64
Neuroscwnce Research, 10 (1991) 64 70 i', 1991 Elsevier Scientific Publishers Ireland, Ltd. 0168-0102/91/$03.50
NEURES 00424
Short Communications
Cyclic AMP elicits biphasic current whose activation is mediated through protein phosphorylation in snail neurons K a z u k o W a t a n a b e and K o z o Funase Department of Physiology, Gifu University School of Medicine, Gifu (Japan) (Received 9 October 1990; Accepted 23 October 1990)
Key words: Cyclic AMP;
Sodium current; Calcium current; Potassium Cyclic AMP-dependent protein phosphorylation; Snail neuron
current;
Protein
kinase;
SUMMARY The involvement of protein phosphorylation in cAMP-induced transmembrane current was tested electrophysiologically and pharmacologically in identified neurons of the Japanese land snail, Euhadra peliomphala. Intracellular injection of cAMP elicited a biphasic transmembrane current (cAMP current) which consisted of inward and outward components. The inward component was blocked with Na+-free, Ca2+-free saline and the outward component abolished by either application of tetraethylammonium or a long-lasting exposure to caffeine in Ca2+-free saline. The cAMP current was completely suppressed by the protein kinase inhibitors, protein kinase inhibitor isolated from rabbit muscle or isoquinoline sulfonamide (H-8). The catalytic subunit of cAMP-dependent protein kinase transiently restored the cAMP current suppressed by H.8 nearly to pre-H-8 level. These findings suggest that protein phosphorylation may be an intermediate step in the activation process of the cAMP current.
There exists a surfeit of evidence on the involvement of cyclic AMP (cAMP) in alterations of neuronal membrane properties. In molluscan neurons, intracellular injec, tion of cAMP or cAMP analogs, under voltage-clamp conditions, causes a variety of specific cAMP-dependent ionic currents, attributing to increased sodium conductance it.a2 and both increased 3,8,20 and decreased 7,19 conductance to K +. In addition, it has been shown that the internal perfusion of Helix neurons with the catalytic subunit (CS) of cAMP-dependent protein kinase could enhance Ca 2+-activated potassium conductance 6,9, suggesting a cAMP-dependent protein phosphorylation-related mechanism for the activation of potassium channels. Similarly, Adams and Levitan ~ have demonstrated in Aplysia neurons that cAMP-mediated increase in the potassium conductance is blocked by intracellular injection of protein kinase inhibitor. On the contrary, the CS has been indicated to close the serotonin-sensitive potassium channels of Aplysia sensory neurons 18 In the present paper, we report a cAMP-elicited biphasic current in identified snail neurons whose activation is mediated through protein phosphorylation. Correspondence." Dr. Kazuko Watanabe, Ph.D., Department of Physiology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500, Japan.
65 Experiments were performed on neurons RC-1 and RC-2, identified in the right caudal cluster of an isolated subesophageal ganglion of the Japanese land snail, Euhadra peliomphala 14. Procedures for dissection and the basic formula for the n o r m a l snail saline were as previously described 16. Na+_free saline was m a d e by substituting Tris-hydroxymethyl a m i n o m e t h a n e for N a ÷, while CaE+-free saline was m a d e b y replacing Ca 2+ with an equimolecular a m o u n t of Co 2 +. The arrangements for the voltage-clamp circuitry have been described elsewhere 15,21. In brief, both recording and current microelectrodes, with acceptable tip resistance ranging from 1 to 5 MI2, were filled with 3 M KC1. A third double-barrel electrode was used for pressure injection. The m e m b r a n e potential of these neurons was usually held at - 4 0 mV. C l a m p current was meausred by a virtual g r o u n d circuit through an I-V converter. C o n s t a n t pressure (1.0 k g / c m 2) injection lasting for 1 s into the neurons was carried out. One barrel of the microelectrode with an outside tip diameter of about 1/~m was filled with 0.1 M KCI ( p H 7.2) containing c A M P (0.1 mM), CS (1.0 m g / m l ) or 5,5'-dethiobis(2-nitrobenzoic acid) (Nbs2)-inactivated CS (1.0 m g / m l ) . The other barrel was filled with 0.1 M KC1 ( p H 7.2) containing CS or c A M P - d e p e n d e n t protein kinase inhibitor (PKI, 0.1 ~ M ) as previously described 14.15. Both CS and protein kinase inhibitor were obtained from bovine heart and from rabbit muscle, respectively, and purified in the laboratory of Dr. M. Terao, w h o also kindly provided Nbs2-inactivated CS. These were backloaded into the tip of an injection microelectrode. To test the effect of the pressure itself, a solution without test material was injected into the neurons. N o significant change in the t r a n s m e m b r a n e current was noted. In RC-1 or RC-2 neurons which normally exhibit regular firing 13,16, intracellular pressure injection of 0.1 m M c A M P caused an increase in discharge frequency with slight m e m b r a n e depolarization, followed by a prolonged hyperpolarization of the m e m b r a n e (Fig. 1A). In the voltage-clamped neurons at - 4 0 mV, c A M P , intracellularly injected in the same way, also elicited a biphasic current which was a t r a n s m e m b r a n e inward current followed by o u t w a r d current (Fig. 1B), resembling so-called ' c A M P - c u r r e n t s ' seen only when a small a m o u n t of c A M P was injected into Helix neurons 11. Therefore, this current
50 mV 10s B
10s Fig. 1. (A) Effect of intracellular injection of 0.1 mM cAMP on Euhadra neurons. Resting membrane potentials ranged between -43 and -40 mV. (B) Transmembrane current (cAMP current) elicited when cAMP (0.1 mM) was injected into the voltage-clamped neurons at -40 mV. The level of the current before injection of cAMP is indicated by a dashed line. Neurons were perfused with normal saline. Pressure injection of 1 kg/cm2 lasting for 1 s was carried out in this and subsequent experiments. Dots, arrow and arrowhead indicate the injection time and the inward and outward components of the cAMP current, respectively.
