The Journal of Neurophysiology Vol. 79 No. 6 June 1998, pp. 3252-3256
Copyright ©1998 by the American Physiological Society

RAPID COMMUNICATION

Modulation of the Ca²⁺-Activated K⁺ Current sI_AHP by aPhosphatase-Kinase Balance Under Basal Conditions inRat CA1 Pyramidal Neurons

Paola Pedarzani¹, Michael Krause¹, Trude Haug², Johan F. Storm², and Walter Stühmer¹

¹ Department of Molecular Biology of Neuronal Signals, Max-Planck-Institute for Experimental Medicine,37075 Gottingen, Germany; and ² Institute of Physiology, University of Oslo, 0317 Oslo, Norway

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Pedarzani, Paola, Michael Krause, Trude Haug, Johan F. Storm, and Walter Stühmer. Modulation of the Ca²⁺-activated K⁺ current sI_AHP by a phosphatase-kinase balance under basal conditions in rat CA1 pyramidal neurons. J. Neurophysiol. 79: 3252-3256, 1998. The slow Ca²⁺-activated K⁺ current, sI_AHP, underlying spike frequency adaptation, was recorded with the whole cell patch-clamp technique in CA1 pyramidal neurons in rat hippocampal slices. Inhibitors of serine/threonine protein phosphatases (microcystin, calyculin A, cantharidic acid) caused a gradual decrease of sI_AHP amplitude, suggesting the presence of a basal phosphorylation-dephosphorylation turnover regulating sI_AHP. Because selective calcineurin (PP-2B) inhibitors did not affect the amplitude of sI_AHP, protein phosphatase 1 (PP-1) or 2A (PP-2A) are most likely involved in the basal regulation of this current. The ATP analogue, ATP--S, caused a gradual decrease in the sI_AHP amplitude, supporting a role of protein phosphorylation in the basal modulation of sI_AHP. When the protein kinase A (PKA) inhibitor adenosine-3',5'-monophosphorothioate, Rp-isomer (Rp-cAMPS) was coapplied with the phosphatase inhibitor microcystin, it prevented the decrease in the sI_AHP amplitude that was observed when microcystin alone was applied. Furthermore, inhibition of PKA by Rp-cAMPS led to an increase in the sI_AHP amplitude. Finally, an adenylyl cyclase inhibitor (SQ22,536) and adenosine 3',5'-cyclic monophosphate-specific type IV phosphodiesterase inhibitors (Ro 20-1724 and rolipram) led to an increase or a decrease in the sI_AHP amplitude, respectively. These findings suggest that a balance between basally active PKA and a phosphatase (PP-1 or PP-2A) is responsible for the tonic modulation of sI_AHP, resulting in a continuous modulation of excitability and firing properties of hippocampal pyramidal neurons.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

The slow Ca²⁺-activated K⁺ current, sI_AHP, underlying the slow afterhyperpolarization and spike frequency adaptation in hippocampal and neocortical neurons (Alger and Nicoll 1980; Hotson and Prince 1980; Lancaster and Adams 1986; Schwartzkroin and Stafstrom 1980) represents one of the best-studied examples of neuromodulation in the vertebrate CNS. Many neurotransmitters, including various monoamines (norepinephrine, serotonin, histamine, and dopamine), acetylcholine, and glutamate, suppress sI_AHP, thereby enhancing neuronal excitability, decreasing spike frequency adaptation (Sah 1996), and most likely contributing to shifts in the overall functional state of the brain (McCormick and Williamson 1989). Monoamine transmitters modulate sI_AHP via adenosine 3',5'-cyclic monophosphate (cAMP) and protein kinase A (PKA) in CA1 pyramidal neurons (Blitzer et al. 1994; Madison and Nicoll 1986; Pedarzani and Storm 1993, 1995; Torres et al. 1995), whereas other kinases have been proposed to mediate the suppression of sI_AHP by muscarinic and metabotropic glutamate receptor agonists (Abdul-Ghani et al. 1996; Gerber et al. 1992; Müller et al. 1992; Pedarzani and Storm 1996; Sim et al. 1992).

