of the activation time course; the slow time constant for current activation was 20.8 _+ 4 ms in control and 20 _ 2 ms in cells injected with AS/7. When 100 #M GABA was applied, control cells respondedwith an average 48% ± 10% (n -- 10) reduction in charge entry. However, in contrast to the results obtained with NE, neurons injected with AS/7 were as responsive to GABA as control cells, showing a comparable 49% ± 1.4% inhibition of charge entry. The proportions of steady-state to kinetic-slowing components, as well as the slow time constant for activation, were also similar in the two groups (Figure 3). These results rule out an action of G~ in GABA-mediated inhibition of N current and further support the notion that the effect of anti-(~ is specific. To test for the involvement of the Go class of proteins in NE- and GABA-induced Ca2+-current inhibition, we injected dorsal root ganglion neurons with G O / l , which recognizes (Zo (Goldsmith et al., 1988). As with AS/7, GO/1 produced no effect on basal Ca 2+currents (Table 2). Unlike AS/7, however, GO/1 interfered with effects of both transmitters, although differences between NE and GABA were observed. The total inhibition produced by GABA in 10 cells injected with GO/1 was 16% ± 6% (compared with 48% ± 10% in the 10 control cells, described above). Virtually all of this inhibition was associated with the steady-state component; the kinetic slowing produced only 0.5% ± 0.7% inhibition on average (Figure 3). Thus, GO/1 reduced the steady-state component by approximately half and eliminated the kinetic-slowing component altogether. When cells were tested with NE, we found that GO/1 again eliminated the transmitter-induced kinetic slowing (similar to results with GABA). Unlike the effects of GABA, however, the steady-state inhibition produced by NE was not reduced in the presence of GO/1. In fact, the steadystate inhibition increased significantly (Figure 3B). The modulatable current was still carried through N channels, as it was all blocked by 1 I.LM co-conotoxin GVIA (data not shown). Somewhat surprisingly, the total inhibition of

control

anti-o~o

anti-o~

20 m s

Figure 2. Anti-G Protein Antibodies Differentially Block the Two Components of Transmitter-Mediated Inhibition of Ca2+Current Each panel shows two superimposed Ca2+ currents evoked by 100 ms step depolarizations from -80 to 0 inV. Only the first portion of each current is shown to enhance visualization of the activation kinetics. Each column shows representative responses of separate cells to extracellular application of 100 I~M NE or GABA or intracellular application of 100 #M GTPyS (as marked). Each row contains responses from uninjected cells (control) or cells injected with a solution containing 20 pg/ml affinity-purified antibodies against e~ or ai2 (AS/7) or (1o(GO/l). Currents have been arbitrarily scaled to approximately equal heights; vertical calibration is therefore not included. The peak current amplitudes ranged from 0.8 to 2.4 nA. The small current illustrating the effect of NE in an anti-(zrinjected neuron was taken from a cell in a plating that exhibited small control currents.

on the mean amplitude of control Ca 2+ current nor on its rates of activation or inactivation (Table 2). We therefore used uninjected cells to establish control responsivity in most experiments and compared their responses with those of antibody-injected cells from the same plating (see Experimental Procedures for additional discussion of this point). As described below, the effects of the antibodies were specific for one or the other modulatory component, further validating the use of uninjected controls and obviating the need to test preimmune serum as a control for specificity. AS/7, which recognizes both (~il and m2 (Falloon et al., 1986), was without effects on basal Ca 2+ current (Table 2); however, the antibody produced a selective reduction in responsivity to NE (Figure 2; Figure 3A). At 100 I~M, NE produced an average 56% ± 7% inhibition in total

A

8O¸

,,5 0 "6

B

8o-

Total Charge Entry

60, (lO)

C

80-

Steady-state Component

Kinetic-slowing Component

(10) 60-

60-

40.

40-

40-

20.

20-

20-

g :6

0

~.~.~

~.~

o

G

8 ~ABA

NE

8 GABA

NE

GABA

NE

Figure 3. Histograms Summarizing the Differential Effects of Anti-G Protein Antibodies on Steady-State Inhibition and Kinetic Slowing Produced by GABA and NE Plotted are the percentage inhibitions of total charge entry (A), the steady-state component (B), and the kinetic-slowing component (C) (separated as described in Experimental Procedures). Error bars represent SEM. In (A), numbers in parentheses indicate total number of cells in each sample; these same cells were used to calculate the percentage inhibition associated with steady-state inhibition and kinetic slowing shown in (B) and (C), respectively.

N-Channel Inhibition 195

A2 g 8°l

A1 ~.

(15)

1.0~ ~ + 0 + 0 2

o 0.7 t • 04

\

~ 20

t 0

250

500 time (s)

BI

B3

B4

B2

~" ~

O~- (51 0.02 0.06 0.2 2 20 P3'concentration (nM)

