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Distinct, convergent second messenger pathways modulate neuronal calcium currents
Neuron,
Vol. 10, 753-760,
April,
1993, Copyright
0 1993 by Cell Press
Distinct, Convergent Second Messenger Pathways Modulate Neuronal Calciun Currents Maria DiversCPierluissi and Kathleen Department of Physiology Tufts University School of Medicine Boston, Massachusetts 02111
Dunlap
Summary Norepinephrine (NE) and y-aminobutyric acid (CABA) inhibit N-typecalcium channels in embryonic chick sensory neurons. We demonstrate here that the modulatory actions of the two transmitters are mediated through distinct biochemical pathways. Intracellular application of the pseudosubstrate inhibitor for protein kinase C blocks the inhibition produced by NE (and the protein kinase C activator oleoylacetylglycerol), but not that produced by CABA. Calcium current inhibition produced by oleoylacetylglycerol occludes inhibition by subsequent application of NE; GABA-mediated inhibition, however, is not eliminated by prior activation of protein kinase C. These results demonstrate that multiple biochemical pathways converge to control N-type calcium channel function.
Introduction A wide variety of neurotransmitters and peptides, including norepinephrine (NE), y-aminobutyric acid (GABA), adenosine, opioids, neuropeptide Y, and luteinizing hormone-releasing hormone, are known to inhibit neuronal high voltage-activated calcium currents (Tsien et al., 1988; Rane and Dunlap, 1990). For certain transmitters, the inhibition is associated with a slowing of the calcium current activation kinetics (Marchetti et al., 1986; Wanke et al., 1987; Grassi and Lux, 1989; lkeda and Schofield, 1989; Kasai and Aosaki, 1989; Elmslie et al., 1990), an effect that can be largely reversed with strong depolarizations (Bean, 1989; Elmslie et al., 1990). In other cases, the transmitter-induced inhibition occurs in theabsenceof any changes in current time course (Dunlap and Fischbach, 1981; Forscher and Oxford, 1985; Bean, 1989; Ewald et al., 1989). The molecular mechanisms responsible for these phenomenologicallydistinct effects of transmitters on calcium current are not fully understood. The involvement of GTP-binding proteins (G proteins) in transmitter-mediated inhibition of calcium channel function is universally accepted (Rane and Dunlap, 1990; Hille, 1992). Intracellular application of hydrolysis-resistant guanine nucleotide analogs enhances (GTPyS) or inhibits (GDPPS) the actions of the transmitters (Holzet al., 1986; Dolphin and Scott, 1987; Wanke et al., 1987; Hescheler et al., 1987). In most cases, treatment of the neurons with pertussis toxin interferes with the calcium current modulation aswell
(Holz et al., 1986; Lewis et al., 1986; Hescheler et al., 1987), suggesting the involvement of G proteins of the G, and/or G, class. Results from experiments in which purified G protein subunits are introduced into pertussis toxin-treated neurons argue strongly that G, mediates the actions of certain transmitters (Ewald et al., 1989; Hescheleret al., 1987). Furthermore, selective inhibition of particular G protein isotypes using antisense oligonucleotides has shown that somatostatininduced inhibitionof calcium current in pituitarycells requirestheactivationofGo2andthatcarbacholinhibits calcium current by activating G,, (Kleuss et al., 1991). Further experimentation is required to determine the significance of such results for G proteinmediated effects on calcium channels in other preparations. The involvement of protein kinase C (PKC) in transmitter-mediated modulation of neuronal calciumcurrents is indicated in some systems. Phorbol esters (activators of PKC) and intracellular injection of PKC enhance calcium current in Aplysia (De Riemer et al., 1985). In other preparations, PKC activators mimic transmitter-induced inhibition of calcium current (Rane and Dunlap, 1986; Strong et al., 1987; Mochida and Kobayashi, 1988; Doerner et al., 1988; Boland et al., 1991), and intracellular application of PKC inhibitors blocks the transmitter effects (Rane et al., 1989; Boland et al., 1991). Finally, down-regulation of PKC activity by long-term treatment with phorbol esters abolishes the inhibitory effects of neuropeptide Y on calcium currents in rat sensory neurons (Ewald et al., 1988). Results of investigations into PKC’s role as a mediator of calcium channel modulation have not all been positive, however. In some preparations, PKC activators do not mimic the actions of the transmitters. In sympathetic neurons, NE inhibits N-type calcium current (Plummer et al., 1991; Mathie et al., 1992), whereas the PKC activator oleoylacetylglycerol (OAG) inhibits both N- and L-type current (Plummer et al., 1991). In these same cells, intracellular application of the pseudosubstrate inhibitor of PKC or long-term treatment with phorbol esters did not interfere with the inhibitory actions of the transmitters (Plummer et al., 1991; Bley and Tsien, 1989; Bernheim et al., 1991). In embryonic chick sensory neurons, NE and GABA both inhibit N-type calcium current (Cox and Dunlap, 1992) via pertussis toxin-sensitive G proteins (Holz et al., 1986). Although the transduction pathways for these two transmitters are similar in certain aspects, we report here that they can be discriminated from one another in terms of their involvement of PKC. These results demonstrate the potential, within a single cell, for complex regulatory control of calcium channel function by multiple biochemical pathways and suggest that comparisons between preparations be made with caution.
Neuron 754
Figure
1. Inhibition
of Chick
Sensory
Neuron
Calcium
Currents
by NE, OAG,
Calcium current was measured using tight-seal, whole-cell recording. for 100 ms. The following agonists were puffer applied: (A) 10 uM NE; uM bicuculline to block the CABA, receptor responses). Current traces Calibration bar: 1 nA (NE and OAG traces), 0.5 nA (GABA traces) and
Results Inhibition of Calcium Current by Transmitters and OAG Tight-seal, whole-cell, voltage-clamp recording was performed on the somata of embryonic chick sensory neurons between 1 and 5 days in vitro. Calcium currents were isolated according to the methods of Cox and Dunlap (1992). Control currents were evoked by 100 ms step depolarizations from a holding potential of -80 mV to 0 mV; currents peaked in -1.7 ms and ranged in amplitude from 0.8 to 4 nA. These calcium currents are 85%-100% w-conotoxin sensitive (Cox and Dunlap, 1992) and are thus defined as N-type. These currents are selectively modulated by NE and GABA. Application of a saturating dose of NE (10 PM; Canfield and Dunlap, 1984) produced a reduction in calcium current in 20120 cells tested (Figure IA). In 75% of these cells, the effect occurred in the absence of measurable changes in current activation kinetics. That is, the average percent reduction in current was the same whether it was measured at the time of peak control current (28% f 7% inhibition) or at the end of the 100 ms test depolarization (26% f 6% inhibition). In the remaining 25% of the cells, the inhibition of calcium current was accompanied by a slowing of the activation phase, as reported in other systems (Marchetti et al., 1986; Wanke et al., 1987; Kasai and Aosaki, 1989; Plummer et al., 1989; Elmslie et al., 1990). In these cells, reduction in peak amplitude was 62% f 7%; that measured at the end of the test pulse was 40% f 5%. Saturating doses of GABA (100 PM; Dunlap, 1981) also inhibited calcium current, but were most often (70% of the time) associated with a slowing in the activation kinetics (Figure IC). At peak current, GABA produced an average36% f 10% reduction in calcium current in 40140 cells tested; by the end of the test pulse, the inhibition subsided to 20% + 7%. To test for a role of PKC in calcium current inhibition produced by NE and GABA, we employed PKC activators and inhibitors. We have repeated certain experiments originally reported by Rane and Dunlap
and GABA
Cells were held at -80 mV and depolarized every 10 s to 0 mV (B) 60 uM OAC; and (C) 100 uM CABA (in the presence of 100 marked with an asterisk were taken in the presence of agonist. 20 ms.
