C (with phorbol ester) activation. amplitudes. receptor-linked G protein (with glutamate, dopamine, F-2268) and by protein kinase

Transcription

1 Journal of Physiology (1993), 468, pp With 10 figures Printed in Great Britain ACTIVATION OF A COMMON POTASSIUM CHANNEL IN MOLLUSCAN NEURONES BY GLUTAMATE, DOPAMINE AND MUSCARINIC AGONIST BY VADIM YU. BOLSHAKOV, SVETLANA A. GAPON, ALEXANDER N. KATCHMAN AND LEV G. MAGAZANIK* From the I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St Petersburg, Russia (Received 23 April 1992) SUMMARY 1. The potassium currents evoked in isolated and identified neurones of molluscan pedal ganglia by either glutamate, dopamine or the muscarinic agonist F-2268 were investigated using voltage and patch clamp techniques. 2. Potassium currents induced by either dopamine or F-2268 could be blocked by pertussis toxin, as well as by a prolonged intracellular injection of the G protein inhibitor, GDP-,8-S. Loading the neurones with the G protein activator, GppNHp, on the other hand, induced a potassium current. This current was not additive to the currents evoked by agonist application. 3. Intracellular injection of the calcium buffer BAPTA failed to affect any of the agonist-induced currents, although it effectively blocked the after-hyperpolarization following directly evoked action potentials. 4. The activity of the potassium channels seen in cell-attached patches was greatly enhanced by application to the bath of either glutamate, dopamine, or F The only effect of an addition of agonists to the bath was to increase the open probability (PO) of the K+ channel already active in the control conditions. The identity of the spontaneously active and agonist-activated channels was concluded from the identity of their channel conductances, rectification properties and current amplitudes. 6. Phorbol-12,13-dibutyrate, when applied to the bath, induced an increase in open time and caused an increase in P., as did the agonists. Staurosporine completely prevented changes of PO induced by the phorbol ester but not those induced by the agonists. 7. The same inwardly rectifying potassium channel may be opened by both the receptor-linked G protein (with glutamate, dopamine, F-2268) and by protein kinase C (with phorbol ester) activation. 8. Strong evidence was obtained against the involvement of any known secondary * To whom correspondence should be addressed at the Laboratory of Biophysics, Sechenov Institute of Evolutionary Physiology and Biochemistry, Thorez pr. 44, St Petersburg, Russia. MS 1324

2 12 V. Yu. BOLSHAKOV AND OTHERS messenger systems (formation of nucleotides, phosphoinositide turnover and subsequent activation of protein kinase C, formation of nitric oxide, metabolism of arachidonic acid) in the transduction mechanism of F-2268-, dopamine- and glutamate-induced responses. 9. Since none of the known secondary messenger systems seems to affect the activation by agonists applied to receptors outside the patch of channels located under the patch electrode, it appears that some as yet undescribed linking system must exist that could connect the spatially separated receptor-g protein complex and the potassium channel. INTRODUCTION The co-existence on a single neurone of receptors to many different neurotransmitters and of more than one receptor subtype to any given transmitter has been shown to be ubiquitous. Furthermore, many different signal transduction pathways have been established (see, for reviews, Birnbaumer, 1990; Nathanson & Harden, 1990; Nicoll, Malenka & Kauer, 1990; Brown, 1991), permitting on the one hand, a given transmitter to have various modes of action, and making it possible, on the other hand, for different receptor-specific cell surface events to converge on common effector pathways. The last mechanism is based mainly on the coupling of receptors to GTP-binding proteins (G proteins). In many cases agonists acting on G proteins evoke an increase in potassium conductance. Accordingly, potassium channels may be a final common target of converging synaptic signals. In molluscan neurones, the convergence of synaptic signals mediated by receptors for different transmitter systems was first suggested by the data of Ascher & Chesnoy-Marchais (1982). They studied neurones of the cerebral ganglion of the Aplysia in which histamine, acetylcholine, and dopamine each elicit K+-dependent responses. A cross-potentiation of the responses was seen at low concentrations of the agonists, whereas at higher concentrations, cross-desensitization was observed. It was shown also that these interactions did not occur at the drug-specific receptor sites, thereby implying a common reaction step further downstream in the receptor-effector chain. Sasaki & Sato (1987) working on the same cells and the same responses, found that a pertussis toxin-sensitive G protein regulates the K+ conductance increase induced by all three of the transmitters. The ability of different G protein-coupled receptors to activate the potassium conductance was observed in both molluscan (Brezina, 1988; Volterra & Siegelbaum, 1990) and vertebrate neurones (Andrade, Malenka & Nicoll, 1986; Christie & North, 1988; Shen, North & Surprenant, 1992). Recently two distinct glutamate receptors (quisqualate and kainate types) controlling potassium channels through the activation of a pertussis toxin-sensitive G protein have been found in identified neurones of the mollusc Planorbarius corneus (Bolshakov, Gapon & Magazanik, 1991; Magazanik, Antonov, Bolshakov, Fedorova & Gapon, 1992; Magazanik & Bolshakov, 1992). No involvement of any known secondary messenger systems in the transduction of quisqualate- and kainateinduced potassium currents could be demonstrated (Bolshakov et al. 1992). We undertook this study (1) to look for other transmitters that might induce an increase in K+ conductance in the same identified neurones, (2) to demonstrate the mediation