One line of evidence supporting the role of protein phosphorylation in the modulation of sI_AHP in hippocampal neurons was provided by experiments in which protein phosphatases were blocked. This resulted in a gradual suppression of sI_AHP, suggesting the presence of an on-going phosphorylation-dephosphorylation turnover regulating sI_AHP in the absence of stimulation by neurotransmitters (Müller et al. 1992; Pedarzani and Storm 1993). A similar basal modulation has been proposed to take place for example in Aplysia sensory neurons (Ichinose and Byrne 1991) and in frog heart, where the L-type Ca²⁺ current and the delayed rectifier current I_K are modulated by a tonic balance between the basal activity of kinases and phosphatases (Frace and Hartzell 1993).

In the present study, we sought to investigate the molecular components involved in the basal modulation of sI_AHP, i.e., in the absence of synaptic stimulation or exogenous neurotransmitter application, in hippocampal neurons.

	METHODS

Abstract Introduction Methods Results Discussion References

Transverse hippocampal slices (400-µm thick) were prepared from 18- to 28-day-old Wistar rats decapitated under halothane anesthesia, transferred to a humidified holding chamber, and allowed to recover for 1 h. During recording, the slices were superfused with extracellular medium (2.5 ml/min) containing (in mM) 125 NaCl, 25 NaHCO₃, 1.25 KCl, 1.25 KH₂PO₄, 2.5 CaCl₂, 1.5 MgCl₂, and 16 glucose and saturated with 95% O₂-5% CO₂ at room temperature (21-24°C). Bicuculline (10 µM), tetrodotoxin (0.5 µM), and tetraethylammonium (TEA; 5 mM) routinely were added to the medium. Whole cell recordings were obtained from CA1 pyramidal cells in the slice, using the "blind" method (Blanton et al. 1989). The patch pipettes were filled with a control solution containing (in mM): 140 potassium gluconate (140 potassium methylsulfate in a subset of experiments), 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 2 Na₂-ATP, 3 MgCl₂, and 0.4 Na₃-GTP (pipette resistance: 5-9 M). ATP--S (0.5-1 mM; Boehringer Mannheim) substituted Na₂-ATP in the corresponding experiments described in RESULTS section. Using an EPC-9 amplifier (Heka Elektronik), the cells were voltage-clamped at 70 mV and depolarizing steps (100-ms long) of sufficient amplitude (typically +60 to +70 mV) to elicit a robust, unclamped Ca²⁺ action current were applied once every 30 s. The access resistance (range 15-35 M) and the amplitude and time course of the Ca²⁺ current during the step were monitored and showed only minimal variations during the recordings included in this study. With the control pipette solution, sI_AHP often showed a time-dependent increase in both amplitude and duration within a few minutes after establishing the whole cell recording mode ("run-up") (see also Zhang et al. 1995). For comparisons, we therefore usually used the traces recorded after the first 10-15 min after achieving the whole cell configuration to allow completion of the run-up. The current traces were filtered at 250 Hz, sampled at 1 kHz, and stored on a Power Macintosh 7100/66 using Pulse v8.00 (Heka Elektronik) or filtered at 10 kHz and stored on videotape. Data analysis was performed using the programs Pulsefit v8.00 (Heka Elektronik) and Igor Pro 2.04 (WaveMetrics). Stock solutions of microcystin LR, calyculin A, FK-506, Ro 20-1724, and rolipram were prepared in dimethyl sulfoxide (DMSO), aliquoted, and kept frozen at 20°C until use. The final concentration of DMSO was invariably <0.5% and did not affect sI_AHP in control recordings. Recordings with drugs and controls were normally intercalated. Microcystin-LR, rolipram, and calcineurin autoinhibitory peptide were obtained from Calbiochem; tetrodotoxin, bicuculline methchloride, 9-(tetrahydro-2-furyl)adenine (SQ22,536), Ro 20-1724, and isoproterenol hydrochloride from Research Biochemicals; calyculin A and cantharidic acid from LC Laboratories; adenosine-3',5'-monophosphorothioate, Rp-isomer (Rp-cAMPS) from BIOLOG Life Science Institute; tetraethylammonium from Sigma. FK-506 was a generous gift of Fujisawa. Dr. Angus Nairn generously provided the PP1 inhibitory peptide, phospho-DARPP-32 peptide [6-39] (Hemmings et al. 1990), which was synthesized in Dr. Nairn's laboratory in Rockefeller University, NY. Values are reported as means ± SE. Two-tailed Student t-test and analysis of variance were used for statistical comparisons between groups ( = 0.05).