(1986) and Rane et al. (1989), which demonstrated that NE-induced inhibition of calcium current required the activation of PKC. Their repetition here allows us to comparedirectlytheactionsof NEwiththoseofGABA in cells from within the same platings. The PKC activator OAG inhibited calcium current by 20% + 7% in 21/21 cells tested (Figure IB). As with the transmitters, the inhibition produced by OAG was specific for N-type calcium current. No effects of OAG were observed on calcium currents recorded in the presence of the specific N channel antagonist w-conotoxin GVIA (data not shown). Control experiments demonstrated that 0.1% dimethylsulfoxide (DMSO; the vehicle for OAG), when applied alone, had no effect on calcium current. Furthermore, dioctanoylglycerol (diC8), a more water-soluble diacylglycerol analog, also inhibited calcium current (by20X) when applied at 50 f.rM (data not shown). The effects of OAG and diC8 were never accompanied by a slowing in the activation kinetics. In this way, modulation produced by the PKC activators resembled more closely the action of NE than that of GABA. Additionally, calcium currents recorded from cells in some platings were found to be resistant to modulation by NE; these cells were also resistant to modulation by OAG. The same cells, however, exhibited normal GABA-mediated inhibition of calcium current, further suggesting that NE and OAG share a common mechanism of action which differs from that underlying the effects of GABA. Effects of the Pseudosubstrate Inhibitor (PKCI 19-31) Introduction of a specific inhibitor of PKC (PKCI 19-31; House and Kemp, 1987) into the cytoplasm of sensory neurons blocked the inhibitoryactionsof both NEand OAG on calcium current without having any effect on the amplitude or kinetics of control currents. The results illustrated in Figure 2 and summarized in Table 1 are from a population study in which cells were recorded using internal solution with or without 3 uM PKCI 19-31 (a maximal dose of the inhibitor). Following 7 min of dialysis to allow the inhibitor to diffuse into the cytosol, cells were tested with OAG or the transmitters. The PKC inhibitor dramatically reduced re-
Calcium 755
Current
Modulation
by C Kinase
Figure 2. The Pseudosubstrate Produced by GABA Sensory neurons were dialyzed acid), and calcium current was from cells dialyzed with vehicle of agonists: (A) 60 PM OAG; (B) bar: 750 pA (all OAG traces), 1
Inhibitor
PKCI 19-31 Blocks
for 7 min with an evoked by 100 ms (top row) or PKCI 10 PM NE; (C) 100 nA (NE and GABA
the Attenuation
of Calcium
Current
internal recording solution containing 3 PM depolarizing pulses to 0 mV from a holding 19-31 (bottom row). Traces marked with an NM GABA + 100 FM bicuculline. Horizontal control traces), 1.4 nA (CABA + PKCI 19-31
sponses to NE (Figure 26) and OAG (Figure 2A). In 25% of the cells tested in the presence of PKCI 19-31, the small NE-induced inhibition remaining exhibited kinetic slowing. By contrast, the reduction in calcium current produced by GABA, as well as the times for onset and recovery from the inhibition, was unaffected by the PKC inhibitor (Figure 2C). These results strongly suggest that NE (and OAG) inhibit sensory neuron calcium current by a mechanism different from that employed by CABA. Occlusion by OAG of Transmitter-induced Responses To explore further the involvement of PKC in the NEand CABA-mediated modulation of calcium current, we tested the ability of OAG to occlude responses to the transmitters. In these experiments, OAG was applied to the neurons at 60 PM (a saturating dose). When the inhibition of calcium current was maximal, a saturating dose of either NE or GABA was applied, in the continual presence of OAG (Figure 3). Preapplication of OAG occluded further inhibition of calcium current by NE (n = 3). This occlusion was complete regardless of the magnitude of the initial OAGinduced inhibition. This suggests that OAG and NE activate a common, saturable pathway. Similar occlusion experiments with OAG and GABA yielded more complex results. In cells that exhibited small initial responses to OAG (IO%-20% reduction in current), GABA responses were not occluded (Figure 3). In fact, GABA-induced inhibition was additive with that produced by OAG under these conditions. In one sample of 5 cells, OAG inhibited calcium current by an average 17% f 8%; a subsequent application of GABA to these cells (in the presence of OAG) evoked an additional inhibition of 35% f 7%. In 5cells not treated with OAG, GABA produced an average 33% + 6% reduction in calcium current compared
Produced
by OAC
and NE but Not That
pseudosubstrate or vehicle (0.5 PM acetic potential of -80 mV. Traces were recorded asterisk were recorded during application calibration bar; 20 ms. Vertical calibration traces), and 3 nA (NE + PKCI 19-31 traces).
with control currents measured prior to transmitter application. These results are consistent with the notion that OAG and GABA do not share a common, saturable pathway. Such additivity was attenuated, however, in cells that produced robust initial responsestoOAG. In5suchcells,theaverageinhibition evoked by OAG was 30% f 8%; subsequent application of GABAfurther inhibited the current by an average 18% f 7% (instead of the 33% + 6% observed in the absence of OAG preapplication). These results suggest that the pathways mediating the PKC-dependent and -independent modulation of N-type calcium channels share a common, saturable element that lies beyond kinase activation. Effects of the Phosphatase inhibitor Okadaic Acid To explore further a potential role of protein phosphorylation in the GABA- and NE-mediated modulation of calcium current, we employed okadaic acid, a fatty acid phosphatase inhibitor isolated from the marine sponge Halichondria. Okadaic acid inhibits protein phosphatase type 1 (ICSO = 20-70 nM) and type 2A (I& = 0.2-0.5 nM) (Bialojan and Takai, 1988; Suganumaet al., 1988) and can effectively increase the cellular level of phosphorylation. Control cells were dialyzed for 7.5-10 min with an internal solution containing vehicle alone (0.1% or 0.5% DMSO), and their responsiveness to GABA was compared with that in
Table 1. Percent and GABA
Inhibition
of Calcium
Agonist
Control Solution
60 PM OAG 10 PM NE 100 PM CABA
16 f 1 (n = 4) 28 f 2.5 (n = 14) 31 f 2 (n = 15)
Current
by OAG,
NE,
Internal 3 PM PKCI
internal
1.5~0.5 (n=7) 7 * 1.25 (n = 12) 33 t 1.25 (n = 15)
Neuron 756
l.OI 0
50
100
150
200
time (s)
containing 100 nM okadaic acid; NE-induced inhibition of calcium current in these cells was compared with that in cells dialyzed with the same internal solution but also containing 3 uM PKCI 19-31. The mean NE response in controls was 63% f 20% (n = 9), and that recorded in the presence of PKCI 19-31 was 3.5% f 1% (n = 7). Thus, all of the inhibition produced by NE, even that potentiated by okadaic acid, is PKC mediated. Consistent with these results, okadaic acid also enhanced OAG-mediated inhibition of calcium current (Figure 4). These results demonstrate the activity of okadaic acid and reinforce the notion that GABA- and NE-mediated modulation of N-type calcium channels is effected through distinct biochemical pathways. Discussion
2.0-c 0
I 40
I 80
I 120
.