66 PKI, nM (o) 0
1
10
100
0.1
1.0
n o ~ 50
L
,~
H-8, pM (e)
Fig. 2. Inhibition of cAMP-dependent protein kinase (PK) activity from Euhadra ganglia by PKI or H-8. PK activity was measured in a 12000× g supernatant prepared from Euhadra ganglion homogenates. The tissue preparation and assay conditions were as described by Onozuka et al. 16. Cyclic AMP-dependent PK activity was measured (26/tg of Euhadra protein per assay) as the difference in activity in the presence and absence of 1.0/~M cAMP. The basal activity measured in the absence of cAMP was 46 pmol of 32p per min/mg of protein. The values on the abscissa are final concentrations of either PKI or H-8 in the assay. Each point represents the mean _+SEM for 5-6 samples. This experiment was repeated 4 times with similar results. will be denoted as ' c A M P current'. The P K I is heat-stable 10-kDa protein that possesses high-affinity binding to CS and is able to block its activity 2,5. In the present experiments, P K I from rabbit skeletal muscle or isoquinoline-sulfonamide (H-8), a m e m b r a n e - p e r m e a ble protein kinase inhibitor, dose-dependently inhibited c A M P - d e p e n d e n t protein phosphorylation in extracts from Euhadra ganglia (Fig. 2). Accordingly, the effect of P K I (0.1 /~M) on the c A M P current was examined by its prior injection into the neurons. As seen in Figure 3B.b, c A M P (0.1 mM), when injected into the neurons wherein P K I had been delivered through the other barrel of the injection electrode, did not p r o d u c e any t r a n s m e m b r a n e current. Similar experiments carried out by perfusion with another inhibitor (H-8) revealed that the c A M P current was also inhibited in a dose-dependent m a n n e r (Fig. 3C.b, c). We further measured the t r a n s m e m b r a n e current when c A M P and CS were simultaneously injected into the neurons which the c A M P current had been completely suppressed by H-8. N o c A M P (Fig. 3D.a) but c A M P together with CS (Fig. 3D.b) restored the H-8-suppressed current nearly to the level which corresponded to the c A M P current (Fig. 3A) without any c A M P - d e p e n d e n t PKI. However, Nbs2-inactivated CS, intracellularly injected in the same manner, was ineffective on the current (data not shown). These results are taken to suggest that c A M P - d e p e n d e n t protein kinase m a y be involved in the c A M P current. In order to evaluate the ionic mechanisms for the generation of the c A M P current, the c A M P currents were examined in either CaE+-free or Na+-free saline, since b o t h N a + and Ca 2÷ influxes contribute to the rising phase of the action potential in Euhadra neurons 13 In Na+-free saline, the c A M P current was reduced by a b o u t 82 + 7% (mean + SD, n = 6) in the inward c o m p o n e n t and b y about 20 + 4% (mean + SD, n = 6) in the o u t w a r d c o m p o n e n t (Fig. 4A.b), respectively. Intracellular injection of c A M P into the neurons in
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10s Fig. 3. Effect of the protein kinase inlfibitors on the c A M P current. (A) Cyclic A M P current in normal saline. (B) Effect of PKI on the c A M P current. Currents were measured before (a) or after c A M P injection (b) into the neuron 30 rain after intracellular application of PKI (0.1 #M). (C) Effect of H-8 on the c A M P current. Current was measured before (a) or after c A M P injection into the neuron 30 rain after perfusion with 0.5 (b) or 1.0 ~ M H-8 (c). (D) Effect of CS on the suppressed c A M P current by H-8. Cyclic A M P (a) or c A M P and CS (b) was injected into the neurons 30 rain after perfusion with 1.0/~M H-8. The level of the current before their injection is indicated by dotted lines. Dots, arrows and arrowheads indicate the injection time and the inward and outward components of the c A M P current. Holding potential (V h), - 4 0 mV.