	RESULTS

Abstract Introduction Methods Results Discussion References

Whole cell recordings (Blanton et al. 1989) were obtained from 80 CA1 pyramidal cells in rat hippocampal slices. In 98% of the cells, a typical slow afterhyperpolarization (AHP) current (sI_AHP) (Lancaster and Adams 1986) followed a Ca²⁺ current elicited by a short depolarizing step. After the initial run-up period (see METHODS and Fig. 1A), sI_AHP remained relatively stable for the duration of the recording, normally 1-2 h (12.6 ± 2.5% peak amplitude decrease after 60-75 min; n = 6; Fig. 1A).

View larger version (24K): [in this window] [in a new window]   FIG. 1. Phosphatase inhibitors microcystin, calyculin A, and cantharidic acid cause run-down of the slow Ca2+-activated K+ current, sIAHP. Under control conditions, the sIAHP amplitude was stable for the duration of the recording, except for the initial increase (run-up) during the 1st 10-15 min (A). In contrast, cells dialyzed with microcystin (B; 10 µM, ; 50 µM, ), calyculin A (C and D; 5 µM, ; 10 µM, ), and cantharidic acid (E and F; 10 µM) exhibited a gradual run-down in the sIAHP amplitude during 30-80 min (13.1 ± 6%; 35.7 ± 3%, and 45.8 ± 12.4% sIAHP left, respectively). On the contrary, intracellular application of the calcineurin (PP-2B) inhibitor FK-506 (G and H; 10 µM, ; 200 µM, ) did not significantly affect the sIAHP amplitude during 60 min (85 ± 3.6%). Time "0 min" in A, B, D, and F indicates the 1st current trace recorded after establishing the whole cell configuration. In C, calyculin A was 10 µM; in G, FK-506 was 200 µM. Scale bars: C, 50 pA and 4 s; E, 20 pA and 2 s; G, 20 pA and 4 s.

As previously shown (Pedarzani and Storm 1993), intracellular application of the phosphatase inhibitor microcystin (5-50 µM) caused a dose-dependent reduction in sI_AHP amplitude (10-50 µM: n = 4; Fig. 1B; 5 µM: n = 2; not shown).

Two other protein phosphatase inhibitors, calyculin A (5-10 µM; n = 4; Fig. 1, C and D) and cantharidic acid (10 µM; n = 3; Fig. 1, E and F), also gradually reduced the sI_AHP amplitude when applied in the patch pipette. At the relatively high concentrations used, microcystin and cantharidic acid can also inhibit the Ca²⁺-calmodulin-dependent protein phosphatase 2B (PP-2B or calcineurin; IC₅₀ = 200 nM for microcystin and 30 µM for cantharidic acid) (Honkanen 1993; Honkanen et al. 1990), beside protein phosphatase 1 (PP-1) and 2A (PP-2A). To investigate the possible involvement of PP-2B in the basal modulation of sI_AHP, we used two specific PP-2B inhibitors, FK-506 and calcineurin autoinhibitory peptide. When intracellularly applied, neither FK-506 (10-200 µM; n = 11; Fig. 1, G and H) nor the calcineurin autoinhibitory peptide (1 mM; n = 6; not shown) produced any effect on sI_AHP, suggesting a lack of involvement of PP-2B in the basal modulation of sI_AHP.

To determine whether the effects of the broad spectrum phosphatase inhibitors were solely a consequence of PP-1 inhibition, we tested a specific PP-1 peptide inhibitor, phospho-DARPP-32 peptide (Hemmings et al. 1990). The PP-1 peptide inhibitor failed to show a significant effect on the sI_AHP (n = 4; not shown).

The involvement of protein phosphorylation in the basal modulation of sI_AHP was further tested with the ATP analogue ATP--S, a substrate for protein kinases causing thiophosphorylation of target proteins, which are then resistant to dephosphorylation by protein phosphatases (Eckstein 1985). ATP--S (0.5-1 mM) caused a gradual run-down in the sI_AHP amplitude (n = 4; P = 0.0002; Fig. 2, A and B), thus supporting an involvement of protein phosphorylation in the basal modulation of sI_AHP.

