Ca2+-Activated K+ conductance in internally perfused snail neurons

Transcription

1 Proc. Natt Acad. Sci. USA Vol. 79, pp , July 98 Neurobiology Ca+-Activated K+ conductance in internally perfused snail neurons is enhanced by protein phosphorylation (K+ channels/cyclic AMP/protein kinase/voltage clamp) JACQUES E. DE PEYER*, ARMAND. CACHELIN*, IRWIN. LEVITANt, AND HARALD REUTER* *Department of Pharmacology, University of erne, erne, Switzerland; and Friedrich Miescher Institut, P.O. ox 7, asel, Switzerland Communicated by Joseph F. Hoffman, March 9, 98 ASTRACT Depolarizing voltage steps induce inward and outward currents in voltage-clamped, internally perfused neurons from the snail Helix roseneri Addition of the catalytic subunit of cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC.7..7) to the internal perfusing medium results in an increase in the net outward current, with no apparent effect on the inward current. Catalytic subunit inactivated by 5,5'-dithiobis(-nitrobenzoic acid) is without effect, indicating that the increase in net outward current results from protein phosphorylation rather than an unspecific effect of protein perfusion. Decreasing the external Ca+ concentration from 0 to mm eliminates the effect of catalytic subunit, suggesting that Ca+ plays an important role in this response. This suggestion is supported by the fact that the stimulation by catalytic subunit can be mimicked by increasing the Ca+ concentration in the internal perfusion medium and can be prevented by intracellular perfusion with 0 mm EGTA. The results are consistent with the hypothesis that cyclic AMP-dependent protein phosphorylation regulates the Ca+-activated K+ conductance in these cells. It has been suggested that cyclic AMP-dependent protein phosphorylation may regulate neuronal electrical activity (, ). Experiments involving the injection ofa specific inhibitor ofcyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC.7..7) into Aplysia neuron R5 have indeed provided evidence that endogenous protein phosphorylation is involved in the regulation of K+ conductance by serotonin (-5). Another approach to study the role ofcyclic AMP-dependent protein phosphorylation is to introduce exogenous catalytic subunit (C subunit) of cyclic AMP-dependent protein kinase into cells. In the case ofaplysia sensory neurons or bag cells, C subunit injection can enhance the action potential (6, 7). In order to investigate whether C subunit can affect specific ion conductance systems, we have internally perfused (8, 9) Helix neurons with physiological concentrations of the enzyme. We have studied two K+ conductance systems, the delayed rectifier and the Ca+-activated K+ current, by using voltage clamp techniques (9, 0). The results indicate that C subunit causes a selective increase in the Ca+-activated K+ current, possibly by increasing the affinity of the K+ channel for Ca". Thus Ca+ and cyclic AMP may interact in these neurons to regulate the activity of a single ion channel. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 8 U. S. C. 74 solely to indicate this fact. 407 MATERIALS AND METHODS Subesophageal ganglia were dissected from land snails (Helix roseneri), and the capsules of left parietal ganglia were opened after brief exposure to 0. % trypsin solution (9). Single unidentified neuronal somata (diameter itm) were used for the experiments. The experimental set-up and the perfusion electrode were similar to those described previously (8, 9). riefly, the suction pipette was pulled from Pyrex glass of mm outside diameter. The tip of the pipette was broken to a diameter of 0-50 tkm and fire polished to a final inside diameter of 5-0 tum. The pipette was fitted onto a Plexiglas chamber containing an inlet and an outlet tube for the perfusion, a platinum/iridium wire for current injection, and an agar/kc microelectrode connected to an Ag/AgCl wire for potential measurement. The voltage and current electrodes were connected to a standard voltage-clamp amplifier. Membrane potential was recorded between the agar microelectrode and an agar bridge placed in the bath solution. Injected current was measured relative to a virtual ground and was compensated for series resistance. All experiments were recorded on-line on a computer (PDP / 04, Digital Equipment, Marlboro, MA). Constant current or voltage steps (56 ms duration; 5 s apart) were delivered by a programmable stimulator (). All current traces obtained from voltage clamp experiments were corrected for linear leak and capacity current (9). C subunit of cyclic AMP-dependent protein kinase from bovine heart was purified to homogeneity () in the laboratory of E. Fischer, University of Washington, Seattle. The activity of this enzyme is independent of Ca+. 