I 160
time (s) Figure
3. OAG
Occludes
the Action
of NE but Not That of GABA
Plots of peak calcium current as a function of time in 2 different sensory neurons. At the time marked by the horizontal bars, 60 uM OAG was applied alone, followed by application of either 100 uMGABA(top)orlOuM NEfbottom) inthecontinual presenceof OAG.
cells dialyzed for the same amount of time with an internal solution containing 100 or 500 nM okadaic acid, respectively. Okadaic acid by itself did not produce any measurable effects on calcium current, suggesting a low level of kinase activity in the basal state. Inhibition of calcium current produced by 100 uM GABA was unaffected by the presence of either concentration of okadaic acid. The data for 100 nM okadaic acid are plotted in Figure 4. In the presence of 500 nM okadaic acid, CABA-mediated decreases in calcium current were 41.3% f 7% (compared with 41% f 8% in cells dialyzed with control internal solution). To test whether the lack of effect of okadaic acid results from a saturation of the modulatory pathway produced by 100 uM GABA, we tested the effect of the phosphatase inhibitor on a subsaturating concentration of GABA (IO PM). Even at this lower GABA concentration, the presence of okadaic acid failed to enhance the action of GABA (n = 5, data not shown). These results argue that GABA-mediated inhibition of N channel function does not involve a phosphorylation event that is sensitive to phosphatase type 1 or type 2A. By contrast with the results for GABA, okadaic acid did potentiate NE-mediated inhibition of calcium current. At 10 FM, NE inhibited calcium current by 56% + 10% in neurons dialyzed with 100 nM okadaic acid compared with 25% f 8% in control cells from the same plating (Figure 4). A separate set of recordings tested whether potentiation of the NE response induced by the presence of okadaic acid involved the activation of PKC. In these experiments
Results reported here demonstrate that transmitterinduced modulation of sensory neuron N-type calcium current involves more than a single biochemical pathway. We have corroborated earlier findings by Rane and Dunlap (1986) that lipid-soluble activators of PKC mimic the effects of NE. In recognition that chloroform can have nonspecific inhibitoryeffectson calcium current when used as a solvent for OAG (Hockberger et al., 1989), we dissolved the OAG in DMSO. The absence of vehicle effects and the efficacy of diC8, a more polar activator of PKC, strongly argue thattheinhibitoryeffectsobservedhereareproduced by OAG. Our results suggest that NE-induced inhibition of calcium current requires the activation of PKC
CON
GABA
Figure 4. Okadaic Acid Not That of GABA
OK.4
NE
Enhances
the Action
CON
OKA
OAG
of NE and OAG but
Sensory neurons were dialyzed for 7.5-10 min with an internal recordingsolutioncontaininglOOnMokadaicacid(O~)orvehicle (CON), and calcium current was evoked every 30 s by 100 ms depolarizing pulses to 0 mV from a holding potential of -80 mV. The bar graph summarizes the average percent inhibition of calcium current produced by NE, OAG, or GABA (as indicated on the abscissa) in a population of neurons. The numbers in parentheses indicate sample sizes, and error bars represent the standard deviation.
Calcium 757
Current
Modulation
by C Kinase
whereas that produced by GABA does not. Based on established conservation of primary sequence in the substrate-binding domain of all PKC isozymes cloned to date, PKCI 19-31 is thought to be effective against all forms of PKC (Bell and Burns, 1991; Orr et al., 1992). Diacylglycerol and phorbol esters have been reported to activate all isoforms with the exception of the I; subspecies (Ono et al., 1989). In light of this evidence, our resultsargue stronglythat GABA-mediated inhibition of N channels does not involve PKC. It is unlikely that chick sensory neurons contain a yet-unidentified isozyme of PKC that is resistant to PKCI 19-31 and can be activated by GABA but not diacylglycerol analogs. Our experiments with okadaic acid suggest that GABA-mediated inhibition of calcium current does not require a phosphorylation event that is sensitive to phosphatase type 1 or type 2A. Okadaic acid (at concentrations of up to 500 nM) failed to alter the response to GABA under conditions in which it potentiated the response to NE. These results also strengthen the conclusions from experiments with the PKC inhibitor. Potentiation byokadaic acid of NEmediated modulation of sensory neuron calcium current implicates the involvement of a phosphorylationdependent signaling pathway. Comparable results have been observed in other systems as well. For example, in cardiac myocytes from guinea pig, extracellular application of okadaic acid enhances the NEinduced increase in L-typecalcium current (Hescheler et al., 1988). Similarly, membranecurrent responses to FMRFamide and serotonin in Aplysia sensory neurons are regulated by phosphatase activity (Ichinose and Byrne, 1991). In other preparations, by contrast, transmitter-induced modulation of ion channel activity may be mediated by dephosphorylation rather than phosphorylation. Somatostatin activates calciumsensitive potassium channels in pituitary tumor cells via a mechanism that is blocked by okadaic acid (White et al., 1991). As the GABA-mediated modulation of current is most often associated with a slowing of the activation kinetics, it is tempting to speculate that kinetic slowing is characteristic of transmitter responses which are independent of phosphorylation. The biochemical mechanism underlying the kinetic slowing is unknown; it can be at least partially reversed, however, with strong depolarizing stimuli (Bean, 1989; Elmslie et al., 1990). In some preparations, this modulatory component may be responsible for tonic inhibition of resting calcium channels (Kasai, 1991) and is thought to underlie facilitation of calcium current observed with stimulus trains (Kasai, 1991; Artalejo et al., 1991). The fact that hydrolysis-resistant analogs of GTP can lead to G protein activation and produce the voltage-dependent inhibition and kinetic slowing of calciumcurrent hasleadsometosuggestadirect interaction between G protein subunits and the calcium channel (Hille, 1992; Yatani et al., 1987). Proof of this hypothesis awaits methods to study N channels successfully in excised patches or planar lipid bilayers.