750

NE

I

PKCl*injected

]3y(30s) Figure 4. PurifiedJ}ySubunitslnhibit Ca2+Current in a Concentration Dependent Manner (A1) Ca2÷current (normalized to the maximum) is plotted as a function of time for 4 different cells, each dialyzed with the concentration of ~y (in riM) shown to the right of each trace, Data have been corrected for the average rate of current rundown determined in control cells dialyzed with the maximum concentration of vehicle (data not shown). The inset shows two superimposed traces to illustrate the inhibition produced after 8 rain of dialysis with 20 nM ~y. Control record was taken 10 s after whole-cell access was achieved, before ~y had much effect on the current. (A2) Histogram summarizing the concentration-response for 137mediated inhibition of total charge entry. Error bars represent SEM; numbers in parentheses indicate total number of cells in each sample. (B1-B4) Currents from sensory neurons dialyzed with normal intracellular solution (B1) or intracellular solution containing 20 nM turkey erythrocyte I}Y (B2-B4). Traces taken in the presence of 100 ~M NE are so marked (B~ and B2). Dialysis of [By (B2-B4). Superimposed traces were taken soon after achieving whole-cell acess (before ~y had much effect on Ca2÷ current) or after I~1'effects were maximal (times indicated in parentheses); cell in B4 had been injected with PKCI 19-31 10 min prior to dialysis with ~'y.

charge entry remained constant; the increased steadystate inhibition was balanced by a reduction in the kineticslowing component. Together, these results suggest that the G proteins mediating NE-induced inhibition of sensory neuron Ca 2+ currents differ in some respects from those mediating GABA-induced inhibition. In addition, they argue that the two G protein classes differentially control the multiple modulatory components.

GTPyS-Induced Inhibition of Ca 2+ Current Is Not Blocked by the Antibodies To control for the possibility that G protein antibodies affect alterations in the susceptibility of N channels to modulation by a mechanism other than block of receptor-mediated G protein activation, we tested the ability of GTPyS to inhibit Ca 2+currents when passively introduced into sensory neurons. The nucleotide analog was added at 100 I~M to the intracellular solution; it diffused into the cells following whole-cell access. As is well known, GTPyS inhibits N currents in a manner indistinguishable from that of the transmitters (Kasai and Aosaki, 1989; Elmslie et

al., 1990; Divers~-Pierluissi and Dunlap, 1994), bypassing receptor-mediated activation of G proteins (for review, see Gilman, 1987). Comparison of GTPyS-mediated inhibition of N currents in the presence or absence of intracellular G protein antibodies revealed no differences in the extent of inhibition (Figure 2) or its rate of onset (data not shown), suggesting that the nucleotide is able to activate antibodybound G proteins directly and that N channels remain susceptible to inhibitory modulation despite the presence of antibody.

Purified J~y Dimers Activate the Steady-State Component Selectively As the antibodies employed in our studies block receptormediated G protein activation (Cerione et al., 1988; McKenzie et al., 1988), results of the injection experiments do not identify which subunit ((~, I}Y, or both) is responsible for the effector response. To address this issue more directly, we have used I}Y subunits purified from turkey erythrocytes (Boyer et al., 1989). The pu rifled protein was added to the patch-pipette solution and introduced into the cytosol by passive diffusion during whole-cell recording. In 40 cells tested, I}7 produced a concentration-dependent inhibition of N current (Figure 4A2). The time required for these effects to reach a steady state varied as an inverse function of concentration (Figure 4A1). The highest concentration studied (20 nM) produced an average maximal inhibition of 65% _ 3% in - 5 min. Interestingly, the inhibition was generally confined to the steady-state component; in 18/20 cells studied with 20 nM 137, no measurable kinetic slowing could be observed (Figure 4A1, inset). In the remaining 2 cells, a small effect on the activation kinetics was seen; double exponential fits to the activation phase revealed a slow component with a "~ = 11.0 ms. (This kinetic slowing comprised 12% of the total inhibition.) No effects of the vehicle were detected at any concentration employed, and heat-inactivation of the 137 preparation (80°C, 30 min) abolished its inhibitory activity (data not shown). To determine whether l~Y and the transmitters inhibit Ca2+current via similar pathways, we first tested the ability of 157to occlude responses to NE and GABA. Cells were dialyzed with internal solution with or without 20 nM 137 for 10 min prior to application of 100 I~M transmitters. Cells dialyzed with control solutions responded to NE with a 40% __. 5% reduction in Ca 2+ current (which averaged 1.0 -+ 0.05 nA prior to NE application). Introduction of I}7 completely eliminated the steady-state inhibition produced by NE. In 7/14 cells tested, however, NE still elicited a slowing of the activation kinetics (-c = 13 ms; Figure 4B2), the same proportion that exhibited kinetic slowing under control conditions (Table 1). Similar results were observed with GABA. Control cells responded to 100 I~M GABA with a 45% -+ 7% inhibition. Responses to GABA were reduced to 10% _+ 4% in the presence of I}Y. Again, the effects of I}7 were confined to the steady-state component; the ~ for kinetic slowing produced by GABA (in the presence of !37) was 13 _+ 5 ms (no different from that measured in control cells). The selective occlusion produced by I}7 implies, but

Neuron 196

does not prove, that the G protein subunit and the transmitters activate common pathways to produce steady-state inhibition. However, interpretation of the results is complicated by the fact that steady-state inhibition saturates at - 6 0 % - 7 0 % (Divers~-Pierluissi and Dunlap, 1994), regardless of the modulatory pathway activated. Maximal activation of one pathway, therefore, occludes inhibition by a second pathway even when the two are biochemically distinct. To provide a more definitive identification of the pathway mediating 6y-induced inhibition, we took advantage of the fact that the pseudosubstrate inhibitor of PKC (PKCI 19-31) selectively blocks the NE-induced but not the GABA-induced steady-state inhibition of N current (Rane et al., 1989; Divers~-Pierluissi and Dunlap, 1993). We first pressure injected a series of neurons with a solution containing 3 I~M PKCI 19-31 (and fluorescein-conjugated dextran), identified the injected cells by fluorescence 10 min later, and studied the effects 20 nM 13y applied by passive diffusion through the patch pipette. In contrast to the average 65% reduction in Ca 2+ current produced by ~, in control cells, no inhibition was observed in cells previously injected with PKCI 19-31 (n = 5; Figure 4B4). Thus, the mutual sensitivity of NE- and ~y-mediated modulation to inhibitors of PKC is the strongest argument that the two share a common pathway. The resistance of GABA-mediated inhibition to PKCI 19-31, on the other hand, argues that 15y and GABA work via separate pathways and that the occlusion we observed is most likely a function of a fixed upper limit imposed on N-channel modulation.