the same way, pretreated with Na+-free saline containing 1.0/~M H-8, produced neither the inward component nor the outward component (Fig. 4A.c). In contrast, the cAMP current in Ca2+-free saline resulted in the generation of transmembrane inward current which was reduced by about 10 _+ 3% (mean _+ SD, n = 6) (Fig. 4B.b), whereas the outward component was attenuated to about 30 -t- 8% (mean +_ SD, n = 6) (arrowhead in Fig. 4B.b). These components were also abolished by pretreatment with the same concentration of H-8 (data not shown). Taken together, it is suggested that cAMP elicits sodium and calcium currents through protein phosphorylation, and that the outward component may be the potassium current linked with Ca 2÷ influx. Since tetraethylammonium (TEA) blocks Ca2+-activated potassium current in Euhadra neurons 15, the latter suggestion was further confirmed by meausuring the cAMP current in the presence of
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Fig. 4. Effect of various salines on the c A M P current. (A) Cyclic A M P current measured in either normal saline (a) or Na+-free saline without (b) or with 1.0 # M H-8 (c). (B) c A M P current measured in normal saline (a) or Ca2÷-free saline (b). (C) Currents either when c A M P was intracellularly injected 10 rain after perfusion with normal saline containing 40 m M TEA (a) or Na+-free, Ca2+-free saline (b) or when c A M P and CS were simultaneously injected into the neurons 30 rain after perfusion with Na+-free, Ca2+-free saline without (c) or with 10 m M caffeine (d). The level of the current before their injection is indicated by dotted lines. Dots, arrows and arrowheads indicate the injection time and the inward and outward components of the c A M P current. Vh= - 4 0 m V .
TEA in the perfusate. As shown in Figure 4C.a, the outward component disappeared about 10 min after perfusion with 40 mM TEA. However, a much weaker outward current of about 0.3 + 0.1 nA (mean + SD, n = 7) persisted even in Na÷-free, Ca2÷-free saline (Fig. 4C.b). A similar but transient outward current was observed when cAMP and CS were simultaneously injected into neurons pretreated with H-8 (Fig. 4C.c); however, their injection after long-lasting exposure of the neurons to 10 mM caffeine no longer had any significant effect (Fig. 4C.d). This outward current is assumed to be carried b y K ÷ which is dependent on the released Ca 2÷ from the intracellular reservoir through cAMP-dependent protein kinase. We have presented experimental evidence in identified Euhadra neurons which demonstrates that protein phosphorylation is a necessary step in the process leading to cAMP-induced activation of transmembrane current. Onozuka et al. 15 have shown in the Euhadra neurons that the protein kinase inhibitors, PKI isolated from rabbit muscle and H-8, abolish dibutyryl cAMP-induced development of negative slope resistance in steady state I-V curve which is dependent on [Na÷]0 and concluded that this second messenger elicits sodium current through protein phosphorylation. Costa and Catterall 4 reported that the sodium channel could be phosphorylated in lyzed synaptosomes by an exogenous catalytic subunit of cAMP-dependent protein kinase and in intact synaptosomes in the presence of 8-bromo-cAMP, which presumably acts by activating an endogenous cAMPdependent protein kinase. Furthermore, they have observed some effects of 8-bromo cAMP-stimulated phosphorylation on the slow influx of radioactive sodium into synaptosomes. On the other hand, the energy-dispersive type of electron probe X-ray mieroanalysis using the same neurons as those used in the present experiments revealed that intraeeltular injection of CS stimulates Ca 2+ release from the intracellular reservoir 15. Considering the present results together with this finding, the outward component of the cAMP