Other signaling pathways responsible for the transduction of GABA-induced inhibition of calcium current must beconsidered. GABAe receptors are known to be coupled to a variety of biochemical pathways. Although they inhibit adenylyl cyclase activity (Bowery et al., 1990), a role for CAMP in the calcium current modulation is considered unlikely (Forscher and Oxford, 1985; Holz et al., 1986). GABAe receptors are also coupled, through G proteins, to activation of phospholipase AZ and the generation of arachidonic acid (Bowery et al., 1990). The role of arachidonate or its metabolites in regulating sensory neuron calcium channels must betested, as they control channel function in other preparations. In Aplysia neurons, for example, metabolites of the lipoxygenase pathway regulate excitability through alterations in potassium channel function (Buttner et al., 1989); similar mechanisms may underlie the action of GABA in hippocampal pyramidal neurons as well (Gage, 1992). It is becoming clear that a single population of calcium channels can be modulated by multiple pathways. In addition to the PKC-dependent and -independent mechanisms reported here, results from other preparations suggest a multiplicity of mechanisms involved in the regulation of high voltage-activated calcium current. In rat sympathetic neurons, N-type calcium currents are inhibited by transmitters that activate both pertussis toxin-sensitive and -resistant pathways (Wanke et al., 1987; Song et al., 1989,199l). The two pathways have been further elucidated by Hille and coworkers. Activation of muscarinic receptors decreases calcium current, employing both fast and slow mechanisms. The fast pathway is membrane delimited, and it has pertussis toxin-sensitive and -resistant components. The slow pathway requires a calcium-dependent second messenger and is voltage independent (Beech et al., 1991; Bernheim et al., 1991; Mathie et al., 1992). NE uses two pathways as wellboth fast-and its effects are voltage dependent and calcium independent (Beech et al., 1992). These results on sympathetic neurons, coupled with those from our own laboratory, indicate that modulation of calcium channel function, even within a single cell, can be quite complex, and any one hypothesis is unlikely to explain the large number of disparate observations that have been reported for transmitter-mediated modulation of high voltageactivated calcium channels. Proposing a model even for chick dorsal root ganglion neurons would be, at this point, premature. The pathway eventually must include pertussis toxin-sensitive G proteins, PKCmediated phosphorylation that is sensitive to okadaic acid (for NE only), and w-conotoxin GVIA-sensitive (N-type) calcium channels. As additional elements are unknown, a variety of potential models are consistent with our data; further experimentation is required, however, before proposing one will be a useful exercise. Sorting out these pathways and their interactions will remain a critical area for continued investigation in the coming decade. It will be particularly important
Neuron 758
to determine whether multiple pathways can converge at the level of a single channel, as suggested in the experiments of Shen and Surprenant (1991). NE and GABA (Cox and Dunlap, 1992), as well as OAG, selectively inhibit oconotoxin-sensitive calcium channels. The degree of channel heterogeneity in this pharmacological class is unknown. The partial occlusion of GABA-mediated inhibition produced in sensory neurons responding robustly to OAC, however, is consistent with the idea that the same channels might be targets for both OAG and GABA. Less likely is the alternative that the saturable element in these occlusion experiments is proximal to the channel (but distal to PKC activation) and that different N channels are targeted by the different transmitters. The evolution of multiple mechanismsforthecontrol of calcium channel function may be one way in which the specificity of receptor-effector interactions is ensured (Hille, 1992). In any case, the coexistence of several pathways for the control of calcium influx underscores the significance of calcium channel modulation in regulating cellular function. Experimental
Procedures
Dorsal root ganglia were dissected from II- to 12day-old chick embryos. Tissue was incubated at 37OC for 50 min in calciumand magnesium-free saline and dissociated by trituration with a fire-polished Pasteur pipette. Cells were plated in collagencoateddishesandgrown in Dulbecco’s modified Eagle’s medium supplemented with heat-inactivated 10% horse serum, 5% chick embryo extract, 50 U/ml penicillin, 50 pglml streptomycin, and nerve growth factor. Cells were used for experiments 3-5 days after plating. For recording calcium currents, the pipette internal solution contained 150 mM CsCI, 10 mM HEPES, 5 Mg-ATP, and 5 mM BAPTA. The external saline solution contained 133 mM NaCI, 1 mM CaC12, 0.8 mM MgC12, 10 mM tetraethylammonium chloride, 25 mM HEPES, 12.5 mM NaOH, 5 mM glucose, and 0.3 PM 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 to 2.5 MCI. A List EPC-7 amplifier (Medical Systems) was used to record calcium currents in the tight-seal, whole-cell configuration (Hamill et al., 1981). Data were acquired at 10 kHz using an Atari Mega 4 STE. Software was obtained from lnstrutech Corporation (Elmont, NY). A standard P/4 protocol was used for leakage subtraction of thecurrents prior to analysis. Currents from population studies are reported as mean + standard deviation. Because cells from different platings varied significantly in their responsiveness to transmitter, population studies were performed on cells within the same plating. Percent decreases in calcium current are reported relative to peak control calcium current. For the occlusion experiments, in which OAG was applied, followed by application of a transmitter (in the continual presence of OAG), percent inhibition produced by the transmitters is calculated relative to peak current measured in the presence of OAG alone. PKCI 19-31 (Bachem) was dissolved to a stock concentration of 1 mM in 0.5 M acetic acid and stored at -8OOC. For experiments, the stock solution was diluted to 3 WM in internal saline solution. Internal solutions used for control cells contained 1.5 mM acetic acid to control for potential effects of the vehicle. OAG was purchased from Avanti Polar Lipids as a chloroform solution. The ampulewas opened, and the chloroform was evap orated under nitrogen. The OAG was redissolved in DMSO as a 2.5 mM stock solution and stored at -80°C in 10 ~1 aliquots. For experiments, fresh 60 PM OAG solutions were prepared each
day by diluting the stock solution into external saline. The final concentration of DMSO was 0.1%. Solutions used to test control cells also contained 0.1% DMSO to control for possible effects of the vehicle. The ammonium salt of okadaic acid (LC Services) was dissolved in DMSO and stored at -80°C as a stock solution of 0.1 mglml. On the day of use, 100 nM okadaic acid solutions were prepared by dilution of the stock solution into the intracellular pipette solution. Internal solutions used for control cells contained 0.1% DMSO to control for possible effects of vehicle. CABA and NE solutions were prepared daily in the extracellular recording solution used to bathe the sensory neurons. Acknowledgments This work was supported by PHS grant NS164B3 and a supple ment to PHS grant NS28815 from the Program for Underrepresented Minorities. 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
October
16, 1992; revised
January
29,1993.