5,5'-Dithiobis(-nitrobenzoic acid) (Nbs)-inactivated C subunit was also supplied by E. Fischer. The C subunit can catalyze the phosphorylation of Helix and Aplysia proteins in vitro, whereas Nbs-inactivated C subunit is without effect (unpublished data; see ref. 6). In order to minimize the amount of solution used, the volume of the perfusion electrode was kept as small as possible (=0.5 ml) and the perfusion solution was recirculated by a pump (Guldener Varia Perpex), using a small centrifuge tube as reservoir. The total volume of the perfusate was.5 ml, and the rate of solution flow was ml/min. Test solution was added by means ofa syringe through a stopper in the reservoir. The concentrations were adjusted such as to add 0 ul of test solution to the.5 ml of internal solution. Except where otherwise indicated, the internal solution contained 5 mm potassium aspartate, mm MgCl, 0.5 mm NaATP, and 0. mm Hepes, ph 7.. Throughout the experiments, the ganglia were continuously superfused with an isotonic solution containing 80 mm NaCl, 4 mm KCl, 0 mm CaCl, mm MgCl, 0 mm NaHCO, and 0. mm NaHPO4, ph 7.4, equilibrated with 5% CO in 0. The temperature was maintained at 7 ± C by means of a Pelletier device. Abbreviations: C subunit; catalytic subunit of cyclic AMP-dependent protein kinase; Nbs, 5,5'-dithiobis(-nitrobenzoic acid); I-V, current-voltage; I,, Ca+-activated K+ current.

2 ~~~~~~~~ Neurobiology: de Peyer etalproc. Nad Acad. Sci. USA 79 (98) outward current. Plots of outward current vs. voltage exhibit a region of negative slope in the current-voltage (I-V) curve (, 4) (Fig. ). In the presence of C subunit (0. MkM), the slow outward current is increased, and the region of negative 0 o 50 ms FIG.. Effect of intracellular perfusion with C subunit on action potentials of snail neurons. The control (trace ) shows the normal repetitive activity evoked by a -na depolarizing current pulse prior to addition of C subunit; traces,, and 4 are traces of responses to the same pulse, after 0, 5, and 55 min of perfusion with 0. PM C subunit, respectively. RESULTS Perfusion of the C Subunit Increases the Outward Current. Perfusion of individual neurons ofhelix roseneri with a solution The Outward Current Involved Is the Ca"-Dependent K+ containing 0.0-,uM C subunit decreases the duration of the Current (Ia). It has been shown that the slow outward current action potential elicited by a constant current pulse and enhances the afterpotential hyperpolarization, even when the a voltage-dependent delayed one and a Ca+-dependent one. in Helix neurons can be separated into two components (, 4), peak of the action potential remains unchanged. The most consistent effect is an increase in the duration ofthe hyperpolariza- in the increase ofoutward current in response to C subunit, we To determine whether one or both ofthose currents is involved tion between two action potentials, often resulting in a complete examined the effects of changing the internal Ca+ concentration. Although we did not add Ca' to the normal internal me- loss of the repetitive activity during a prolonged depolarizing current pulse (Fig. ). In order to investigate the mechanism dium, it contained about um as determined by atomic absorption spectrophotometry. Ifthe action of the C subunit is to of this response, we analyzed it by using voltage clamp techniques. Stepping the voltage from a holding potential (-40 mv) enhance IC, it might be possible to mimic the effect of the C to increasing depolarizing voltages induces a typical pattern of subunit by increasing the internal Ca+ concentration. Fig. 4 current responses (Fig. A), an inward current, which is insensitive to tetrodotoxin, and a fast and a slow component of changes in the current records and I-V relationship similar to shows that perfusion ofa solution containing 0,uM Ca+ causes A C f ah>===; 40 I ', 50 ms 00._~ Anf 80-4* U FIG.. Voltage clamp analysis of C subunit effect. (A) Superimposed traces of current responses to depolarizing voltage steps (0-mV increments up to 00 mv). Holding potential -40 mv. Set, prior to the addition of C subunit; sets and, 5 and 55 min, respectively, after the addition slope is displaced to more positive potentials (Fig. A and ). Moreover, after 0 min ofperfusion with a higher concentration (,um) of C subunit, the two components of outward current can no longer be distinguished and the steady-state I-V relationship becomes linear for all positive potentials (Fig. C). In addition, the kinetics of the currents are changed. The C subunit increases the rate ofactivation of the outward current even at voltages at which the steady-state current is not affected (Fig. A) Ṡimilar results were obtained consistently in five cells with 0. /im C subunit and in four cells with,/m; in contrast, perfusion with,um Nbs-inactivated C subunit was without effect in four cells. The slow time course of these effects of C subunit is almost certainly due to the slowness ofthe perfusion system, including diffusion of the protein from the tip of the pipette to its site of action. Even perfusion with K+ or C- in the millimolar range takes several minutes before a steady state is reached (8). of 0. A.M C subunit to the perfusion medium. () Current-voltage (I-V) relationships, showing the peak inward current and the maximal outward current for the same three sets of traces. (C) I-V relationship obtained from another cell before perfusion (trace ) and during perfusion (trace, 5 min; trace, 0 min) with utm C subunit. The C subunit increases the outward current. This increase becomes most obvious for larger depolarizing steps. The apparent decrease in outward current seen at very positive potentials is also observed in the absence of C subunit and is probably due to a slow run-down of the preparation during perfusion (see Fig. 5).