Discussion Our results demonstrate that transmitter-mediated modulation of N-type Ca 2÷ channels in embryonic chick dorsal root ganglion neurons involves multiple biochemical pathways selectively activated by distinct G protein types. This finding enhances our understanding of the observation that kinetic slowing and steady-state inhibitioh can be separated on the basis of their voltage dependence; the steady-state inhibition is voltage independent, whereas kinetic slowing is reversed at depolarized potentials (Luebke and Dunlap, 1994). Despite the fact that these two inhibitory components show similar rates for onset, recovery, and desensitization, they likely involve distinct biochemical pathways as shown here. In other words, the biophysically distinct mechanisms of modulation produced by a single transmitter appear to result from the simultaneous activation of multiple pathways that converge to inhibit N-channel function.

How Many Pathways Inhibit N-Channel Function? The two biophysically separable components we have identified appear themselves to be heterogeneous, as our results argue for a minimum of three different biochemical pathways. The results are summarized schematically in Figure 5. First is a steady-state inhibition produced by G~-mediated activation of PKC. In embryonic chick dorsal root ganglion neurons, this pathway is activated by NE

depolarization e

GO~1

"~

Go - - ?'~ke kinetic slowing

0~2 Receptors AS~7

G - - R-~_ D~-- Dk'r steady-state ,,~ . L v - - . . . . -- inhibition purified protein from turkey erythroeyte

GO/1

GABAs Receptors

"~

depolarization

G° - - "~'~

kinetic slowing steady-state inhibition

Figure 5. Schematic Diagram Summarizing the Three lnhibitory Pathways That Modulate N-Channel Function in Chick Sensory Neurons Reagents or protocols used to block selectively or activate particular pathways are shown in italics.

but not GABA. Our experiments with purified turkey erythrocyte ~, imply further that the activation of PKC likely results from a 13y-induced activation of phospholipase C (and the consequent production of diacylglycerol), as has been reported in many preparations (Boyer et al., 1989, 1992; Blank et al., 1992). This pathway may be expressed in a variety of other cell types as well, where P KC activators (such as phorbol esters and diacylglycerol analogs) inhibit HVA Ca 2+ current without altering its activation kinetics (Doerner et al., 1988; Ewald et al., 1988; Boland et al., 1991). The second pathway is a steady-state inhibition independent of G~ and PKC and appears to require, at least in part, the activation of Go. The fact that injection of anti-m or PKC inhibitors eliminates only NE-induced inhibition of Ca 2÷ current (leaving GABA responses unaffected) suggests that this second pathway is selectively activated by GABA. It is voltage independent as well (Luebke and Dunlap, 1994) and may be similar to the PKC-independent inhibition that persists even at depolarized potentials in a number of preparations (Grassi and Lux, 1989; Elmslie et al., 1990; Beech et al., 1992; Boland and Bean, 1993; Formenti et al., 1993; Zhu and Ikeda, 1993). As GABA and NE responses are both partially inhibited by GO/l, it is curious that voltage-independent, steady-state inhibition was observed only for GABA. This might suggest the involvement of two different Go subtypes (both recognized by GO/l) that are selectively coupled to the different receptors (Kleuss et al., 1991). Alternatively, m3 is known to exhibit slight cross-reactivity with GO/l; it is possible, therefore, that G~3 mediates part of the modulatory response produced by one, but not both, of the transmitters. A third pathway is activated by both NE and GABA, producing a Go-mediated slowing of the activation kinetics. This inhibition is voltage dependent (Kasai and Aosaki, 1989; Luebke and Dunlap, 1994). It is also PKC independent and may be the most commonly observed pathway to modulate neuronal Ca 2+currents. A number of transmitters and peptides (including, but not limited to, dopamine, acetylcholine, luteinizing hormone releasing hormone, and adenosine) have been reported to evoke this type of