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Vol. 10, 753-760,
April,
1993, Copyright
0 1993 by Cell Press
Distinct, Convergent Second Messenger Pathways Modulate Neuronal Calciun Currents Maria DiversCPierluissi and Kathleen Department of Physiology Tufts University School of Medicine Boston, Massachusetts 02111
Dunlap
Summary Norepinephrine (NE) and y-aminobutyric acid (CABA) inhibit N-typecalcium channels in embryonic chick sensory neurons. We demonstrate here that the modulatory actions of the two transmitters are mediated through distinct biochemical pathways. Intracellular application of the pseudosubstrate inhibitor for protein kinase C blocks the inhibition produced by NE (and the protein kinase C activator oleoylacetylglycerol), but not that produced by CABA. Calcium current inhibition produced by oleoylacetylglycerol occludes inhibition by subsequent application of NE; GABA-mediated inhibition, however, is not eliminated by prior activation of protein kinase C. These results demonstrate that multiple biochemical pathways converge to control N-type calcium channel function.
Introduction A wide variety of neurotransmitters and peptides, including norepinephrine (NE), y-aminobutyric acid (GABA), adenosine, opioids, neuropeptide Y, and luteinizing hormone-releasing hormone, are known to inhibit neuronal high voltage-activated calcium currents (Tsien et al., 1988; Rane and Dunlap, 1990). For certain transmitters, the inhibition is associated with a slowing of the calcium current activation kinetics (Marchetti et al., 1986; Wanke et al., 1987; Grassi and Lux, 1989; lkeda and Schofield, 1989; Kasai and Aosaki, 1989; Elmslie et al., 1990), an effect that can be largely reversed with strong depolarizations (Bean, 1989; Elmslie et al., 1990). In other cases, the transmitter-induced inhibition occurs in theabsenceof any changes in current time course (Dunlap and Fischbach, 1981; Forscher and Oxford, 1985; Bean, 1989; Ewald et al., 1989). The molecular mechanisms responsible for these phenomenologicallydistinct effects of transmitters on calcium current are not fully understood. The involvement of GTP-binding proteins (G proteins) in transmitter-mediated inhibition of calcium channel function is universally accepted (Rane and Dunlap, 1990; Hille, 1992). Intracellular application of hydrolysis-resistant guanine nucleotide analogs enhances (GTPyS) or inhibits (GDPPS) the actions of the transmitters (Holzet al., 1986; Dolphin and Scott, 1987; Wanke et al., 1987; Hescheler et al., 1987). In most cases, treatment of the neurons with pertussis toxin interferes with the calcium current modulation aswell
(Holz et al., 1986; Lewis et al., 1986; Hescheler et al., 1987), suggesting the involvement of G proteins of the G, and/or G, class. Results from experiments in which purified G protein subunits are introduced into pertussis toxin-treated neurons argue strongly that G, mediates the actions of certain transmitters (Ewald et al., 1989; Hescheleret al., 1987). Furthermore, selective inhibition of particular G protein isotypes using antisense oligonucleotides has shown that somatostatininduced inhibitionof calcium current in pituitarycells requirestheactivationofGo2andthatcarbacholinhibits calcium current by activating G,, (Kleuss et al., 1991). Further experimentation is required to determine the significance of such results for G proteinmediated effects on calcium channels in other preparations. The involvement of protein kinase C (PKC) in transmitter-mediated modulation of neuronal calciumcurrents is indicated in some systems. Phorbol esters (activators of PKC) and intracellular injection of PKC enhance calcium current in Aplysia (De Riemer et al., 1985). In other preparations, PKC activators mimic transmitter-induced inhibition of calcium current (Rane and Dunlap, 1986; Strong et al., 1987; Mochida and Kobayashi, 1988; Doerner et al., 1988; Boland et al., 1991), and intracellular application of PKC inhibitors blocks the transmitter effects (Rane et al., 1989; Boland et al., 1991). Finally, down-regulation of PKC activity by long-term treatment with phorbol esters abolishes the inhibitory effects of neuropeptide Y on calcium currents in rat sensory neurons (Ewald et al., 1988). Results of investigations into PKC’s role as a mediator of calcium channel modulation have not all been positive, however. In some preparations, PKC activators do not mimic the actions of the transmitters. In sympathetic neurons, NE inhibits N-type calcium current (Plummer et al., 1991; Mathie et al., 1992), whereas the PKC activator oleoylacetylglycerol (OAG) inhibits both N- and L-type current (Plummer et al., 1991). In these same cells, intracellular application of the pseudosubstrate inhibitor of PKC or long-term treatment with phorbol esters did not interfere with the inhibitory actions of the transmitters (Plummer et al., 1991; Bley and Tsien, 1989; Bernheim et al., 1991). In embryonic chick sensory neurons, NE and GABA both inhibit N-type calcium current (Cox and Dunlap, 1992) via pertussis toxin-sensitive G proteins (Holz et al., 1986). Although the transduction pathways for these two transmitters are similar in certain aspects, we report here that they can be discriminated from one another in terms of their involvement of PKC. These results demonstrate the potential, within a single cell, for complex regulatory control of calcium channel function by multiple biochemical pathways and suggest that comparisons between preparations be made with caution.
Neuron 754
Figure
1. Inhibition
of Chick
Sensory
Neuron
Calcium
Currents
by NE, OAG,
Calcium current was measured using tight-seal, whole-cell recording. for 100 ms. The following agonists were puffer applied: (A) 10 uM NE; uM bicuculline to block the CABA, receptor responses). Current traces Calibration bar: 1 nA (NE and OAG traces), 0.5 nA (GABA traces) and
Results Inhibition of Calcium Current by Transmitters and OAG Tight-seal, whole-cell, voltage-clamp recording was performed on the somata of embryonic chick sensory neurons between 1 and 5 days in vitro. Calcium currents were isolated according to the methods of Cox and Dunlap (1992). Control currents were evoked by 100 ms step depolarizations from a holding potential of -80 mV to 0 mV; currents peaked in -1.7 ms and ranged in amplitude from 0.8 to 4 nA. These calcium currents are 85%-100% w-conotoxin sensitive (Cox and Dunlap, 1992) and are thus defined as N-type. These currents are selectively modulated by NE and GABA. Application of a saturating dose of NE (10 PM; Canfield and Dunlap, 1984) produced a reduction in calcium current in 20120 cells tested (Figure IA). In 75% of these cells, the effect occurred in the absence of measurable changes in current activation kinetics. That is, the average percent reduction in current was the same whether it was measured at the time of peak control current (28% f 7% inhibition) or at the end of the 100 ms test depolarization (26% f 6% inhibition). In the remaining 25% of the cells, the inhibition of calcium current was accompanied by a slowing of the activation phase, as reported in other systems (Marchetti et al., 1986; Wanke et al., 1987; Kasai and Aosaki, 1989; Plummer et al., 1989; Elmslie et al., 1990). In these cells, reduction in peak amplitude was 62% f 7%; that measured at the end of the test pulse was 40% f 5%. Saturating doses of GABA (100 PM; Dunlap, 1981) also inhibited calcium current, but were most often (70% of the time) associated with a slowing in the activation kinetics (Figure IC). At peak current, GABA produced an average36% f 10% reduction in calcium current in 40140 cells tested; by the end of the test pulse, the inhibition subsided to 20% + 7%. To test for a role of PKC in calcium current inhibition produced by NE and GABA, we employed PKC activators and inhibitors. We have repeated certain experiments originally reported by Rane and Dunlap
and GABA
Cells were held at -80 mV and depolarized every 10 s to 0 mV (B) 60 uM OAC; and (C) 100 uM CABA (in the presence of 100 marked with an asterisk were taken in the presence of agonist. 20 ms.