3 POTASSIUM CHANNEL ACTIVATED BY AGONISTS of those responses by a pertussis toxin-sensitive G protein and to study the possible involvement of any known secondary messenger in the transduction mechanism, and (3) to identify and compare the K+ channel(s) activated by the various transmitters. It is shown here that in two identified neurones (Ped-8 and Ped-9) glutamate, dopamine and a muscarinic agonist all induce G protein-mediated K+-dependent responses, and that increased activity of a single type of K+ channel underlies the response to the three different receptor systems, as well as that induced by the phorbol ester, phorbol-12,13-dibutyrate. Agonists did not cause the appearance of single channel currents distinct from those seen prior to their application. None of the currently known second messenger systems seem to be involved in the manifestation of these responses. 13 METHODS Techniques for the identification and subsequent isolation of neurones RPed-8 and RPed-9 from the right pedal ganglion of the fresh-water mollusc Planorbarius corneus were essentially the same as those described previously (Bolshakov, Gapon & Magazanik, 1991). Neurones were immersed in saline solution containing (mm): NaCl, 50; KCl, 1-6; CaCl2, 4; MgCl2, 8; Tris-base, 1; ph adjusted to 7-5 with H2So4. The temperature was maintained at 'C. In the case of conventional voltage clamp experiments, agonists were applied by microsuperfusion from a drug-filled pipette with a tip of about jtm in diameter. With this method, a change in solution required 100 ms. In some of the voltage clamp experiments and in all of patch clamp ones, agonists were added slowly to the bath perfusion in order to maintain stability of the patch. This method of agonist application resulted in a s delay between switching the solutions and the complete change of bath saline. Some drugs (GppNHp, GDP-/l-S, and BAPTA, each dissolved in 2-5 M KCl) were applied into the neurones ionophoretically from the recording electrode. Recording. A routine two-microelectrode method of voltage clamp was used for recording of whole-cell currents. Recordings of single channel currents from isolated RPed-8 neurones were made from cell-attached patches. During cell-attached patch recordings, no other electrodes penetrated the cell, so the cell membrane potential was neither recorded nor clamped. Consequently, the potentials indicated in the single channel recordings refer to the estimated potential of patch membrane obtained after having compensated for the assumed resting potential by applying the appropriate potential to the pipette. A resting potential of -41 mv was assumed, and no corrections were made for the effects of agonists on this assumed whole-cell potential. The standard convention of outward currents directed upwards and inward currents directed downwards has been used both for whole-cell and single channel currents. Patch pipettes were filled with a solution containing (mm): KCl, 50; BaCl2 20; tetraethylammonium chloride (TEA), 10; CaCl2, 0-1; EGTA, lt (pca 8); Hepes buffer, 2-5; ph 7 2. Single channel recordings were low-pass filtered at Hz (-3 db, Bessel response) and stored on tape for subsequent analysis. Records were digitized by computer at 5 khz. Step durations of short events were corrected for limited time resolution as proposed by Colquhoun & Sigworth (1983) and used for calculation of probability density functions of selected events. Distributions of open and closed times were fitted by the sum of exponentials using the maximal likelihood methods. Drugs. L-Glutamic acid and isobutylmethylxantine (IBMX) were obtained from Serva, Heidelberg. The sodium salt of 5-guanylylimidodiphosphate (GppNHp), trilithium guanosine 5'-O- (2-thio)diphosphate (GDP-,/-S), phorbol- 12,13-dibutyrate (PhDBu), 8-bromo-guanosine 3',5'- cyclic monophosphate (8-Br-cGMP), forskolin, staurosporine, indomethacin, S(-)-sulpiride and apomorphine were obtained from Sigma, St Louis, MO, USA. Dopamine was purchased from Fluka, Ronkonkoma, NY, USA. The muscarinic agonist, 2-methyl-4-dimethylaminomethyl-1,3- dioxolane methiodide (F-2268, dioxolane) was a kind gift from Dr G. P. Sokolov (Riga, Latvia). Ergotamine was obtained from SPOFA, Czechoslovakia. PhDBu was dissolved in ethanol. Indomethacin, arachidonic acid (from Nu-Chek-Prep, Elysian, Minnesota, USA), nordihydroguaiaretic acid (NDGA; from Biomol, Plymouth Meeting, PA, USA), staurosporine and forskolin

4 14 V. Yu. BOLSHAKOV AND OTHERS A L-GIU (100pM) B F-2268 (0-1 jim) C 10 mv 5s DA (1 M) Fig. 1. Inhibitory effect of glutamate (L-Glu), a muscarinic agonist (F-2268), and dopamine (DA) on the resting potential and spontaneous activity of RPed-8 neurones. The resting potentials were -43 mv (A), -45 mv (B) and -40 mv (C). The bar below each trace represents the period of agonist application from a drug-filled pipette. were dissolved in dimethylsulphoxide; 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA) was dissolved in 2-5 M KCl titrated with 0 5 M KOH. RESULTS Comparison of responses to glutamate, dopamine and the muscarinic agonist F-2268 Figure 1 shows an isolated RPed-8 neurone which responded to fast focal application of 100 /M glutamate, 0-1 JM F-2268 (a muscarinic agonist) and 1 /LM dopamine with approximately equivalent hyperpolarizations (10-15 mv in amplitude), which lead to inhibition of neuronal firing. When the neurone was clamped at the resting potential (-41V0 mv), it responded to these agonists with an outward current. This current was similar in many respects to the slow glutamate responses described