N-Channel Inhibition 197

inhibition of HVA Ca 2+ currents (Marchetti et al., 1986; Wanke et al., 1987; Bean, 1989; Grassi and Lux, 1989; Kasai and Aosaki, 1989; Elmslie et al., 1990). A potential role for PKC as a mediator of these transmitter-activated pathways has historically been ruled out in such cases by demonstrating the inefficacy of PKC inhibitors against transmitter-mediated inhibition (Plummer et al., 1991 ; Bley and Tsien, 1989; Bernheim et al., 1991). Abundant evidence has implicated the involvement of Go in HVA Ca2÷-channel modulation. The first G protein reconstitution experiments demonstrated that Go is more effective than G~ in restoring opioid-induced inhibition of Ca 2÷ currents in pertussis toxin-treated neuroblastoma cells (Hescheler et al., 1987). Similarly, no is more effective than ~ in reconstituting functional neuropeptide Y-mediated inhibition of Ca 2÷ current in rat sensory neurons (Ewald et al., 1989) and rat sympathetic neurons (Caulfield et al., 1994). Antibodies to no blocked inhibitory modulation in NG108-15 cells (McFadzean et al., 1989), and intranuclear injection of Go-selective antisense oligonucleotides into rat pituitary GH3 cells showed that specific subtypes of Go mediate the inhibition of dihydropyridinesensitive Ca 2÷ current by somatostatin and carbachol (Kleuss et al., 1991). In more recent reports, N channels have been noted as a primary target for Go (MenonJohansson et al., 1993). The possible coexistence of multiple pathways that modulate N current, involving more than one type of G protein, has not been considered previously. The variable components of these pathways may not be limited to G proteins and PKC. It is unknown, for example, whether the activation of G protein subtypes occurs through a single GABA or NE receptor type (i.e., involving receptor-G protein crosstalk) or whether the GABAB (Dunlap, 1981) and a2-adrenergic (Canfield and Dunlap, 1984) receptors that mediate these responses are themselves heterogeneous. Subtypes of GABAB receptor identified by their sensitivities to antagonists have been described (Bowery et al., 1990), and the structural and pharmacological heterogeneity among the ~2-adrenergic receptors is well known (Summers and McMartin, 1993; MacKinnon et al., 1994). Likewise, multiple, structurally distinct N-type Ca 2÷ channels may mediate the different modulatory responses separately. Multiple transcripts have been described for class-B, Ca2÷-channel ~ subunits; when expressed in mammalian cells, the resultant currents are sensitive to (o-conotoxin GVIA, identifying them as N-type (Williams et al., 1992; Fujita et al., 1993). It can be further speculated that subunit composition and/or posttranslational modification of the Ca2÷-channel complex predisposes the N channels to one type of modulation or another. Analysis of these possibilities at a physiological level awaits the development of agonists or antagonists that can selectively discriminate among N-channel and receptor subtypes.

Identification of the Active G-Protein Subunits The activation of multiple modulatory pathways appears to involve both ~ and 15"ysubunits. Our results argue that the PKC-dependent, steady-state inhibition is mediated by 157, most likely acting via phospholipase C (Boyer et

al., 1989, 1992; Blank et al., 1992). As the effects of 15~, on N current are eliminated by PKCI 19-31, we conclude that ~7's action is mediated entirely through the phospholipase C/PKC pathway, rather than through a direct effect on N channels, as has been suggested for the acetylcholine-mediated modulation of inward rectifier potassium channels in cardiac myocytes (Logothetis et al., 1987; Ito et al., 1992). The fact that only the 15~,associated with a~ (and not that associated with ~o) evokes PKC-dependent, steady-state inhibition further suggests selectivity in the ~,-phospholipase C interaction. Scant support for this notion can be found in the biochemical literature, however. Studies of phospholipase C activation by 13~'in vitro indicate little difference between a wide variety of 15~,subunit combinations (Boyer et al., 1994). Despite this, it remains possible that investigations on intact cells may reveal a level of specificity that is disrupted in the biochemical reconstitution experiments. In addition to a possible variable efficacy among the different 15~,subunits, the differential action of the two antibodies used in our studies may imply that localization of (or selective coupling between) particular receptors, G proteins, and effector molecules provides a mechanism for specificity. Which subunits mediate the PKC-independent modulation? Identification of the a and ~, subunits present in the G proteins expressed in chick sensory neurons will allow direct tests of their respective roles. The selectivity of turkey erythrocyte 157(a mixture of 151~'7and 152~'~)for the PKCdependent, steady-state inhibition suggests that attenuation of Ca 2+current by NE and GABA is likely not mediated exclusively by 137 subunits. Unless the modulatory pathways for Ca 2+ current involve targets for 137that are differentially recognized by the various subunit combinations, therefore, our results would suggest a role for ~ subunits as mediators of kinetic slowing and PKC-independent, steady-state inhibition.

Interactions of Modulatory Pathways Our results further suggest that kinetic slowing and steadystate inhibition can each be activated in isolation. Their effects on Ca 2÷ channels are not entirely independent, however, as selective elimination of one type of inhibition can potentiate the efficacy of the remaining inhibition. For example, we found that the steady-state inhibition produced by NE was enhanced under conditions in which kinetic slowing was blocked by anti-no (Figure 3B). The mechanism underlying this enhancement is unknown, but interactions of the modulatory mechanisms at the level of the channels seems likely. In other words, biochemical modifications of N channels associated with one pathway may reduce the susceptibility of the channel to further modification via an alternative pathway.