69 current is probably the potassium current linked with both the intracellularly released Ca 2÷ and the influxed Ca 2÷ which were arbitrated by cAMP-dependent protein phosphorylation. Kaczemarek et al. 10 showed that microinjection of CS broadens calcium action potentials of Aplysia bag cell neurons. An enhancement of calcium current by intracellular injection of CS has also been shown by Osterrier et al. 17 for cardiac myocytes. Furthermore, Levitan et al. 1,6.9 have suggested that the Ca2+-dependent potassium channel in Helix neurons is modulated by cAMP dependently phosphorylating either the potassium channel itself or something intimately associated with the channel. However, our recent observations regarding the molecular mechanism of the Ca 2÷activated potassium channel in Euhadra neurons suggest that the TEA-sensitive outward component of the cAMP current suppressed by H-7, a general protein kinase inhibitor, is transiently restored by intracellular injection of Ca2+/calmodulin-dependent protein kinase II, but not when CS is intracellularly applied in the same way (unpublished data), thus implying the possibility that the activation of this component may be finally mediated by Ca2+/calmodulin-dependent protein phosphorylation. In conclusion, we suggest that protein phosphorylation may be a necessary step in the activation process of the cAMP-elicited current in Euhadra neurons. Further studies will focus on elucidating the phosphoproteins involved in channel opening. ACKNOWLEDGEMENTS
This work was supported in part by grants from the Tokai Foundation for the Encouragement of Academic Work and the Narishige Neuroscience Trust. REFERENCES 1 Adams, W.B. and Levitan, I.B., Intracellular injection of protein kinase inhibitor blocks the serotonin-induced increase in K + conductance in Aplysia neuron R15, Proc. Natl. Aead. Sci. USA, 79 (1982) 3877-3880. 2 Ashby, C.D. and Walsh, D.A., Characterization of the interaction of a protein inhibitor with 3',5'-monophosphate-dependent protein kinase, J. Biol. Chem., 247 (1972) 6637-6642. 3 Benson, J.A. and Levitan, I.B., Serotonin increases an anomalously rectifying K + current in the Aplysia neuron R 15, Proc. Natl. Acad. Sci. USA, 80 (1983) 3522-3525. 4 Costa, M.R., Casnellie, J.E. and Catterall, W.A., Selective phosphorylation of the a subunit of the sodium channel by cAMP-dependent protein kinase, J. Biol. Chem., 257 (1982) 7918-7921. 5 Demaille, J.G., Perers, K.A. and Fisher, E.H., Isolation and properties of the rabbit skeletal muscle protein inhibitor of adenosine 3',5'-monophosphate dependent protein kinase, Biochemstry, 16 (1977) 3080-3086. 6 Depeyer, J.E., Cachelin, A.B., Levitan, I.B. and Reuter, H., Ca2+-activated K + conductance in internally perfused snail neurons is enhanced by protein phosphorylation, Proc. Natl. Acad. Sci. USA, 79 (1982) 4207-4211. 7 Deterre, P., Paupardin-Tritsch, D., Bockhaert, J. and Gerschenfeld, H.M., Role of cyclic AMP in a serotonin-evoked slow inward current in snail neurons, Nature, 290 (1981) 783-785. 8 Drummond, A., Benson, J. and Levitan, I.B., Serotonin-induced hyperpolarization of an identified Aplysia neuron is mediated by cyclic AMP, Proc. Natl. Acad. Sci. USA, 77 (1980) 5013-5017. 9 Ewald, D., Williams, A. and Levitan, I.B., Modulation of single Ca++-dependent K ÷ channel activity by protein phosphorylation, Nature, 315 (1985) 503-506. 10 Kaczmarek, L.K., Jennings, K.R., Sturmwasser, F., Nairn, A.C., Walter, N.U., Wilson, F.D. and Greengard, P., Microinjection of catalytic subunit of cyclic AMP-dependent protein kinase enhances calcium action potentials of bag cell neurons in cell culture, Proc. Natl. Acad. Sci. USA, 77 (1980) 7487-7491. 11 Kononenko, N.I., Kostyuk, P.C. and Shcherbatko, A.D., The effect of intracellular cAMP injection on stationary membrane conductance and voltage- and time-dependent ionic currents in identified snail neurons, Brain Res., 268 (1983) 321-338. 12 McCroham, C.R. and Gillette, R., Cyclic AMP-stimulated sodium current in identified feeding neurons of Lymaea stagnalis, Brain Res., 438 (1988) 115-123.