5 POTASSIUM CHANNEL ACTIVATED BY AGONISTS earlier (Bolshakov et al. 1991). Dopamine- (Ascher, 1972; Matsumoto, Sasaki, Takashima & Sato, 1987) and F-2268-induced (Katchman, Ger & Zeimal, 1980) currents also appeared after a delay (about 1 s) and developed slowly (over several seconds). An inversion of the current was not obtained in spite of hyperpolarization of the neurone to -110 or -120 mv. The level of membrane potential when the 15 A B ~~~~~~~~~~~~~~~~~~ 0****l DA (1 M) V(mV) I/(nA) C Control (-)-Sulpiride (50,uM) DA (1 M) 0.1 na L Fig. 2. Dopamine (DA) responses and the inhibitory effect of sulpiride. A, DA-induced currents recorded at different holding potentials (values are shown, in mv). The interval between subsequent applications was not less than 3 min. B, I-V plot of these responses. C, control response and after 10 min treatment with sulpiride. Here and in subsequent figures, the upward and downward deflections in the traces reflect outward and inward currents, respectively. Holding potential, -30 mv. response disappeared was taken as the estimated equilibrium potential (Er) value. An increase of [K+]O shifted Er in a more positive direction and permitted reversal of the currents by hyperpolarization (for details, see Bolshakov et al. 1991). Long-lasting application of agonists (especially, in the case of bath application) led to a prominent decrease in current amplitude. The I-V curves for F-2268 and dopamine responses (Fig. 2A and B) were non-linear, which is in agreement with observations made earlier by Ginsborg & Kado (1975) and by Matsumoto et al. (1987) for the K+ responses mediated by the comparable receptors in Aplysia neurones. The F induced current disappeared at mv (n = 12) and the dopamine-induced current at I1 mv (n = 16), both values quite close to the potassium 20s

6 16 V. Yu. BOLSHAKOV AND OTHERS equilibrium potential (EK = -87 mv) as estimated using direct intracellular measurements of potassium activity ( mm, activity coefficient ) in neurones of Planorbarius corneus (Kostyuk, 1968). A twofold increase in [K+]. (3-2 mm) shifted the Er of both responses by a factor similar to that predicted by the Nernst equation ( mv (n = 3) for dopamine and mv (n = 3) for F- A B C _ f-n L-GIu (5 mm) DA (1 im) F-2268 (0-1 gm) D DA (1 gm) E F-2268 (0-1 pm) 0.2 na 20 s L-GIu (5 mm) L-GIu (5 mm) Fig. 3. Non-additive interaction of dopamine or F-2268 and maximal glutamate responses. A, the maximal glutamate responses obtained with a glutamate concentration of 5 mm. B and C, test responses to DA and F D and E, superimposed application of DA or F-2268 on the height of L-Glu responses failed to induce an additional potassium current. All drugs were applied focally. Holding potential, -40 mv. 2268). The assumed reversal potentials for the dopamine and F-2268 responses as well as the shifts in these reversal potentials in increased [K+]o were similar to those obtained previously (Bolshakov et al. 1991) for the glutamate response induced in the same cells (-846±+ 2-6 mv, n = 22 and mv, n = 3, respectively). Pharmacological characterization of the dopamine receptor Pharmacological characteristics of the glutamate (Bolshakov et al. 1991) and acetylcholine (Kehoe, 1972; Ger & Zeimal, 1977; Katchman et al. 1980) receptors which mediate potassium conductance increases in molluscan neurones have been previously described. The classification of the type of dopamine receptor activating potassium channels in molluscs has been based mainly on the antagonism between dopamine and the drugs ergotamine and sulpiride (Ascher, 1972; Matsumoto, Sasaki, Sato, Shozushima & Takashima, 1988). In our studies we found apomorphine (a partial agonist of D1 type of dopamine receptor) to be ineffective up to 100 fim (n = 6), whereas a 10 min application of ergotamine (01-1P0 M, n = 5) completely inhibited the dopamine response (data not shown), as did sulpiride, a selective

7 A GppNHp a F (0-5 pm) b r ~~~~~~~~~~~~~~0-2 nal POTASSIUM CHANNEL ACTIVATED BY AGONISTS F-2268 (0.5,uM) 17 DA (1 pm) 2 rmin B GDP-,B-S a -. 3 min I F-2268 (0-5 lim) 15 min 35 min b 3 min 0.1 na L 20 s 15 min 35 min DA (1 AM) Fig. 4. Effects of intracellular injection of GppNHp (A) and GTP-,3-S (B). Aa and b, ionophoretic injection from the recording microelectrode contained 10 mm GppNHp. F (a) and DA (b) were applied at the beginning of the injection and at the height of the GppNHp-induced potassium current. Ba and b, ionophoretic injection from the recording microelectrode contained 10 mm GDP-,/-S. F-2268 (a) and DA (b) were applied at different times (values are shown) after the beginning of GDP-,l-S injection. Holding potential in A and B, -30 mv. inhibitor of D2 type of the dopamite receptor (see Fig. 2 C; 10,tM, n = 3, min bath application). Non-additive interaction of glutamate, dopamine and muscarinic responses The maximal response to L-glutamate was obtained with a 5 mm concentration (Fig. 3A). The mean amplitude of this current was na in seven experiments. When test doses of dopamine (1 sum, Fig. 3B) or F-2268 (01 /LM, Fig. 3C) were used at the moment of maximal glutamate response, no increment of current was observed (Fig. 3D and E).