Physiological Significance of Multiple Modulatory Mechanisms Steady-state and kinetic-slowing components might be expected to play distinct roles in regulating cellular function and/or intercellular communication. For example, because of its voltage dependence (Bean, 1989; Elmslie et al., 1990), transmitter-mediated kinetic slowing would be

Neuron 198

p r e d i c t e d to be p h a s i c u n d e r c o n d i t i o n s o f repetitive firing, s u b s i d i n g with s u c c e s s i v e stimulation. B y contrast, s t e a d y - s t a t e inhibition, by virtue of its v o l t a g e i n d e p e n d e n c e , w o u l d likely persist, e v e n u n d e r c o n d i t i o n s o f high f r e q u e n c y stimulation. T h u s , the s e l e c t i v e activation o f individual p a t h w a y s by p a r t i c u l a r t r a n s m i t t e r s a n d the differential e x p r e s s i o n of the p a t h w a y s in individual cells a r e both potential m e c h a n i s m s t o p r o v i d e e i t h e r a tonic or p h a s i c p h y s i o l o g i c a l bias. T h e r e is p r e c e d e n t for both o f t h e s e a l t e r n a t i v e s in emb r y o n i c chick d o r s a l root g a n g l i o n n e u r o n s . T h e inhibition o f N c u r r e n t s by a d e n o s i n e , f o r e x a m p l e , h a s b e e n d e m o n s t r a t e d to be p u r e l y v o l t a g e d e p e n d e n t , with c o m p l e t e rev e r s a l of t r a n s m i t t e r action at d e p o l a r i z e d p o t e n t i a l s (Kasai a n d A o s a k i , 1989). By contrast, o u r w o r k (Divers~Pierluissi a n d D u n l a p , 1993; L u e b k e a n d D u n l a p , 1994) d e m o n s t r a t e s that s o m e s e n s o r y n e u r o n s r e s p o n d to N E a n d G A B A s o l e l y with s t e a d y - s t a t e inhibition, as if s u c h cells l a c k e d the kinetic s l o w i n g p a t h w a y . T h e d i f f e r e n t i a l e x p r e s s i o n of t o n i c a n d p h a s i c m o d u l a t o r y c o m p o n e n t s m a y thus o f f e r an i m p o r t a n t d e g r e e o f flexibility in the regulation o f C a 2 + - d e p e n d e n t p h y s i o l o g i c a l p r o c e s s e s s u c h as n e u r o t r a n s m i t t e r release. Electrical activity p r o v i d e s the o n / o f f switch to t r i g g e r the p h y s i o l o g i c a l event, but the s a t u r a b l e , m o d u l a t o r y p a t h w a y s act as d i m m e r s w i t c h e s to m o d i f y r e s p o n s e m a g n i t u d e .

Experimental Procedures Cell Culture Dorsal root ganglia were dissected from 11- to 12-day-old chick embryos. Tissue was incubated at 37°C for 50 min in Ca2÷-and Mg2+-free saline and dissociated by trituration with a fire-polished Pasteur pipette. Cells were plated in plastic, collagen-coated 35 mm tissue culture dishes (Falcon 3001) and grown in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 100/0 heat-inactivated horse serum, 5% chick embryo extract, 50 U/ml penicillin, 50 i~g/ml streptomycin, and nerve growth factor. Injection Methods G protein antibodies were affinity purified according to published methods (Gierschik et al., 1986) and stored at -70°C as a stock solution of 0.8 mg/ml. Injection solutions were made by dilution of the stock solution into standard internal recording solution (see below) containing 0.1% fluorescein-conjugated dextran. Injection pipettes were back-filled with - 3 I~1 of the injection solution (containing 20 p~g/ml antibody) and mounted on an Eppendorf 5171 microprocessorcontrolled manipulator. Cells on the stage of a Zeiss IM inverted microscope were visualized with phase-contrast optics, and an Eppendorf 5272 automated microinjection system was employed to inject sensory neuron cell bodies with the antibody. Precise quantitation of injected volumes was not made; standardized injection pressures (30-50 HPa) and times (0.3 s) were employed to minimize potential variation in injection volumes between cells and experiments. Electrophysiological experiments were performed 1 hr later on injected cells identified by epifluorescence microscopy. Little quantitative difference was observed between Ca2. currents recorded from uninjected control cells and cells injected with G protein antibodies (see Table 2); the injected cells were, however, more fragile than their uninjected neighbors, reducing the frequency of successful recordings. Significant interplating variability in the magnitude of transmitter-mediated inhibition required that responses from several control cells ( - 5) be taken for comparison with the experimental group on each day of recording. As the frequency of success in recording was diminished by injection alone, and since the antibody per se was innocuous against basal Ca 2* currents, we chose to use uninjected cells for control data to maximize the number of cells from which data could be collected on any given day.