(1986) and Rane et al. (1989), which demonstrated that NE-induced inhibition of calcium current required the activation of PKC. Their repetition here allows us to comparedirectlytheactionsof NEwiththoseofGABA in cells from within the same platings. The PKC activator OAG inhibited calcium current by 20% + 7% in 21/21 cells tested (Figure IB). As with the transmitters, the inhibition produced by OAG was specific for N-type calcium current. No effects of OAG were observed on calcium currents recorded in the presence of the specific N channel antagonist w-conotoxin GVIA (data not shown). Control experiments demonstrated that 0.1% dimethylsulfoxide (DMSO; the vehicle for OAG), when applied alone, had no effect on calcium current. Furthermore, dioctanoylglycerol (diC8), a more water-soluble diacylglycerol analog, also inhibited calcium current (by20X) when applied at 50 f.rM (data not shown). The effects of OAG and diC8 were never accompanied by a slowing in the activation kinetics. In this way, modulation produced by the PKC activators resembled more closely the action of NE than that of GABA. Additionally, calcium currents recorded from cells in some platings were found to be resistant to modulation by NE; these cells were also resistant to modulation by OAG. The same cells, however, exhibited normal GABA-mediated inhibition of calcium current, further suggesting that NE and OAG share a common mechanism of action which differs from that underlying the effects of GABA. Effects of the Pseudosubstrate Inhibitor (PKCI 19-31) Introduction of a specific inhibitor of PKC (PKCI 19-31; House and Kemp, 1987) into the cytoplasm of sensory neurons blocked the inhibitoryactionsof both NEand OAG on calcium current without having any effect on the amplitude or kinetics of control currents. The results illustrated in Figure 2 and summarized in Table 1 are from a population study in which cells were recorded using internal solution with or without 3 uM PKCI 19-31 (a maximal dose of the inhibitor). Following 7 min of dialysis to allow the inhibitor to diffuse into the cytosol, cells were tested with OAG or the transmitters. The PKC inhibitor dramatically reduced re-
Calcium 755
Current
Modulation
by C Kinase
Figure 2. The Pseudosubstrate Produced by GABA Sensory neurons were dialyzed acid), and calcium current was from cells dialyzed with vehicle of agonists: (A) 60 PM OAG; (B) bar: 750 pA (all OAG traces), 1
Inhibitor
PKCI 19-31 Blocks
for 7 min with an evoked by 100 ms (top row) or PKCI 10 PM NE; (C) 100 nA (NE and GABA
the Attenuation
of Calcium
Current
internal recording solution containing 3 PM depolarizing pulses to 0 mV from a holding 19-31 (bottom row). Traces marked with an NM GABA + 100 FM bicuculline. Horizontal control traces), 1.4 nA (CABA + PKCI 19-31
sponses to NE (Figure 26) and OAG (Figure 2A). In 25% of the cells tested in the presence of PKCI 19-31, the small NE-induced inhibition remaining exhibited kinetic slowing. By contrast, the reduction in calcium current produced by GABA, as well as the times for onset and recovery from the inhibition, was unaffected by the PKC inhibitor (Figure 2C). These results strongly suggest that NE (and OAG) inhibit sensory neuron calcium current by a mechanism different from that employed by CABA. Occlusion by OAG of Transmitter-induced Responses To explore further the involvement of PKC in the NEand CABA-mediated modulation of calcium current, we tested the ability of OAG to occlude responses to the transmitters. In these experiments, OAG was applied to the neurons at 60 PM (a saturating dose). When the inhibition of calcium current was maximal, a saturating dose of either NE or GABA was applied, in the continual presence of OAG (Figure 3). Preapplication of OAG occluded further inhibition of calcium current by NE (n = 3). This occlusion was complete regardless of the magnitude of the initial OAGinduced inhibition. This suggests that OAG and NE activate a common, saturable pathway. Similar occlusion experiments with OAG and GABA yielded more complex results. In cells that exhibited small initial responses to OAG (IO%-20% reduction in current), GABA responses were not occluded (Figure 3). In fact, GABA-induced inhibition was additive with that produced by OAG under these conditions. In one sample of 5 cells, OAG inhibited calcium current by an average 17% f 8%; a subsequent application of GABA to these cells (in the presence of OAG) evoked an additional inhibition of 35% f 7%. In 5cells not treated with OAG, GABA produced an average 33% + 6% reduction in calcium current compared
Produced
by OAC
and NE but Not That
pseudosubstrate or vehicle (0.5 PM acetic potential of -80 mV. Traces were recorded asterisk were recorded during application calibration bar; 20 ms. Vertical calibration traces), and 3 nA (NE + PKCI 19-31 traces).
with control currents measured prior to transmitter application. These results are consistent with the notion that OAG and GABA do not share a common, saturable pathway. Such additivity was attenuated, however, in cells that produced robust initial responsestoOAG. In5suchcells,theaverageinhibition evoked by OAG was 30% f 8%; subsequent application of GABAfurther inhibited the current by an average 18% f 7% (instead of the 33% + 6% observed in the absence of OAG preapplication). These results suggest that the pathways mediating the PKC-dependent and -independent modulation of N-type calcium channels share a common, saturable element that lies beyond kinase activation. Effects of the Phosphatase inhibitor Okadaic Acid To explore further a potential role of protein phosphorylation in the GABA- and NE-mediated modulation of calcium current, we employed okadaic acid, a fatty acid phosphatase inhibitor isolated from the marine sponge Halichondria. Okadaic acid inhibits protein phosphatase type 1 (ICSO = 20-70 nM) and type 2A (I& = 0.2-0.5 nM) (Bialojan and Takai, 1988; Suganumaet al., 1988) and can effectively increase the cellular level of phosphorylation. Control cells were dialyzed for 7.5-10 min with an internal solution containing vehicle alone (0.1% or 0.5% DMSO), and their responsiveness to GABA was compared with that in
Table 1. Percent and GABA
Inhibition
of Calcium
Agonist
Control Solution
60 PM OAG 10 PM NE 100 PM CABA
16 f 1 (n = 4) 28 f 2.5 (n = 14) 31 f 2 (n = 15)
Current
by OAG,
NE,
Internal 3 PM PKCI
internal
1.5~0.5 (n=7) 7 * 1.25 (n = 12) 33 t 1.25 (n = 15)
Neuron 756
l.OI 0
50
100
150
200
time (s)
containing 100 nM okadaic acid; NE-induced inhibition of calcium current in these cells was compared with that in cells dialyzed with the same internal solution but also containing 3 uM PKCI 19-31. The mean NE response in controls was 63% f 20% (n = 9), and that recorded in the presence of PKCI 19-31 was 3.5% f 1% (n = 7). Thus, all of the inhibition produced by NE, even that potentiated by okadaic acid, is PKC mediated. Consistent with these results, okadaic acid also enhanced OAG-mediated inhibition of calcium current (Figure 4). These results demonstrate the activity of okadaic acid and reinforce the notion that GABA- and NE-mediated modulation of N-type calcium channels is effected through distinct biochemical pathways. Discussion
2.0-c 0
I 40
I 80
I 120
.