8 18 V. Yu. BOLSHAKOV AND OTHERS Effects of a protein activation and inhibition on responses to dopamine and F-2268 It has been shown that the potassium currents induced in a Ped-8 neurone by glutamate, quisqualate or kainate application can be mimicked by an intracellular A B +100 JL u C 5.0 I (pa) <JC. A C < C V(MV) +40 -< C <w<c < C -so - _ < C -5 _-I 5pA 0.3 0*3-~~~~~~ 0.2 PO 120 ms.01 7 V(mV) Fig. 5. Cell-attached recording of a potassium channel in the RPed-8 neurone. A, typical traces at different patch membrane potentials (mv). C indicates the closed state of the channel. B, I-V plot obtained from the membrane patch illustrated in A. The slope conductance measured on the linear part of the curve was 81 ps. C, the effects of the patch potential on the open probability (PO) of the channel from the same membrane patch. injection of irreversible activators of G protein (GTP-y-S or GppNHp) or inhibited by pretreatment with pertussis toxin (Bolshakov et al. 1991). Intracellular ionophoretic injection of GppNHp over min from the tip of a

9 POTASSIUM CHANNEL ACTIVATED BY AGONISTS micropipette (filled with 10 mm GppNHp added to 2-5 M KCl solution) caused a potassium current, which slowly reached maximal amplitude ( na, n = 8, at a holding potential of -30 mv). Application of 01X-05 /M F-2268 (n = 4) or 1 ftm dopamine (n = 4) on neurones loaded with GppNHp failed to evoke additional potassium currents (Fig. 4A). In Ped-8 neurones voltage clamped at -40 mv pertussis toxin (PTX), a bacterial toxin that blocks the activation of distinct types of G proteins through ADP ribosylation, significantly reduced the responses to dopamine (by 81P2 %, n = 9) and to the muscarinic agonist F-2268 (by 85 %, n = 10). Treated and control preparations were tested after h of contact with 2,cg/ml PTX or with the normal solution respectively. It was shown previously that the same procedure of PTX treatment of Ped-9 neurones did not affect the glutamate-evoked chloride current (Bolshakov et al. 1991). Similar results were obtained when another G protein inhibitor, a non-hydrolysable GDP analogue, GDP-fl-S, was used (Fig. 4B). Prolonged intracellular (35-40 min) injection from 10 mm GDP-,f-S-filled pipettes decreased the amplitude of the 0 5,UM F-2268 response to % (n = 4) and the 1,UM dopamine response to '5 % (n = 3) of their respective control values. Effects of glutamate, dopamine and F-2268 on single channel currents When a Grl seal was made on an isolated RPed-8 cell using a patch pipette filled with 50 mm KCl, noise-like current fluctuations were recorded. In contrast, when Na+ replaced K+ in the pipette, these current fluctuations were no longer observed at any patch membrane potential. It appears therefore that the 'current noise' seen using the KCl solution reflected the superposition of a large number of single potassium channel currents. The addition of potassium channel blocking agents to the 50 mm KCl pipette solution (10 mm TEA and either mm Ba2" or Cs2+) markedly decreased the number of active channels in the patch, and permitted the resolution of single events. As can be seen in Fig. 5A, single channel currents recorded under these conditions at any given potential of the patch membrane had an uniform amplitude. This was confirmed by amplitude histograms for at least events (see below), revealing that a homogeneous population of potassium channels was activated in the patch studied. The I-V dependence of this channel was almost linear at patch membrane potentials more negative than 50 mv (see Fig. 5B). The single channel current reversed at an assumed patch membrane potential of mv (n = 29), consistent with predictions from the Nernst equation when using 50 mm KCl in the patch pipette and when using previous estimates (Kostyuk, 1968) of potassium intracellular activity ( mm) and the activity coefficient (0-73) in Planorbarius neurones. The average slope conductance was ps (n = 24) in the linear part of the I-V plot, but the I-V relation showed an inward-going rectification: the linearity of the I-V relation was lost at voltages positive to mv. Under the experimental conditions used, the open probability of the channel (P.) was relatively low in the region close to the reversal potential, and was significantly increased at more positive patch membrane potentials (Fig. 5C). 19

10 20 V. Yu. BOLSHAKOV AND OTHERS A Control B 5.0- L-GIu (1 mm) /(pa) // O/ V(mV) Wash -5.0 C Control D I,I I Ii I <C F-2268 (1 tm) /(pa) / 0 E Wash IIll L il I Control 7.0 pa Dopamine (5pM) Wash <C <C 20s L <c F 0 I (pa) V(mv) V(mV) J Fig. 6. Effects of bath-applied glutamate (A and B), F-2268 (C and D) and dopamine (E and F). Cell-attached recordings of the single channel currents before, in the presence of agonist (2 min), and 2 min after washing out the agonist. Patch membrane potential was + 30 mv. B, D and F, I-V plots for the single channel currents obtained in the presence

11 POTASSIUM CHANNEL ACTIVATED BY AGONISTS In the experiments shown in Fig. 6A, channel activity was recorded in the cellattached patches before, during and after bath application of glutamate, dopamine or F After application of the agonist solution (seven neurones with glutamate, six neurones with F-2268 and five neurones with dopamine) the channel activity increased markedly (4-10 times) and reached a new steady level of frequency in TABLE 1. Effects of agonists and phorbol ester on single channel current parameters* Concentration Mean amplitude Open time, rt Open probability, Drug (UM) n (pa) (ms) PI Control Glutamate F Dopamine Phorbol ester * At a holding potential of 30 mv. about 2 min. In some experiments an overlapping of the openings of two or three channels could be observed. Wash-out of neurones by the standard solution induced a fast decrease in PO. I-V plots showed that the agonists change neither the conductance of the channels nor the position of the potential range of the inwardgoing rectification (Fig. 6B, D and F). As is shown in Fig. 6A, the unit amplitude of the single channel current did not change after application of agonists. This observation was verified by a comparison of the amplitude histograms in control conditions with those obtained after application of any of the agonists (Table 1). The results of a representative experiment, where 1 mm glutamate was applied, are shown in Fig. 7. The position of the main peak of the amplitude histogram remained the same. The small second mode appearing in some experiments as a result of agonist action reflected the overlapping of two channel openings due to increase of their open probability. This observation indicated that the channel conductance was independent of whether the receptors were occupied by agonists or not. As mentioned above, the whole-cell potential was not clamped, so any hyperpolarization induced by agonist action outside the patch would affect the potential of the patch membrane and a marked change in the single current amplitude would be expected. This was not the case, however; the agonist effect was not reflected in the amplitude of the currents in the patch. One plausible interpretation is that in the experiments described in this study the hyperpolarization induced by the agonists was relatively small at the moment of single current recording, due to desensitization. Actually, bath application of high doses of agonist induced a marked transient effect, but after a 2 min contact with the agonist only a small fraction of the channels was still active. The open and closed time distributions were analysed to estimate the effects of the agonist being studied (glutamate in the example shown) on the kinetic properties of the potassium channel currents. The histogram of open times showed a single of agonists from the same experiments as in A, C and E, respectively. The slope conductance measured on the linear part of the curve was 79 ps in B, 81 ps in D and 82 ps in F (compare with control I-V plot in Fig. 5B). PHY