Electrophysiological Methods Cells were used for recording 1-3 days after plating. The pipette internal solution contained 150 mM CsCI, 10 mM HEPES, 5 mM MgATP, and 5 mM bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA). The external saline solution contained 133 mM NaCI, 1 mM CaCI2, 0.8 mM MgCI2, 10 mM tetraethylammonium chloride, 25 mM HEPES, 12.5 mM NaOH, 5 mM glucose, and 0.3 p.M tetrodotoxin (Sigma). All salts were obtained from Fluka. Pipettes were prepared from Fisher microhematocrit capillary tubes. Pipette resistances prior to forming high resistance seals ranged from 1.5-2.5 MQ. A List EPC-7 amplifier (Medical Systems) was used to record Ca 2+ currents in the tight-seal, whole-cell configuration. Data were acquired at 10 kHz using an Atari Mega 4 STE computer (software from Instrutech Corporation, Great Neck, NY). A standard P/4 protocol was used for leakage subtraction of the currents prior to analysis. Currents from population studies are reported as the mean _+ standard error. Data Analysis Digitized currents were analyzed on a Macintosh computer using 4gor (Wave Metrics, Lake Oswego, OR). Two methods were employed to separate kinetic slowing from steady-state inhibition produced by the transmitters and GTP'yS. The activation phases of the currents were characterized by fitting them to an exponential function of the form I(t) = A exp -~T, where the rate of activation is inversely proportional to ~. Two exponential components were required to describe currents that exhibited transmitter-induced kinetic slowing; these currents were best fit to an equation of the form I(t)= A~ exp-"~1+ A2exp ~2. The fast component (~1) was indistinguishable from ~ of control cells. The magnitude of kinetic slowing was proportional to ~2. This slow component was 95% complete in - 6 0 ms. In the second method, control currents were normalized to transmitter-modulated currents at 95 ms (when the kinetic-slowing component was largely completed). The integral of the difference between the control and normalized currents represents the charge (nC) associated with the steady-state component, while the difference between the normalized and modulated currents represents the charge associated with the kinetic-slowing component. Pharmaceuticals and Their Application Purified turkey erythrocyte ~,y (Jos~ Boyer and T. Kendall Harden, University of North Carolina) was supplied in a buffer containing 20 mM Tris, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM benzamidine/phenylmethylsulfonyl fluoride, 0.05% Lubrol, and 200 mM NaCI. Dilutions were made into standard intracellular saline, and the protein was applied intracellularly by passive diffusion from the recording pipette. Staurosporine (LC Services) was prepared as a stock solution in dimethylsulfoxide and stored in aliquots at -20°C. GTPyS (Fluka), GABA (Sigma), and NE (D,L-arterenol, Sigma) were prepared as stock solutions in distilled water. GTPyS (100 raM) and GABA (10 mM) were stored at -20°C and 4°C, respectively, while norepinephrine stocks (10 mM) were prepared fresh on the day of the experiment. Experimental solutions were prepared prior to each experiment by dilution of the stocks into intracellular saline. GTPyS (100 I~M)was applied intracellulady through patch pipettes; the transmitters (100 I~M) were applied extracellularly by pressure ejection from puffer pipettes of 2-3 p.m tip diameter. Staurosporine (10 p.M) was bath-applied, and responses of neurons exposed to staurosporine were compared with those of control cells bathed in normal external solution. Acknowledgments The authors are grateful to Allen Spiegel for numerous helpful conversations and review of the manuscript, to T. Kendall Harden and Josd Boyer for generously providing the purified J]T, and to Michael Goy, Jennifer Luebke, and Timothy Turner for their critical comments on the manuscript. This work was supported by NS16483 (K. D.). All correspondence should be addressed to K. D. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC Section 1734 solely to indicate this fact. Received July 11, 1994; revised August 29, 1994.

N-Channel Inhibition 199

References

Bean, B. P. (1989). Neurotransmitter inhibition of neuronal calcium currents by changes in channel voltage dependence. Nature 340, 153156. Beech, D.J., Bern helm, L., Mathie, A., and Hille, B. (1991). Intracellular Ca 2+ buffers disrupt muecarinic suppression of Ca 2÷ current and M current in rat sympathetic neurons. Proc. Natl. Acad. Sci. USA 88, 652-656. Beech, D. J., Bernheim, L, and Hille, B. (1992). Pertussis toxin and voltage dependence distinguish multiple pathways modulating calcium channels of rat sympathetic neurons. Neuron 8, 97-106. Bernheim, L., Beech, D. J., and Hille, B. (1991). A diffusible second messenger mediates one of the pathways coupling receptors to calcium channels in rat sympathetic neurons. Neuron 6, 859-867. Blank, J. L., Brattain, K. A., and Exton, J. H. (1992). Activation of cytosolic phosphoinositide phospholipase C by G-protein ~y subunits. J. Biol. Chem. 267, 23069-23075. Bley, K. R., and Tsien, R. W. (1989). Inhibition of Ca2+and K+ channels in sympathetic neurons by neuropeptides and other ganglionic transmitters. Neuron 4, 379-391. Boland, L. M., and Bean, B. P. (1993). Modulation of N-type calcium channels in bullfrog sympathetic neurons by luteinizing hormone releasing hormone: kinetics and voltage dependence. J. Neurosci. 13, 516-533. Boland, L. M., Allen, A., and Dingledine, R. (1991). Inhibition by bradykinin of voltage-activated barium current in a rat dorsal root ganglion cell line: role of protein kinase C. J. Neurosci. 11, 1140-1149. Bowery, N. G., Knott, C., Moratalla, R., and Pratt, G. D. (1990). GABAB receptors and their heterogeneity. Adv. Biochem. Psychopharmacol. 21,395-402. Boyer, J. L., Waldo, G. L., Evans, T., Northup, J. K., Downes, P. C., and Harden, T. K. (1989). Modification of AIF -4 and receptor-stimulated phospholipase C activity by G-protein ~y subunits. J. Biol. Chem. 264, 13917-13922. Boyer, J. L., Waldo, G. L., and Harden, T. K. (1992). 13~,-subunitactivation of G-protein-regulated phospholipase C. J. Biol. Chem. 267, 25451-25456. Boyer, J. L., Graber, S. G., Waldo, G. L., Harden, T. K., and Garrison, J. C. (1994). Selective activation of phospholipase C by recombinant G-protein a and 1~7subunits. J. Biol. Chem. 269, 2814-2819. Canfield, D. R., and Dunlap, K. (1984). Pharmacological characterization of amine receptors on embryonic chick sensory neurones. Br. J. Pharmacol. 82, 557-563. Caulfield, M. P., Jones, S., Vallis, Y., Buckley, N. J., Kim, G.-D, Milligan, G., and Brown, D. A. (1994). Muscarinic M-current inhibition via Gc{q~l~and ct-adrenoceptor inhibition of Ca 2÷current via G(lo in rat sympathetic neurones. J. Physiol. 477, 415-422. Cerione, R. A., Kroll, S., Rajaram, R., Unson, C., Goldsmith, P., and Spiegel, A. (1988). An antibody directed against the carboxy-terminal decapeptide of the retinal GTP-binding protein, transducin: effects on transducin function. J. Biol. Chem. 263, 9345-9352. Cox, D. H., and Dunlap, K. (1992). Pharmacological discrimination of N-type from L-type calcium current and its selective modulation by transmitters. J. Neurosci. 12, 906-914. Delcour, A. H., and Tsien, R. W. (1993). Altered prevalence of gating modes in neurotransmitter inhibition of N-type calcium channels. Science 259, 980-984. Divers~-Pierluissi, M., and Dunlap, K. (1993). Distinct, convergent second messenger pathways modulate neuronal calcium currents. Neuron 10, 753-760. Diverse-Pierluissi, M., and Dunlap, K. (1994). Interaction of convergent pathways that inhibit N-type calcium currents in sensory neurons. Neuroscience, in press. Doerner, D., AbdeI-Latif, M., Rogers, T. B., and Alger, B. E. (1988). Protein kinase C-dependent and-independent effects of phorbol esters on hippocampal calcium channel current. J. Neurosci. 10, 1669-1706. Dunlap, K. (1981). Two types of ~,-aminobutyric acid receptor on embryonic sensory neurones. Br. J. Pharmacol. 74, 579-585.