I 160
time (s) Figure
3. OAG
Occludes
the Action
of NE but Not That of GABA
Plots of peak calcium current as a function of time in 2 different sensory neurons. At the time marked by the horizontal bars, 60 uM OAG was applied alone, followed by application of either 100 uMGABA(top)orlOuM NEfbottom) inthecontinual presenceof OAG.
cells dialyzed for the same amount of time with an internal solution containing 100 or 500 nM okadaic acid, respectively. Okadaic acid by itself did not produce any measurable effects on calcium current, suggesting a low level of kinase activity in the basal state. Inhibition of calcium current produced by 100 uM GABA was unaffected by the presence of either concentration of okadaic acid. The data for 100 nM okadaic acid are plotted in Figure 4. In the presence of 500 nM okadaic acid, CABA-mediated decreases in calcium current were 41.3% f 7% (compared with 41% f 8% in cells dialyzed with control internal solution). To test whether the lack of effect of okadaic acid results from a saturation of the modulatory pathway produced by 100 uM GABA, we tested the effect of the phosphatase inhibitor on a subsaturating concentration of GABA (IO PM). Even at this lower GABA concentration, the presence of okadaic acid failed to enhance the action of GABA (n = 5, data not shown). These results argue that GABA-mediated inhibition of N channel function does not involve a phosphorylation event that is sensitive to phosphatase type 1 or type 2A. By contrast with the results for GABA, okadaic acid did potentiate NE-mediated inhibition of calcium current. At 10 FM, NE inhibited calcium current by 56% + 10% in neurons dialyzed with 100 nM okadaic acid compared with 25% f 8% in control cells from the same plating (Figure 4). A separate set of recordings tested whether potentiation of the NE response induced by the presence of okadaic acid involved the activation of PKC. In these experiments
Results reported here demonstrate that transmitterinduced modulation of sensory neuron N-type calcium current involves more than a single biochemical pathway. We have corroborated earlier findings by Rane and Dunlap (1986) that lipid-soluble activators of PKC mimic the effects of NE. In recognition that chloroform can have nonspecific inhibitoryeffectson calcium current when used as a solvent for OAG (Hockberger et al., 1989), we dissolved the OAG in DMSO. The absence of vehicle effects and the efficacy of diC8, a more polar activator of PKC, strongly argue thattheinhibitoryeffectsobservedhereareproduced by OAG. Our results suggest that NE-induced inhibition of calcium current requires the activation of PKC
CON
GABA
Figure 4. Okadaic Acid Not That of GABA
OK.4
NE
Enhances
the Action
CON
OKA
OAG
of NE and OAG but
Sensory neurons were dialyzed for 7.5-10 min with an internal recordingsolutioncontaininglOOnMokadaicacid(O~)orvehicle (CON), and calcium current was evoked every 30 s by 100 ms depolarizing pulses to 0 mV from a holding potential of -80 mV. The bar graph summarizes the average percent inhibition of calcium current produced by NE, OAG, or GABA (as indicated on the abscissa) in a population of neurons. The numbers in parentheses indicate sample sizes, and error bars represent the standard deviation.
Calcium 757
Current
Modulation
by C Kinase
whereas that produced by GABA does not. Based on established conservation of primary sequence in the substrate-binding domain of all PKC isozymes cloned to date, PKCI 19-31 is thought to be effective against all forms of PKC (Bell and Burns, 1991; Orr et al., 1992). Diacylglycerol and phorbol esters have been reported to activate all isoforms with the exception of the I; subspecies (Ono et al., 1989). In light of this evidence, our resultsargue stronglythat GABA-mediated inhibition of N channels does not involve PKC. It is unlikely that chick sensory neurons contain a yet-unidentified isozyme of PKC that is resistant to PKCI 19-31 and can be activated by GABA but not diacylglycerol analogs. Our experiments with okadaic acid suggest that GABA-mediated inhibition of calcium current does not require a phosphorylation event that is sensitive to phosphatase type 1 or type 2A. Okadaic acid (at concentrations of up to 500 nM) failed to alter the response to GABA under conditions in which it potentiated the response to NE. These results also strengthen the conclusions from experiments with the PKC inhibitor. Potentiation byokadaic acid of NEmediated modulation of sensory neuron calcium current implicates the involvement of a phosphorylationdependent signaling pathway. Comparable results have been observed in other systems as well. For example, in cardiac myocytes from guinea pig, extracellular application of okadaic acid enhances the NEinduced increase in L-typecalcium current (Hescheler et al., 1988). Similarly, membranecurrent responses to FMRFamide and serotonin in Aplysia sensory neurons are regulated by phosphatase activity (Ichinose and Byrne, 1991). In other preparations, by contrast, transmitter-induced modulation of ion channel activity may be mediated by dephosphorylation rather than phosphorylation. Somatostatin activates calciumsensitive potassium channels in pituitary tumor cells via a mechanism that is blocked by okadaic acid (White et al., 1991). As the GABA-mediated modulation of current is most often associated with a slowing of the activation kinetics, it is tempting to speculate that kinetic slowing is characteristic of transmitter responses which are independent of phosphorylation. The biochemical mechanism underlying the kinetic slowing is unknown; it can be at least partially reversed, however, with strong depolarizing stimuli (Bean, 1989; Elmslie et al., 1990). In some preparations, this modulatory component may be responsible for tonic inhibition of resting calcium channels (Kasai, 1991) and is thought to underlie facilitation of calcium current observed with stimulus trains (Kasai, 1991; Artalejo et al., 1991). The fact that hydrolysis-resistant analogs of GTP can lead to G protein activation and produce the voltage-dependent inhibition and kinetic slowing of calciumcurrent hasleadsometosuggestadirect interaction between G protein subunits and the calcium channel (Hille, 1992; Yatani et al., 1987). Proof of this hypothesis awaits methods to study N channels successfully in excised patches or planar lipid bilayers.