3 A 50 ms FIG.. Superimposed records of current traces after different times of perfusion with,um C subunit (A) and 0 gm CaCl (). (A) Trace, control; traces and, 5 and 0 min, respectively, after addition of C subunit. () Trace, control; traces and, 40 and 60 min, respectively, after introduction of Ca+ in the perfusion medium. In both cases the rate of activation of outward current increases. Mge Neurobiology: de Peyer et al those produced by C subunit at AM (Fig. C). Again, as in the case of C subunit (see above), a relatively long perfusion time is required before the effects of internal Ca' become apparent. To determine whether the C subunit was selectively affecting IC) it was necessary to separate the two outward currents. One way to isolate the voltage-dependent delayed current is to add EGTA to the internal solution (9, 4). Addition of the C subunit to an internal perfusate containing 0 mm EGTA has no effect on the remaining outward current (Fig. 5), indicating that the voltage-dependent delayed K+ conductance is not affected by the C subunit. The slight decrease in outward current is due to slow deterioration of the cell during perfusion and is not an effect of C subunit. In contrast, C subunit is still fully effective with mm internal EGTA, which only partially suppresses Ic. Are the Changes Coupled to an Increased Influx of Ca+? Decreasing the external Ca+ concentration reduces the amount of IC [by decreasing Ca+ entry (, 4)] and also lessens, or even suppresses, the effect of C subunit. Lowering the external Ca+ concentration to mm in the presence of 5 mm inhibited the augmentation of outward current by C subunit, even though a small I,, was still evident. Nevertheless, it is unlikely that the increase in outward current by the C subunit is due to an increased influx of Ca+ during the depolarizing pulses. In none of 5 experiments with the C subunit did we observe a significant increase in inward current. Plots of those inward currents show a decrease with time, even in cases in which C subunit has no effect on the outward current (Fig. 5). DISCUSSION The possibility that ion channels in nerve cells may be subject to long-term metabolic regulation is a subject of wide current CD Proc. Natl. Acad. Sci. USA 79 (98) 409 interest. Recently a number of investigators have taken advantage of the favorable properties of molluscan neurons to investigate the possible role of protein phosphorylation in regulation of neuronal electrical activity (4, 6, 7). Intracellular injection of C subunit into neurosecretory bag cells (6) or an identified sensory neuron (7) from Aplysia causes an enhancement of the action potential. On the basis of previous voltage clamp experiments on the sensory neuron (5), it seems likely that this effect results from a decrease in K+ conductance. In Aplysia neuron R5, on the other hand, protein phosphorylation produces an increase in K+ conductance, because injection of a specific protein kinase inhibitor (6) into voltage-clamped neuron R5 blocks the K+ conductance increase normally elicited in this cell by serotonin (, 4). The present results using nonidentified Helix neurons support the notion that protein phosphorylation can cause an increase in K+ conductance. The outward current produced by depolarizing voltage clamp steps in these neurons is enhanced when as little as 0.0 /.M C subunit is added to the internal perfusing medium, and the effect is observed consistently with 0. gm or more. The total intracellular concentration of C subunit (both free and bound) is thought to be about ,uM in several different mammalian tissues (), indicating that the concentrations used in the present experiments are well within the physiological range. In addition, the availability of Nbs-inactivated C subunit provides an excellent control. The reaction of Nbs with sulfhydryl groups of the C subunit represents only a minor modification in the structure of the protein but completely eliminates its enzymatic activity (ref. ; unpublished data). Thus the fact that Nbs-inactivated C subunit, added to the perfusate at concentrations as high as um, is completely without effect on the cell's electrical properties, provides strong evidence that the enhancement of outward current produced by active C subunit is in fact due to protein phosphorylation rather than to some nonspecific effect of the protein