70 13 Onozuka, M., Furuichi, H., Kishii, K. and lmai, S., Membrane properties and intracellular biochemical processes during vasopressin-induced bursting activity in snail neurons, Neurosci. Res., 4 (1986) 37-50. 14 Onozuka, M., Imai, S., Deura, S., Nishiyama, K. and Ozono, S., Stimulation of sodium current by cyclic AMP is mediated through protein phosphorylation in Euhadra neurons, Experientia, 44 (1988) 996-998. 15 Onozuka, M., lmai, S., Kubo, K., Deura, S., Nishiyama, K. and Ozono, S., Cyclic AMP-dependent protein phosphorylation is involved in activation of the potassium current associated with endogenous cellular calcium in Euhadra neurons, Brain Res.~ 473 (1988) 401-405. 16 Onozuka, M., Kishii, K., Furuichi, H. and Sugaya, E., Behavior of intracellular cyclic nucleotide and calcium in pentylenetetrazole-induced bursting activity in snail neurons, Brain Res., 269 (1983) 277-286. 17 Osterrieder, W., Brum, G., Hescheter, J. and Trautwein, W., Injection of subunit of cyclic AMP-dependent protein kinase into cardiac myocytes modulates Ca 2+ current, Nature, 298 (1982) 576-578. 18 Schuster, M.J., Camardo, J.S., Siegelbaum, S.A. and Kandel, E.R., Cyclic AMP-dependent protein kinase closes the serotonin-sensitive K + channels of Aplysia sensory neurons in cell-free membrane patches, Nature, 313 (1985) 392-395. 19 Siegelbaum, S.A., Camardo, J.S. and Kandel, E.R., Serotonin and cyclic AMP close single K ~ channels in Aplysia sensory neurones, Nature, 299 (1982) 413-417. 20 Treistman, S.N., Effect of adenosin 3',5'-monophosphate neuronal pacemaker activity: a voltage clamp analysis, Science, 211 (1981) 59-61. 21 Watanabe, K. and Gola, M., Forskolin interaction with voltage-dependent K channels in Helix is not mediated by cyclic nucleotides, Neurosci. Left., 78 (1987) 211-216.
Neuroscwnce Research, 10 (1991) 64 70 i', 1991 Elsevier Scientific Publishers Ireland, Ltd. 0168-0102/91/$03.50
NEURES 00424
Short Communications
Cyclic AMP elicits biphasic current whose activation is mediated through protein phosphorylation in snail neurons K a z u k o W a t a n a b e and K o z o Funase Department of Physiology, Gifu University School of Medicine, Gifu (Japan) (Received 9 October 1990; Accepted 23 October 1990)
Key words: Cyclic AMP;
Sodium current; Calcium current; Potassium Cyclic AMP-dependent protein phosphorylation; Snail neuron
current;
Protein
kinase;
SUMMARY The involvement of protein phosphorylation in cAMP-induced transmembrane current was tested electrophysiologically and pharmacologically in identified neurons of the Japanese land snail, Euhadra peliomphala. Intracellular injection of cAMP elicited a biphasic transmembrane current (cAMP current) which consisted of inward and outward components. The inward component was blocked with Na+-free, Ca2+-free saline and the outward component abolished by either application of tetraethylammonium or a long-lasting exposure to caffeine in Ca2+-free saline. The cAMP current was completely suppressed by the protein kinase inhibitors, protein kinase inhibitor isolated from rabbit muscle or isoquinoline sulfonamide (H-8). The catalytic subunit of cAMP-dependent protein kinase transiently restored the cAMP current suppressed by H.8 nearly to pre-H-8 level. These findings suggest that protein phosphorylation may be an intermediate step in the activation process of the cAMP current.
There exists a surfeit of evidence on the involvement of cyclic AMP (cAMP) in alterations of neuronal membrane properties. In molluscan neurons, intracellular injec, tion of cAMP or cAMP analogs, under voltage-clamp conditions, causes a variety of specific cAMP-dependent ionic currents, attributing to increased sodium conductance it.a2 and both increased 3,8,20 and decreased 7,19 conductance to K +. In addition, it has been shown that the internal perfusion of Helix neurons with the catalytic subunit (CS) of cAMP-dependent protein kinase could enhance Ca 2+-activated potassium conductance 6,9, suggesting a cAMP-dependent protein phosphorylation-related mechanism for the activation of potassium channels. Similarly, Adams and Levitan ~ have demonstrated in Aplysia neurons that cAMP-mediated increase in the potassium conductance is blocked by intracellular injection of protein kinase inhibitor. On the contrary, the CS has been indicated to close the serotonin-sensitive potassium channels of Aplysia sensory neurons 18 In the present paper, we report a cAMP-elicited biphasic current in identified snail neurons whose activation is mediated through protein phosphorylation. Correspondence." Dr. Kazuko Watanabe, Ph.D., Department of Physiology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500, Japan.