Dunlap, K., and Fischbach, G. (1981). Neurotransmitters decrease the calcium conductance activated by depolarization of embryonic chick sensory neurons. J. Physiol. 317, 519-535. Elmslie, K. S., Zhou, W., and Jones, S. W. (1990). LHRH and GTP..~,-S modify calcium current activation in bullfrog sympathetic neurons. Neuron 5, 75-80. Elmslie, K. S., Kammermeier, P. J., and Jones, S. W. (1994). Reevaluation of Ca 2+channel types and their modulation in bullfrog sympathetic neurons. Neuron 13, 217-228. Ewald, D., Sternweis, P., and Miller, R. J. (1988). Guanine nucleotidebinding protein Go-induced coupling of neuropeptide Y receptors to Ca 2+ channels in sensory neurons. Proc. Natl. Acad. Sci. USA 85, 3633-3637. Ewald, D., Pang, I.-H., Sternweis, P. C., and Miller, R. J. (1989). Differential G protein-mediated coupling of neurotransmitter receptors to Ca 2+ channels in rat dorsal root ganglion neurons in vitro. Neuron 2, 1185-1193. Falloon, J., Malech, H., Milligan, G., Unson, C. G., Kahn, R., Goldsmith, P., and Spiegel, A. (1986). Detection of a major pertussis toxin substrate of human leukocytes with antisera raised against synthetic peptides. FEBS Lett. 209, 352-356. Formenti, A., Arrigoni, E., and Mancia, M. (1993). Two distinct modulatory effects on calcium channels in adult rat sensory neurons. Biophys. J. 64, 1029-1037. Fujita, Y., Mynlieff, M., Dirksen, R. T., Kim, M.-S., Niidome, T., Nakai, J., Friedrich, r., Iwabe, N., Miyata, T., Furuichi, T., Furutama, D., Mikoshiba, K., Mori, Y., and Beam, K. G. (1993). Primary structure and functional expression of the e)-conotoxin-sensitive N-type calcium channel from rabbit brain. Neuron 10, 585-598. Gierschik, P., Milligan, G., Pines, M., Goldsmith, P., Codina, J., Klee, W., and Spiegel, A. (1986). Use of specific antibodies to quantitate the guanine nucleotide-binding protein Go in brain. Proc. Natl. Acad. Sci. USA 83, 2258-2262. Gilman, A. G. (1987). G proteins: transducers of receptor-generated signals. Annu. Rev. Biochem. 56, 615-650. Golard, A., and Siegelbaum, S. A. (1993). Kinetic basis for the voltage dependent inhibition of N-type calcium current by somatostatin and norepinephrine in chick sympathetic neurons. J. Neurosci. 13, 38843894. Goldsmith, P., Backlund, P. S., Rossiter, K., Carter, A., Milligan, G., Unson, C. G., and Spiegel, A. (1988). Purification of heterotrimeric GTP-binding proteins from brain: identification of a novel form of Go. Biochemistry 27, 7085-7090. Grassi, F., and Lux, H. D. (1989). Voltage-dependent GASA-induced modulation of calcium currents in chick sensory neurons. Neurosci. Lett. 105, 113-119. Hescheler, J., Rosenthal, W., Trautwein, W., and Schultz, G. (1987). The GTP-binding protein, Go, regulates neuronal calcium currents. Nature 325, 445-447. Hille, B. (1992). G protein-coupled mechanisms and nervous signaling. Neuron 9, 187-195. Holz, G. G., Rane, S. G., and Dunlap, K. (1986). GTP-binding proteins mediate transmitter inhibition of voltage-dependent calcium channels. Nature 319, 670-672. Ito, H., Tung, R. T., Sugimoto, T., Kobayashi, I., Takahashi, K., Katada, T., Ui, M., and Kurachi, Y. (1992). On the mechanism of G protein J3~, subunit-activation of the muscarinic K÷ channel in guinea pig atrial cell membrane: comparison with the ATP-sensitive K÷ channel. J. Gen. Physiol. 99, 961-983. Kasai, H., and Aosaki, T. (1989). Modulation of calcium channel current by an adenosine analog mediated by a GTP-binding protein in chick sensory neurons. Pfl0gers Arch. 414, 145-149. Kasai, H., Aosaki, T., and Fukuda, J. (1987). Presynaptic Ca antagonist (o-conotoxin irreversibly blocks N-type Ca-channels in chick sensory neurons. Neurosci. Res. 4, 228-235. Kleuss, C., Hescheler, J., Ewel, C., Rosenthal, W., Schultz, G., and Wittig, B. (1991). Assignment of G-protein subtypes to specific receptors inducing inhibition of calcium channels. Nature 353, 43-48. Logothetis, D. E., Kurachi, Y., Galper, J., Neer, E. J., and Clapham,