12 22 V. Yu. BOLSHAKOV AND OTHERS exponential distribution function both in control conditions (zr = 9-6 ms) and in the presence of glutamate (T0 = 9-4 ms). The open times did not differ under these two conditions (Fig. 7A and B and Table 1). The histograms of closed times were best fitted by the sum of at least two exponentials. The duration of the faster time C C C c -o -~~~~~~~~~~~~~~~~~60 ID ) 0) 0) 0)~~~~100 4)0) 0) 5 0) 0) z z z z ~~~~ Amplitude (pa) Closed time (ms) Open time (ms) Burst duration (ms) B 600 ~~~~~~~~~~~ C C~~~~ ~80 ~400 0.~~~~~~~~~~~~6 C 0)~~~~~~~~~~~~~200- > ( and- 80 >0D) z z Z Z 20 Fi.7C itgamofapiue,coe ie Contieadus drain60 oe A) n 0 ote0d 0 se Amplitude (pa) Closed time (ins) Open time (is) Burst duration (mis) Fig. 7. Histograms of amplitude, closed time, open time and burst duration before (A) and in the presence of 1 mm glutamate (B). Data obtained from the experiment shown in Fig. 6A. The last bin in the closed time histograms gives the number of all events longer than 60 (A) and 80 ms (B). Note that the current amplitude and open time is not changed by glutamate. For other details see text. constant (Tci) in the presence of the agonist was decreased only by about one-third (5-4 ms in control and 3-6 ms in the presence of glutamate), but the slower component (rs2) was shortened more markedly at the height of the glutamate effect (from 400 ms in control to 89 is, respectively). Changes Of Tc2and the area under the exponential correlated directly with the increase of open probability. Contiguous openings were defined as a burst when the closed time separating them was less than 3 X Tcl- The histogram of burst lifetime was best fitted by one exponential Orb) which was increased from 29-8 to 38-9 ms in the presence of glutamate (Fig. 7A and B). The results obtained in the presence of dopamine or F-2268 were practically the same as those obtained with glutamate.

13 POTASSIUM CHANNEL ACTIVATED BY AGONISTS These data strongly suggest that the channels activated by the agonists are identical to those present in the patch under control conditions, since the spontaneously active and agonist-induced channels were shown to have identical current amplitudes, channel conductances, single channel open times, and rectification properties. At the concentrations used, the effect of the agonists was limited to increasing the open probability, PF, of the potassium channel (see Table 1). Effects of a phorbol ester on single channel currents The ability of protein kinase C to modulate potassium channel activity has been shown previously (Shearman, Sekiguchi & Nishizuka, 1989, for review). Activation of protein kinase C by phorbol esters or synthetic analogues of diacylglycerol has been shown to induce an outward potassium current in the molluscan neurones studied (Bolshakov et al. 1992). Application of 5 fm phorbol-12,13-dibutyrate is shown in Fig. 8A to induce a marked increase in channel openings (in all eight experiments). The amplitude of the single channel current was the same in control records and in records obtained after exposure to the phorbol ester (Fig. 8A). This finding was verified by a comparison of amplitude histograms (n = 5, not shown). A marked effect on P. developed over time (Fig. 8B). The phorbol ester changed neither the I-V dependence for a single channel (compare Figs 5B and 8C), the slope conductance ( ps, n = 3) nor the inward-going rectification properties of the channel (Fig. 8C). The open and closed time distributions were analysed (n = 5) to estimate the effects of the phorbol ester on the kinetic properties of the potassium channel currents. In the representative experiment shown in Fig. 8A, the histogram of open times was best fitted by one exponential with the time constant 14-2 ms (151 % of control value). The histogram of the closed times was best fitted by two exponentials r,l = 3-2 ms and 'r12 = 430 ms (48 and 10% of control values respectively). A marked change of area under the exponential TC2 reflected mainly the pronounced increase of frequency of channel opening. In two experiments the neurones were pretreated over min with 5 /SM staurosporine, a specific inhibitor of protein kinase C. Subsequent application of 5/M of the phorbol ester completely failed to influence P.. These results suggest that the channel activated by the phorbol ester is identical to that seen in control conditions and in the presence of the three agonists studied. The possible involvement of secondary messengers in the transduction of dopamine and F-2268 action. It has been shown previously that application of glutamate, quisqualate or kainate to neurones treated with a phorbol ester induces smaller current responses than those in untreated preparations. However, staurosporine effectively prevented this inhibitory effect, as it does the outward potassium current evoked by the phorbol ester itself. These facts were interpreted as evidence that protein kinase C could not be involved in the transduction mechanism for the glutamate-induced potassium current (Bolshakov et al. 1992). A 5-10 min exposure to 3 /SM phorbol-12,13- dibutyrate reduced the amplitude of potassium currents (measured at -30 mv) evoked by 0 5 /M F-2268 and 1 /SM dopamine by (n = 3) and % (n = 3) respectively (Fig. 9A and B). The voltage dependence and reversal potential of