65 Experiments were performed on neurons RC-1 and RC-2, identified in the right caudal cluster of an isolated subesophageal ganglion of the Japanese land snail, Euhadra peliomphala 14. Procedures for dissection and the basic formula for the n o r m a l snail saline were as previously described 16. Na+_free saline was m a d e by substituting Tris-hydroxymethyl a m i n o m e t h a n e for N a ÷, while CaE+-free saline was m a d e b y replacing Ca 2+ with an equimolecular a m o u n t of Co 2 +. The arrangements for the voltage-clamp circuitry have been described elsewhere 15,21. In brief, both recording and current microelectrodes, with acceptable tip resistance ranging from 1 to 5 MI2, were filled with 3 M KC1. A third double-barrel electrode was used for pressure injection. The m e m b r a n e potential of these neurons was usually held at - 4 0 mV. C l a m p current was meausred by a virtual g r o u n d circuit through an I-V converter. C o n s t a n t pressure (1.0 k g / c m 2) injection lasting for 1 s into the neurons was carried out. One barrel of the microelectrode with an outside tip diameter of about 1/~m was filled with 0.1 M KCI ( p H 7.2) containing c A M P (0.1 mM), CS (1.0 m g / m l ) or 5,5'-dethiobis(2-nitrobenzoic acid) (Nbs2)-inactivated CS (1.0 m g / m l ) . The other barrel was filled with 0.1 M KC1 ( p H 7.2) containing CS or c A M P - d e p e n d e n t protein kinase inhibitor (PKI, 0.1 ~ M ) as previously described 14.15. Both CS and protein kinase inhibitor were obtained from bovine heart and from rabbit muscle, respectively, and purified in the laboratory of Dr. M. Terao, w h o also kindly provided Nbs2-inactivated CS. These were backloaded into the tip of an injection microelectrode. To test the effect of the pressure itself, a solution without test material was injected into the neurons. N o significant change in the t r a n s m e m b r a n e current was noted. In RC-1 or RC-2 neurons which normally exhibit regular firing 13,16, intracellular pressure injection of 0.1 m M c A M P caused an increase in discharge frequency with slight m e m b r a n e depolarization, followed by a prolonged hyperpolarization of the m e m b r a n e (Fig. 1A). In the voltage-clamped neurons at - 4 0 mV, c A M P , intracellularly injected in the same way, also elicited a biphasic current which was a t r a n s m e m b r a n e inward current followed by o u t w a r d current (Fig. 1B), resembling so-called ' c A M P - c u r r e n t s ' seen only when a small a m o u n t of c A M P was injected into Helix neurons 11. Therefore, this current
50 mV 10s B
10s Fig. 1. (A) Effect of intracellular injection of 0.1 mM cAMP on Euhadra neurons. Resting membrane potentials ranged between -43 and -40 mV. (B) Transmembrane current (cAMP current) elicited when cAMP (0.1 mM) was injected into the voltage-clamped neurons at -40 mV. The level of the current before injection of cAMP is indicated by a dashed line. Neurons were perfused with normal saline. Pressure injection of 1 kg/cm2 lasting for 1 s was carried out in this and subsequent experiments. Dots, arrow and arrowhead indicate the injection time and the inward and outward components of the cAMP current, respectively.
66 PKI, nM (o) 0
1
10
100
0.1
1.0
n o ~ 50
L
,~
H-8, pM (e)
Fig. 2. Inhibition of cAMP-dependent protein kinase (PK) activity from Euhadra ganglia by PKI or H-8. PK activity was measured in a 12000× g supernatant prepared from Euhadra ganglion homogenates. The tissue preparation and assay conditions were as described by Onozuka et al. 16. Cyclic AMP-dependent PK activity was measured (26/tg of Euhadra protein per assay) as the difference in activity in the presence and absence of 1.0/~M cAMP. The basal activity measured in the absence of cAMP was 46 pmol of 32p per min/mg of protein. The values on the abscissa are final concentrations of either PKI or H-8 in the assay. Each point represents the mean _+SEM for 5-6 samples. This experiment was repeated 4 times with similar results. will be denoted as ' c A M P current'. The P K I is heat-stable 10-kDa protein that possesses high-affinity binding to CS and is able to block its activity 2,5. In the present experiments, P K I from rabbit skeletal muscle or isoquinoline-sulfonamide (H-8), a m e m b r a n e - p e r m e a ble protein kinase inhibitor, dose-dependently inhibited c A M P - d e p e n d e n t protein phosphorylation in extracts from Euhadra ganglia (Fig. 2). Accordingly, the effect of P K I (0.1 /~M) on the c A M P current was examined by its prior injection into the neurons. As seen in Figure 3B.b, c A M P (0.1 mM), when injected into the neurons wherein P K I had been delivered through the other barrel of the injection electrode, did not p r o d u c e any t r a n s m e m b r a n e current. Similar experiments carried out by perfusion with another inhibitor (H-8) revealed that the c A M P current was also inhibited in a dose-dependent m a n n e r (Fig. 3C.b, c). We further measured the t r a n s m e m b r a n e current when c A M P and