Neuron 2OO

D. E. (1987). The ~, subunits of GTP-binding proteins activate the muscarinic K÷ channel in heart. Nature 325, 321-326. Luebke, J. I., and Dunlap, K. (1994). Sensory neuron N-type calcium currents are inhibited by both voltage-dependent and -independent mechanisms. Pflegers Arch. 428, 499-609. Marchetti, C., Carbone, E., and Lux, H. D. (1986). Effects of dopamine and noradrenaline on Ca channels of cultured sensory and sympathetic neurons of chick. PflSgers Arch. 406, 104-111. MacKinnon, A. C., Spedding, M., and Brown, C. M. (1994). ~2adrenoceptors: more subtypes but fewer functional differences. Trends Pharmacol. Sci. 15, 119-123. McFadzean, I., Mullaney, I., Brown, D. A., and Milligan, G. (1989). Antibodies to the GTP binding protein, Go, antagonize noradrenalineinduced calcium current inhibition in NG108-15 hybrid cells. Neuron 3, 177-182. McKenzie, F. R., Kelly, E. C., Unson, C. G., Spiegel, A. M., and Milligan, G. (1988). Antibodies which recognize the C-terminus of the inhibitory guanine nucleotide binding protein (G~) demonstrate that opioid peptides and foetal calf serum stimulate the high affinity GTPase activity of two separate pertussis toxin substrates. Biochem. J. 249, 653659. Menon-Johansson, A. S., Berrow, N., and Dolphin, A. C. (1993). Go transduces GABA~-receptor modulation of N-type calcium channels in cultured dorsal root ganglion neurons. Pfl0gers Archiv. 425, 335343. Plummer, M. R., Logothetis, D. E., and Hess, P. (1989). Elementary properties and pharmacological sensitivities of calcium channels in mammalian peripheral neurons. Neuron 2, 1453-1463. Plummer., M R., Rittenhouse, A., KanevskY, M., and Hess, P. (1991). Neurotransmitter modulation of calcium channels in rat sympathetic neurons. J. Neurosci. 11, 2339-2348. Rane, S. G., and Dunlap, K. (1986). Kinase C activator 1,2oleoylacetylglycerol attenuates voltage-dependent calcium current in sensory neurons. Proc. Natl. Acad. Sci. USA 83, 184-188. Rane, S. G., and Dunlap, K. (1990). G-protein and protein kinase C mediated regulation of voltage-dependent calcium channels. In G-Proteins, R. lyengar and L. Birnbaumer, eds. (San Diego, California: Academic Press, Inc.), pp. 107-115. Rane, S. G., Walsh, M. P., McDonald, J. R., and Dunlap, K. (1989). Specific inhibitors of protein kinase C block transmitter-induced modulation of sensory neuron calcium current. Neuron 3, 239-245. Regan, L. J., Sah, D. W. Y., and Bean, B. P. (1991). Ca2-- channels in rat central and peripheral neurons: high-threshold current resistant to dihydropyridine blockers and (~-conotoxin. Neuron 6, 269-280. Spiegel, A. (1990). Immunologic probes for heterotrimeric GTPbinding proteins. In G-Proteins, R. lyengar and L. Birnbaumer, eds. (San Diego, California: Academic Press, Inc.), pp. 115-136. Summers, R. J., and McMartin, L. R. (1993). Adrenoceptors and their second messengers. J. Neurochem. 60, 10-23. Tsien, R. W., Lipscombe, D., Madison, D. V., Bley, K. R., and Fox, A. P. (1988). Multiple types of calcium channels and their selective modulation. Trends Neurosci. 14, 46-51. Wanke, E., Ferroni, A., Ambrosini, A., Pozzan, T., and Meidolessi, J. (1987). Activation of a muscarinic receptor selectively inhibits a rapidly inactivated Ca2+current in rat sympathetic neurons. Proc. Natl. Acad. Sci. USA 84, 4313-4321. Williams, M. E., Brust, P. F., Feldman, D. H., Patthi, S., Simerson, S., Maroufi, A., McCue, A. F., Veli(;elebi, G., Ellis, S. B., and Harpold, M. M. (1992). Structure and functional expression of an e)-conotoxinsensitive human N-type calcium channel. Science 257, 389-395. Zhu, Y., and Ikeda, S. R. (1993). Adenosine modulates voltage-gated Ca 2÷channels in adult rat sympathetic neurons. J. Neurophysiol. 70, 610-620.