14 24 V. Yu. BOLSHAKOV AND OTHERS F or dopamine-induced potassium currents remained unaltered. Application of staurosporine (3 ftm, min) did not affect the response to subsequent applications of F-2268 (five neurones) or dopamine (six neurones), but prevented the effect of phorbol esters on the F or dopamine-induced potassium current (Fig. A Control PhDBu (5 gm, 2 min) PhDBu (5pM, 4 min) -C c < C N <C omwiiimilni-im, < C B somoaf- I- A< tva0-3s< 3.5 pa <C.0 0Cu Co 0. 0._ 0 X C I (pa) V(mV) Time (min) '-2.0 J Fig. 8. Effects of bath-applied phorbol-12,13-dibutyrate (PhDBu). A, cell-attached recordings of the single channel currents before and at different times after PhDBu application. B, increase in time of open probability (PO) induced by PhDBu. C, I-V plot for the single channel currents obtained in the presence (4 min) of PhDBu from the same experiments as in A. The slope conductance measured on the linear part of the curve was 79pS. 9C). Similar results were obtained when 10 /tm oleoylacetylglycerol (n = 1) was used instead of phorbol ester. Treatment of neurones with a mixture of the phosphodiesterase inhibitor, IBMX (100,UM), and adenylate cyclase activator, forskolin (20 /tm), did not influence the F (n = 3) or dopamine-induced (n = 3) responses. Bath application of 1 mm of the membrane permeant cyclic nucleotide analogue, 8-Br-cGMP, did not affect (n = 3) the amplitude of F or dopamine-induced potassium currents. The intracellular concentration of cyclic nucleotides was obviously enhanced by these

15 POTASSIUM CHANNEL ACTIVATED BY AGONISTS procedures, as evidenced by the appearance of a cyclic nucleotide-mediated inwardgoing current frequently observed in molluscan neurones (Kononenko, Kostyuk, Shcherbatko, 1983; Kehoe, 1990; Brown & McCrohan, 1992). Our data contrast, however, with those of other authors (Kehoe, 1985; Brezina, 1988) who found that camp blocks the cholinergic K+-dependent response in Aplysia neurones. 25 A B Control PhDBu (3 lzm) Control PhDBu (3 /am) -30.J ' JN F-2268 (0.5 pm) F-2268 (0.5 AM) DA (1 um) DA (1uM) C Staurosporine (3 ym) Control Staurosporine (3 pm) PhDBu (3#M) ) F-2268 (0.5 pm) F-2268 (0.5 pm) F-2268 (0.5 pm) 0.1 na L 20 s _ -50 Z -50 _ DA(1 M) DA(1,uM) DA(1pM) Fig. 9. Effects of phorbol ester and staurosporine on the responses evoked by F-2268 and dopamine. Potassium currents induced by F-2268 (A) and dopamine (B) before and 5-10 min after the treatment of neurones with PhDBu. C, absence of staurosporine effect on F and dopamine-induced responses and preventive action of staurosporine against PhDBu effects. Calibration is the same for all records. Application of 50 #M arachidonic acid induced a slowly rising outward current. This effect was reversible by washing with standard solution. Treatment of neurones (at least 120 min, n = 4) with a mixture of the 12-lipoxygenase and 5-lipoxygenase inhibitor, NDGA (50 #tsm), and the cyclo-oxygenase inhibitor, indomethacin (50 #M), prevented the current induced by arachidonic acid but failed to influence the F-2268 and dopamine responses. Previously it has been shown (Bolshakov et al. 1991, 1992) that intracellular

16 26 V. Yu. BOLSHAKOV AND OTHERS injection of the calcium chelators EGTA and BAPTA into molluscan neurones failed to affect any of the glutamate responses. Similar results were obtained in experiments (n = 4) where BAPTA was injected intracellularly from the recording electrode. In spite of the failure of such injections to affect the glutamate response, the intracellular calcium concentration dropped significantly, as indicated by a prominent decrease of hyperpolarization which followed action potentials evoked by passing current through a second intracellular microelectrode. Thus all our results provide strong evidence against the involvement of any known second messenger systems in the mediation of the muscarinic and dopamine responses, as has previously been concluded for the glutamate response (Bolshakov et al. 1992). Whatever the second messengers involved, the potassium channel activated through the three receptor systems has been shown to be the same. DISCUSSION Pharmacological properties of the receptors The present results suggest that in the neurones studied here, in addition to two previously described glutamate receptors (Bolshakov et al. 1991; Magazanik & Bolshakov, 1992), there are at least two other receptors (one dopaminergic and one muscarinic) which likewise induce a slow potassium current. There are several pharmacological distinctions between muscarinic receptors in neurones of molluscs and vertebrates (Kehoe, 1972; Ger & Zeimal, 1977). Molluscan muscarinic receptors are not blocked by atropine and differ in their sensitivity to a number of specific agonists (arecoline, methylfurmetide, acetyl-,6-methylcholine, F- 2268). Since F-2268 (dioxolane) is much more potent than the other agonists for eliciting the cholinergic K+-dependent response on Planorbarius neurones (Ger & Zeimal, 1977) it was selected for activating of muscarinic receptors in neurones studied. The dopamine receptors mediating the K+-dependent response has been shown in Aplysia neurones to co-exist with other dopamine receptors, thereby yielding multicomponent responses (Ascher, 1972; Matsumoto et al. 1987). In Ped-8 and Ped- 9 neurones of Planorbarius corneus, dopamine elicited only the K+-dependent response. Apomorphine, which is known as an agonist of the D1 type of dopamine receptors had practically no effect on this response, but sulpiride, a selective inhibitor of D2 type (Kebabian & Calne, 1979; Matsumoto et al. 1988), completely blocked the dopamine response. This slow outward current could also be blocked by ergotamine, which was used by Ascher (1972) for blocking the inhibitory effect of dopamine on Aplysia neurones. It appears that the dopamine receptor studied here belongs to D2 type of receptor. Ionic basis of dopamine and F-2268 responses Both agonists induced outward currents in RPed-8 neurones clamped at resting potential level. The reversal potentials of responses were quite close to the potassium equilibrium potential and practically coincided with the Er of glutamate responses, estimated earlier (Bolshakov et al. 1991). The shift of the Er of the dopamine and F responses (about 17 mv) induced by a twofold increase of [K+]. was, as in the