CS were simultaneously injected into the neurons which the c A M P current had been completely suppressed by H-8. N o c A M P (Fig. 3D.a) but c A M P together with CS (Fig. 3D.b) restored the H-8-suppressed current nearly to the level which corresponded to the c A M P current (Fig. 3A) without any c A M P - d e p e n d e n t PKI. However, Nbs2-inactivated CS, intracellularly injected in the same manner, was ineffective on the current (data not shown). These results are taken to suggest that c A M P - d e p e n d e n t protein kinase m a y be involved in the c A M P current. In order to evaluate the ionic mechanisms for the generation of the c A M P current, the c A M P currents were examined in either CaE+-free or Na+-free saline, since b o t h N a + and Ca 2÷ influxes contribute to the rising phase of the action potential in Euhadra neurons 13 In Na+-free saline, the c A M P current was reduced by a b o u t 82 + 7% (mean + SD, n = 6) in the inward c o m p o n e n t and b y about 20 + 4% (mean + SD, n = 6) in the o u t w a r d c o m p o n e n t (Fig. 4A.b), respectively. Intracellular injection of c A M P into the neurons in
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10s Fig. 3. Effect of the protein kinase inlfibitors on the c A M P current. (A) Cyclic A M P current in normal saline. (B) Effect of PKI on the c A M P current. Currents were measured before (a) or after c A M P injection (b) into the neuron 30 rain after intracellular application of PKI (0.1 #M). (C) Effect of H-8 on the c A M P current. Current was measured before (a) or after c A M P injection into the neuron 30 rain after perfusion with 0.5 (b) or 1.0 ~ M H-8 (c). (D) Effect of CS on the suppressed c A M P current by H-8. Cyclic A M P (a) or c A M P and CS (b) was injected into the neurons 30 rain after perfusion with 1.0/~M H-8. The level of the current before their injection is indicated by dotted lines. Dots, arrows and arrowheads indicate the injection time and the inward and outward components of the c A M P current. Holding potential (V h), - 4 0 mV.
the same way, pretreated with Na+-free saline containing 1.0/~M H-8, produced neither the inward component nor the outward component (Fig. 4A.c). In contrast, the cAMP current in Ca2+-free saline resulted in the generation of transmembrane inward current which was reduced by about 10 _+ 3% (mean _+ SD, n = 6) (Fig. 4B.b), whereas the outward component was attenuated to about 30 -t- 8% (mean +_ SD, n = 6) (arrowhead in Fig. 4B.b). These components were also abolished by pretreatment with the same concentration of H-8 (data not shown). Taken together, it is suggested that cAMP elicits sodium and calcium currents through protein phosphorylation, and that the outward component may be the potassium current linked with Ca 2÷ influx. Since tetraethylammonium (TEA) blocks Ca2+-activated potassium current in Euhadra neurons 15, the latter suggestion was further confirmed by meausuring the cAMP current in the presence of
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Fig. 4. Effect of various salines on the c A M P current. (A) Cyclic A M P current measured in either normal saline (a) or Na+-free saline without (b) or with 1.0 # M H-8 (c). (B) c A M P current measured in normal saline (a) or Ca2÷-free saline (b). (C) Currents either when c A M P was intracellularly injected 10 rain after perfusion with normal saline containing 40 m M TEA (a) or Na+-free, Ca2+-free saline (b) or when c A M P and CS were simultaneously injected into the neurons 30 rain after perfusion with Na+-free, Ca2+-free saline without (c) or with 10 m M caffeine (d). The level of the current before their injection is indicated by dotted lines. Dots, arrows and arrowheads indicate the injection time and the inward and outward components of the c A M P current. Vh= - 4 0 m V .
TEA in the perfusate. As shown in Figure 4C.a, the outward component disappeared about 10 min after perfusion with 40 mM TEA. However, a much weaker outward current of about 0.3 + 0.1 nA (mean + SD, n = 7) persisted even in Na÷-free, Ca2÷-free saline (Fig. 4C.b). A similar but transient outward current was observed when cAMP and CS were simultaneously injected into neurons pretreated with H-8 (Fig. 4C.c); however, their injection after long-lasting exposure of the neurons to 10 mM caffeine no longer had any significant effect (Fig. 4C.d). This outward current is assumed to be carried b y K ÷ which is dependent on the released Ca 2÷ from the intracellular reservoir through cAMP-dependent protein kinase. We have presented experimental evidence in identified Euhadra neurons which demonstrates that protein phosphorylation is a necessary step in the process leading to cAMP-induced activation of transmembrane current. Onozuka et al. 15 have shown in the Euhadra neurons that the protein kinase inhibitors, PKI isolated from rabbit muscle and H-8, abolish dibutyryl cAMP-induced development of negative slope resistance in steady state I-V curve which is dependent on [Na÷]0 and concluded