per se. The outward current component enhanced by C subunit appears to be the I,, because the effect requires extracellular Ca+, is blocked by internal perfusion with 0 mm EGTA, and can be mimicked by adding 0,uM free Ca' to the internal perfusate. This is consistent with the observation that repetitive spiking is reduced by C subunit, because the Ca+-activated K+ channel is thought to play a role in regulation of such repetitive activity (7). It is unlikely that C subunit increases this current by increasing the entry of Ca+ during the depolarizing voltage clamp steps, because the total inward current is not noticeably altered by C subunit treatment. However, a small increase in the Ca+ component of the total inward current cannot be excluded, because it has not been measured directly. Another possibility is that C subunit releases free Ca+ from intracellular stores. However, the fact that buffering the internal Ca+ concentration with mm EGTA has little effect on the action of the C subunit is not easily reconciled with this possibility. A more likely explanation for the present results is that C subunit phosphorylates a regulatory component of the K+ channel such as to increase its affinity for Ca. This explanation is consistent with our finding that C subunit displaces the N-shaped region of the I-V curve to more positive voltages and often eliminates it entirely; if the affinity of the channel for Ca+ is increased, one would expect to see greater activation with depolarizing steps to voltages at which relatively little Ca+ can enter the cell. Furthermore, the observation that the K+ current is activated more rapidly in the presence ofc subunit is also consistent with an increase in affinity ofthe Ca+-activated K+ channel for Ca. However, we cannot entirely exclude other explanations for these complex effects. Patch clamp experiments (8) can be performed to investigate more directly the mechanism of the

4 40 Neurobiology: de Peyer et al. Proc. Natl. Acad. Sci. USA 79 (98) A ms < ' / Membrane potential, mv FIG. 4. Effect of perfusion with 0 /im Ca". Sets of current traces (A) and I-V relationships () as in Fig.. The sequence of events is similar to that appearing in the perfusion with C subunit. Measurements were made prior to the introduction (set ) and 40 (set ) and 60 (set ) min after the introduction of the 0 /LM Ca+ into the perfusion medium. effect of C subunit. It should be pointed out that low and high CaO sensitivity ofthe Ca+-activated K+ permeability has been 0 / reported before in the case of erythrocyte membranes (9). In / this system the different Ca+ sensitivities may also depend on X / cell metabolism. 00 / One general principle that is emerging from recent studies // f of oon molluscan neurons is that the activity ofnormal K+ channels may be regulated by protein phosphorylation. In the present 80 study the channel affected appears to be the Ca+-activated K+ channel, whereas in Aplysia neuron R5 it is the K+ channel responsible for anomalous rectification (unpublished data). In ^ 60 other neurons C subunit can decrease K+ conductance (6, 7); in one ofthese cases the Ca+-activated K+ channel is inhibited (0), whereas in the other () it may be a K+ channel distinct 40 from those that have previously been described in these cells (0). Thus it is becoming apparent that in different cells protein phosphorylation may regulate the activity of different ion chan- 0 * nels, * and in different ways. It will be particularly important to identify the phosphorylated channel components that are responsible for these changes in neuronal electrical properties. o We are grateful to Dr. E. Fischer for providing us with the C subunit Membrane potential, mv used in this study; the financial support by the Swiss National Science Foundation is gratefully acknowledged. FIG. 5. I-V curves for inward and outward current of a neuron perfused with C subunit (0.,uM) in the presence of 0 mm EGTA.. Kuo, J. F. & Greengard, P. (969) Proc. Natt Acad. Sci. USA 64, Under these conditions I, is absent and the C subunit has no effect on the residual voltage-dependent slow outward current. Curve, control;. Greengard, P. (978) Science 99, curves and, 5 and 60 min, respectively, after the addition of C. Levitan, I.. & Adams, W.. (98) Adv. Cyclic Nucleotide Res. subunit. 4,

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