17 POTASSIUM CHANNEL ACTIVATED BY AGONISTS case of glutamate, in accordance with the Nernst equation predictions. Evidently all these outward currents are mediated by a selective increase in potassium conductance. Since neither the dopamine nor F-2268 were able to produce additional current when applied at the moment of a maximal glutamate response, it would seem that all agonists studied exerted their effects through the same population of potassium channels. These channels are not sensitive to [Ca21]i since the intracellular injection of a fast chelator of Ca2" ions, BAPTA, failed to affect the dopamine or F-2268 responses. The involvement of G protein in the transduction of dopamine and F-2268 responses Several pieces of evidence were obtained that proved the indispensable involvement of G proteins at an early stage of the generation of dopamine and F responses. Intracellular ionophoretic injection of irreversible activators of the G proteins, GTP-y-S or GppNHp, in RPed-8 induced the outward potassium current. These findings are similar to those obtained on Aplysia neurones (Treistman & Levitan, 1976; Sasaki & Sato, 1987; Brezina, 1988; Volterra & Siegelbaum, 1990). Responses studied in the present experiments to glutamate (see also Bolshakov et al. 1991), dopamine, F-2268, and to the non-hydrolysable guanine-nucleotide analogues were not additive. Pertussis toxin (PTX), which causes receptors to uncouple from G proteins, and the GDP analog, GDP-,8-S, which competes with GTP for binding to the az-subunit (Eckstein, Cassel, Levkovitz, Lowe & Selinger, 1979), both prevented responses to dopamine and F A similar effect of PTX on the glutamate response in the same molluscan neurone has been described previously (Bolshakov et al. 1991). Thus at least four types of receptors (dopamine, muscarinic, kainate and quisqualate) in the RPed-8 neurone are coupled to the same population of potassium channels through PTX-sensitive type of G protein. Properties of potassium channels activated by agonists The activity of single channels was observed in a RPed-8 neurone by patch recordings in the cell-attached mode. The following pieces of evidence support the view that the basal activity (that seen in the absence of any agonists) observed in these neurones is generated by potassium channels. (1) When Na+ replaced K+ in the pipette, current fluctuations were no longer observed at any holding potential. (2) In the present experiments the reversal potential of single channel currents was in good agreement with Nernst equation predictions for the solutions used (50 mm KCl inside the pipette). (3) The predicted shift ofer due to a decrease ofpotassium concentration in 'intracellular' solution from 50 to 10 mm was observed in experiments on cell-free inside-out patches which were pulled from the same neurones (Katchman, Samoilova & Snetkov, 1989), and the channels showed the same type of inward-going rectification as did the channels studied in the cell-attached mode. Their open probability, like that of the channels studied in cell-attached patches, was dependent on membrane potential. The data strongly suggest that the basal activity and the agonist-induced currents are generated by the same potassium channel. This idea is supported by the following 27

18 28 V. Yu. BOLSHAKOV AND OTHERS observations. (1) The current-voltage relationships obtained before and during agonist application are quite similar, including a characteristic inward-going rectification. The slope conductance measured on the linear part of the I-V plot did not change in the presence of any agonist. (2) The open time of unitary events was not influenced by agonists. (3) The changes in the distributions of the other kinetic parameters (closed times, burst durations) are also consistent with the view that the main effect of the agonists is the increase in channel open probability. (4) The amplitude histograms were the same in the absence or presence of any agonist. The last results are consistent with the premise that desensitization limits the persistence of hyperpolarization of the whole-cell membrane induced by bath-applied agonists. Potassium channels can also be activated by treatment of the same neurone, RPed-8, with phorbol esters (Bolshakov et al. 1992). A similar set of evidence was used to prove that phorbol- 12,13-dibutyrate induced an increase of open probability of the same potassium channels as those activated by the agonists. Analysis of patch recordings in the cell-attached configuration showed that treatment with the phorbol ester had no effect on either the slope or the rectifying properties of the I-V relationship whereas it drastically increased PO, mainly through a shortening of closed times. The marked increase of open times (1P5 times), which was not observed during agonist action, may result from the effect of the phorbol ester on protein kinase C, because it can be prevented by staurosporine, a potent inhibitor of this enzyme (Ruegg & Burgess, 1989). Thus, we conclude that the same type of potassium channel is activated in RPed- 8 by several different agonists (dopamine, F-2268, quisqualate and kainate), by phorbol esters or by depolarization of the patch membrane. All these procedures induce the prominent increase of open probability, PO, but the mechanism of their effect is not necessarily the same. In particular, there is some difference in the influence of agonists and the phorbol ester on the open time of the single channel current which suggests the possibility of distinct mechanisms. Does any secondary messenger take part in the transduction of the dopamine- and F induced potassium currents? Two main types of transduction mechanisms can be considered: (1) G proteininduced stimulation (or inhibition) of specific enzymatic activity which results in changes of some low-molecular weight diffusible substances; and (2) direct interaction between the G protein oc-subunit and a channel protein (Pfaffinger & Siegelbaum, 1990, for review). So far the conclusions about direct coupling of G proteins and channels have been based mainly on unsuccessful attempts to elucidate the involvement of any known secondary messengers (Andrade et al. 1986; Bosma, Bernheim, Leibowitz, Pfaffinger & Hille, 1990; Selyanko, Smith & Zidichouski, 1990; Gerschenfeld, Paupardin-Tritsch & Yakel, 1991). Previously we used a number of standard approaches to evaluate the possible involvement of any secondary messengers in the transduction mechanism for glutamate-induced potassium current (Bolshakov et al. 1992). But these experiments did not unveil the activation of any enzymatic systems known to be involved in pathways of secondary messenger synthesis or destruction. The same experimental procedures were used in the present investigation of the potassium currents evoked