that this second messenger elicits sodium current through protein phosphorylation. Costa and Catterall 4 reported that the sodium channel could be phosphorylated in lyzed synaptosomes by an exogenous catalytic subunit of cAMP-dependent protein kinase and in intact synaptosomes in the presence of 8-bromo-cAMP, which presumably acts by activating an endogenous cAMPdependent protein kinase. Furthermore, they have observed some effects of 8-bromo cAMP-stimulated phosphorylation on the slow influx of radioactive sodium into synaptosomes. On the other hand, the energy-dispersive type of electron probe X-ray mieroanalysis using the same neurons as those used in the present experiments revealed that intraeeltular injection of CS stimulates Ca 2+ release from the intracellular reservoir 15. Considering the present results together with this finding, the outward component of the cAMP
69 current is probably the potassium current linked with both the intracellularly released Ca 2÷ and the influxed Ca 2÷ which were arbitrated by cAMP-dependent protein phosphorylation. Kaczemarek et al. 10 showed that microinjection of CS broadens calcium action potentials of Aplysia bag cell neurons. An enhancement of calcium current by intracellular injection of CS has also been shown by Osterrier et al. 17 for cardiac myocytes. Furthermore, Levitan et al. 1,6.9 have suggested that the Ca2+-dependent potassium channel in Helix neurons is modulated by cAMP dependently phosphorylating either the potassium channel itself or something intimately associated with the channel. However, our recent observations regarding the molecular mechanism of the Ca 2÷activated potassium channel in Euhadra neurons suggest that the TEA-sensitive outward component of the cAMP current suppressed by H-7, a general protein kinase inhibitor, is transiently restored by intracellular injection of Ca2+/calmodulin-dependent protein kinase II, but not when CS is intracellularly applied in the same way (unpublished data), thus implying the possibility that the activation of this component may be finally mediated by Ca2+/calmodulin-dependent protein phosphorylation. In conclusion, we suggest that protein phosphorylation may be a necessary step in the activation process of the cAMP-elicited current in Euhadra neurons. Further studies will focus on elucidating the phosphoproteins involved in channel opening. ACKNOWLEDGEMENTS
This work was supported in part by grants from the Tokai Foundation for the Encouragement of Academic Work and the Narishige Neuroscience Trust. REFERENCES 1 Adams, W.B. and Levitan, I.B., Intracellular injection of protein kinase inhibitor blocks the serotonin-induced increase in K + conductance in Aplysia neuron R15, Proc. Natl. Aead. Sci. USA, 79 (1982) 3877-3880. 2 Ashby, C.D. and Walsh, D.A., Characterization of the interaction of a protein inhibitor with 3',5'-monophosphate-dependent protein kinase, J. Biol. Chem., 247 (1972) 6637-6642. 3 Benson, J.A. and Levitan, I.B., Serotonin increases an anomalously rectifying K + current in the Aplysia neuron R 15, Proc. Natl. Acad. Sci. USA, 80 (1983) 3522-3525. 4 Costa, M.R., Casnellie, J.E. and Catterall, W.A., Selective phosphorylation of the a subunit of the sodium channel by cAMP-dependent protein kinase, J. Biol. Chem., 257 (1982) 7918-7921. 5 Demaille, J.G., Perers, K.A. and Fisher, E.H., Isolation and properties of the rabbit skeletal muscle protein inhibitor of adenosine 3',5'-monophosphate dependent protein kinase, Biochemstry, 16 (1977) 3080-3086. 6 Depeyer, J.E., Cachelin, A.B., Levitan, I.B. and Reuter, H., Ca2+-activated K + conductance in internally perfused snail neurons is enhanced by protein phosphorylation, Proc. Natl. Acad. Sci. USA, 79 (1982) 4207-4211. 7 Deterre, P., Paupardin-Tritsch, D., Bockhaert, J. and Gerschenfeld, H.M., Role of cyclic AMP in a serotonin-evoked slow inward current in snail neurons, Nature, 290 (1981) 783-785. 8 Drummond, A., Benson, J. and Levitan, I.B., Serotonin-induced hyperpolarization of an identified Aplysia neuron is mediated by cyclic AMP, Proc. Natl. Acad. Sci. USA, 77 (1980) 5013-5017. 9 Ewald, D., Williams, A. and Levitan, I.B., Modulation of single Ca++-dependent K ÷ channel activity by protein phosphorylation, Nature, 315 (1985) 503-506. 10 Kaczmarek, L.K., Jennings, K.R., Sturmwasser, F., Nairn, A.C., Walter, N.U., Wilson, F.D. and Greengard, P., Microinjection of catalytic subunit of cyclic AMP-dependent protein kinase enhances calcium action potentials of bag cell neurons in cell culture, Proc. Natl. Acad. Sci. USA, 77 (1980) 7487-7491. 11 Kononenko, N.I., Kostyuk, P.C. and Shcherbatko, A.D., The effect of intracellular cAMP injection on stationary membrane conductance and voltage- and time-dependent ionic currents in identified snail neurons, Brain Res., 268 (1983) 321-338. 12 McCroham, C.R. and Gillette, R., Cyclic AMP-stimulated sodium current in identified feeding neurons of Lymaea stagnalis, Brain Res., 438 (1988) 115-123.
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