Other signaling pathways responsible for the transduction of GABA-induced inhibition of calcium current must beconsidered. GABAe receptors are known to be coupled to a variety of biochemical pathways. Although they inhibit adenylyl cyclase activity (Bowery et al., 1990), a role for CAMP in the calcium current modulation is considered unlikely (Forscher and Oxford, 1985; Holz et al., 1986). GABAe receptors are also coupled, through G proteins, to activation of phospholipase AZ and the generation of arachidonic acid (Bowery et al., 1990). The role of arachidonate or its metabolites in regulating sensory neuron calcium channels must betested, as they control channel function in other preparations. In Aplysia neurons, for example, metabolites of the lipoxygenase pathway regulate excitability through alterations in potassium channel function (Buttner et al., 1989); similar mechanisms may underlie the action of GABA in hippocampal pyramidal neurons as well (Gage, 1992). It is becoming clear that a single population of calcium channels can be modulated by multiple pathways. In addition to the PKC-dependent and -independent mechanisms reported here, results from other preparations suggest a multiplicity of mechanisms involved in the regulation of high voltage-activated calcium current. In rat sympathetic neurons, N-type calcium currents are inhibited by transmitters that activate both pertussis toxin-sensitive and -resistant pathways (Wanke et al., 1987; Song et al., 1989,199l). The two pathways have been further elucidated by Hille and coworkers. Activation of muscarinic receptors decreases calcium current, employing both fast and slow mechanisms. The fast pathway is membrane delimited, and it has pertussis toxin-sensitive and -resistant components. The slow pathway requires a calcium-dependent second messenger and is voltage independent (Beech et al., 1991; Bernheim et al., 1991; Mathie et al., 1992). NE uses two pathways as wellboth fast-and its effects are voltage dependent and calcium independent (Beech et al., 1992). These results on sympathetic neurons, coupled with those from our own laboratory, indicate that modulation of calcium channel function, even within a single cell, can be quite complex, and any one hypothesis is unlikely to explain the large number of disparate observations that have been reported for transmitter-mediated modulation of high voltageactivated calcium channels. Proposing a model even for chick dorsal root ganglion neurons would be, at this point, premature. The pathway eventually must include pertussis toxin-sensitive G proteins, PKCmediated phosphorylation that is sensitive to okadaic acid (for NE only), and w-conotoxin GVIA-sensitive (N-type) calcium channels. As additional elements are unknown, a variety of potential models are consistent with our data; further experimentation is required, however, before proposing one will be a useful exercise. Sorting out these pathways and their interactions will remain a critical area for continued investigation in the coming decade. It will be particularly important
Neuron 758
to determine whether multiple pathways can converge at the level of a single channel, as suggested in the experiments of Shen and Surprenant (1991). NE and GABA (Cox and Dunlap, 1992), as well as OAG, selectively inhibit oconotoxin-sensitive calcium channels. The degree of channel heterogeneity in this pharmacological class is unknown. The partial occlusion of GABA-mediated inhibition produced in sensory neurons responding robustly to OAC, however, is consistent with the idea that the same channels might be targets for both OAG and GABA. Less likely is the alternative that the saturable element in these occlusion experiments is proximal to the channel (but distal to PKC activation) and that different N channels are targeted by the different transmitters. The evolution of multiple mechanismsforthecontrol of calcium channel function may be one way in which the specificity of receptor-effector interactions is ensured (Hille, 1992). In any case, the coexistence of several pathways for the control of calcium influx underscores the significance of calcium channel modulation in regulating cellular function. Experimental
Procedures
Dorsal root ganglia were dissected from II- to 12day-old chick embryos. Tissue was incubated at 37OC for 50 min in calciumand magnesium-free saline and dissociated by trituration with a fire-polished Pasteur pipette. Cells were plated in collagencoateddishesandgrown in Dulbecco’s modified Eagle’s medium supplemented with heat-inactivated 10% horse serum, 5% chick embryo extract, 50 U/ml penicillin, 50 pglml streptomycin, and nerve growth factor. Cells were used for experiments 3-5 days after plating. For recording calcium currents, the pipette internal solution contained 150 mM CsCI, 10 mM HEPES, 5 Mg-ATP, and 5 mM BAPTA. The external saline solution contained 133 mM NaCI, 1 mM CaC12, 0.8 mM MgC12, 10 mM tetraethylammonium chloride, 25 mM HEPES, 12.5 mM NaOH, 5 mM glucose, and 0.3 PM 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 to 2.5 MCI. A List EPC-7 amplifier (Medical Systems) was used to record calcium currents in the tight-seal, whole-cell configuration (Hamill et al., 1981). Data were acquired at 10 kHz using an Atari Mega 4 STE. Software was obtained from lnstrutech Corporation (Elmont, NY). A standard P/4 protocol was used for leakage subtraction of thecurrents prior to analysis. Currents from population studies are reported as mean + standard deviation. Because cells from different platings varied significantly in their responsiveness to transmitter, population studies were performed on cells within the same plating. Percent decreases in calcium current are reported relative to peak control calcium current. For the occlusion experiments, in which OAG was applied, followed by application of a transmitter (in the continual presence of OAG), percent inhibition produced by the transmitters is calculated relative to peak current measured in the presence of OAG alone. PKCI 19-31 (Bachem) was dissolved to a stock concentration of 1 mM in 0.5 M acetic acid and stored at -8OOC. For experiments, the stock solution was diluted to 3 WM in internal saline solution. Internal solutions used for control cells contained 1.5 mM acetic acid to control for potential effects of the vehicle. OAG was purchased from Avanti Polar Lipids as a chloroform solution. The ampulewas opened, and the chloroform was evap orated under nitrogen. The OAG was redissolved in DMSO as a 2.5 mM stock solution and stored at -80°C in 10 ~1 aliquots. For experiments, fresh 60 PM OAG solutions were prepared each
day by diluting the stock solution into external saline. The final concentration of DMSO was 0.1%. Solutions used to test control cells also contained 0.1% DMSO to control for possible effects of the vehicle. The ammonium salt of okadaic acid (LC Services) was dissolved in DMSO and stored at -80°C as a stock solution of 0.1 mglml. On the day of use, 100 nM okadaic acid solutions were prepared by dilution of the stock solution into the intracellular pipette solution. Internal solutions used for control cells contained 0.1% DMSO to control for possible effects of vehicle. CABA and NE solutions were prepared daily in the extracellular recording solution used to bathe the sensory neurons. Acknowledgments This work was supported by PHS grant NS164B3 and a supple ment to PHS grant NS28815 from the Program for Underrepresented Minorities. 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
October
16, 1992; revised
January
29,1993.
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