19 POTASSIUM CHANNEL ACTIVATED BY AGONISTS in the same neurone by two other agonists (dopamine and F-2268), and led to similar conclusions. Our experiments rule out the involvement of cyclic nucleotides such as cyclic AMP or cyclic GMP because the application of the permeable analogue of cgmp and/or the activator of adenylate cyclase, forskolin, in combination with an inhibitor of phosphodiesterase, IBMX, failed to affect the dopamine or F-2268 responses. Also no indication of the functional role of protein kinase C was found. Responses to phorbol esters, in contrast to responses to dopamine or F-2268, may be inhibited by the specific inhibitor of protein kinase C, staurosporine. Thus the mechanism of channel activation appears to be different. The increased open time (which was not observed during agonist action) suggests phosphorylation as a possible mechanism of phorbol ester-induced channel activation (Shearman et al. 1989). The independence of the dopamine and F-2268 responses on intracellular calcium content rules out the involvement of inositol 1,4,5-trisphosphate (Rana & Hokin, 1990, for review) and Ca2+-calmodulin-dependent protein kinase (Doroshenko, Kostyuk & Luk'yanetz, 1988; Gerschenfeld et al. 1991). Arachidonic acid (Piomelli & Greengard, 1990), the main product of phospholipid hydrolysis, activated a potassium current in the neurones studied, but the inhibitors of its metabolism (indomethacin and nordihydroguaiaretic acid (NDGA)), failed to affect the dopamine and F-2268 responses. A functional role of nitric oxide (Moncada, Palmer & Higgs, 1989) is also doubtful because cgmp is not obviously involved in the transduction mechanism. Thus, in the present experiments, just as was seen for glutamate, quisqualate and kainate (Bolshakov et al. 1992), no involvement of the well-established secondary messengers in the activation of the same population of G protein-operated potassium channel by dopamine and F-2268 could be demonstrated. G protein-potassium channel coupling mechanisms The cell-attached mode of single channel current recording is commonly used to answer the question of whether a G protein controls the channel by direct interaction of the a-subunit with the channel, or indirectly, with the help of some intracellular diffusible factor, i.e. a secondary messenger. If the receptor-g protein complex activates the channel directly, the channel may respond to agonists applied only via the patch electrode, but not to agonists added to the neuronal membrane outside of the patch (Soejima & Noma, 1984). One of the most striking findings made in the present study is the apparent contradiction between the ability of bath-applied agonists to activate the single channel currents recorded in the cell-attached configuration and the failure to implicate known second messenger pathways. The tight seal between the pipette and neuronal membrane should prevent access of the applied agonists to the receptor and channels in the patch membrane. The existence of some other 'signal service' which is able to overcome the distance between the receptor-g protein complex located outside and potassium channels located inside the patch must therefore be postulated. At least two possibilities must be discussed: either an unknown second messenger system may be involved, or it must be assumed that the a-subunit of the G protein is very hydrophilic, and thus can migrate from the receptor complex to the 29

20 30 V. Yu. BOLSHAKOV AND OTHERS channel. However, in order to play the second messenger-like role, the a-subunit must be able to move with sufficient velocity across a distance comparable to the patch diameter. Unfortunately, there is no direct evidence about the maximal length of the a-subunit functional run. Taking into account the size of a-subunits Glu ACh DA PhDBu Fig. 10. Scheme of convergence on the same potassium channel of signals mediated through different receptors or protein kinase C activation. Cell-attached recording of single channel currents and bath application of agonists. R, receptors; K+, potassium channel; PKC, protein kinase C; a, fi and y, subunits of GTP-binding proteins. (39-40 kda) such a possibility seems doubtful. The paradoxial situation found in the present study suggests that attention should be focused on the study of some other cytosol (or membrane) components interposed between G proteins and potassium channels. Multiplicity of ways to activate potassium channels The potassium channel studied in these molluscan neurones may be modulated by the activation of at least four membrane receptors (kainate and quisqualate subtypes of the glutamate receptor, a muscarinic and a dopamine receptor) operated through a G protein. Independently this channel is a target of an intracellular enzymatic system such as protein kinase C (Fig. 10). Convergence of action of various neurotransmitter receptors onto potassium channels has been shown repeatedly in experiments on different neurones (Ascher & Chesnoy-Marchais, 1982; Andrade et al. 1986; Sasaki & Sato, 1987; Christie & North, 1988; Shen, North & Surprenant, 1992; see for review Nicoll et al. 1990). What is the physiological significance of multiple pathways to a common population of potassium channels? The exact answer is not known yet. One of the widely discussed possibilities is based on the idea that receptors are located on different confined spots of neuronal membrane and thereby can be specifically activated through different synaptic inputs (Nicoll et al. 1990). Another level of convergence was demonstrated in Aplysia sensory neurones, where FMRF-amide and serotonin receptors, through different G proteins (sensitive and

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