View larger version (24K): [in this window] [in a new window]   FIG. 2. In A and B, substitution of ATP by ATP--S (0.5-1 mM) in the intracellular solution led to a gradual partial inhibition of the sIAHP (58.5 ± 3.5% current left after 30-40 min). A: ATP--S was 0.5 mM. B: summary of the data obtained with ATP--S from 4 cells. C-E: cell dialysation with microcystin (10 µM) and the selective protein kinase A inhibitor Rp-cAMPS (500 µM) led to the maintainance of a stable sIAHP amplitude during 30-60 min (C and D; 89.9 ± 3% sIAHP left). E: summary of all data obtained with 10 µM microcystin (MC) and 10 µM microcystin + 500 µM Rp-cAMPS (MC + Rp), compared with control data. F: in cells dialyzed with the kinase inhibitor Rp-cAMPS (500 µM), the mean peak amplitude of sIAHP, measured 15-20 min after achieving the whole cell configuration, is larger than in controls. Scale bars: A, 20 pA, 2 s; C, 20 pA and 4 s; .

To identify the protein kinase responsible for the basal phosphorylation, we tried to prevent sI_AHP run-down caused by phosphatase inhibitors by coapplying the specific protein kinase A (PKA) inhibitor Rp-cAMPS (500 µM) (Botelho et al. 1988). When this inhibitor was applied intracellularly together with microcystin (10 µM), no run-down in the sI_AHP was observed (n = 13; Fig. 2, C-E), in contrast to the marked reduction of sI_AHP produced by the same dose of microcystin applied alone (P = 0.0003; Figs. 1B and 2C). This suggests that a balance exists between the basal activity of PKA and of a phosphatase (PP-1 or PP-2A) modulating sI_AHP in the absence of a exogenous stimulation of the cAMP cascade. This conclusion is consistent with the larger amplitude of sI_AHP observed in cells loaded with Rp-cAMPS (40.6 ± 8.1 pA; n = 5) in comparison with control cells (26.2 ± 2.6 pA; n = 21; P = 0.04; Fig. 2F). Also with methylsulfate as main anion in the pipette solution, the amplitude of sI_AHP was more than twice as large (246%) in cells recorded with Rp-cAMPS (n = 18) as in control cells (n = 12; P = 0.0005; not shown). To further test if the basal activity of the cAMP cascade tonically modulates the sI_AHP amplitude, we tried to inhibit production and breakdown of cAMP. Application of the adenylyl cyclase inhibitor SQ22,536 induced a small but consistent increase in the size of sI_AHP (n = 4; P = 0.06; Fig. 3, A and C), suggesting that sI_AHP is inhibited tonically by cAMP produced by basally active adenylyl cyclase. Furthermore, two inhibitors of cAMP-phosphodiesterase type IV (Beavo and Reifsnyder 1990), Ro 20-1724 (200 µM; n = 2; Fig. 3B) and rolipram (250 µM; n = 5; Fig. 3B and D), caused a gradual decline of sI_AHP (P = 0.02). The effect of rolipram was observed in four of five cells tested. Taken together, these observations support the hypothesis of a steady-state basal modulation of sI_AHP due to a tonic level of activity of the cAMP-PKA system in CA1 neurons.

View larger version (22K): [in this window] [in a new window]   FIG. 3. A and C: extracellular application of the adenylyl cyclase inhibitor SQ22,536 (100 µM) led to a small but significant increase of the sIAHP amplitude (23.6 ± 11.7%). C: summary of the data from 4 cells after SQ22,536 application. B and D: cells dialyzed with the adenosine 3',5'-cyclic monophosphate phosphodiesterase inhibitors Ro 20-1724 (200 µM) and rolipram (250 µM) exhibited a gradual run-down of the sIAHP amplitude during 10-60 min, amounting to 26.5 ± 0.1% and 32.3 ± 4.1%, respectively. D: summary of the results obtained with rolipram from 5 cells. Scale bars: A and B, 10 pA, 2 s.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

The results presented in this study provide converging lines of evidence that basal phosphorylation and dephosphorylation activities exert a tonic modulatory effect on sI_AHP in the absence of exogenous stimulation in CA1 hippocampal pyramidal neurons. The basal phosphorylation activity is provided by the cAMP signaling cascade activating PKA, whereas the dephosphorylation is most likely provided by PP-1 or PP-2A.

Considering the different chemical structure of calyculin A, cantharidic acid, and microcystin, the results obtained with these protein phosphatase inhibitors argue against the possibility that sI_AHP inhibition is due to some unspecific effects of these drugs on sI_AHP or Ca²⁺ channels. We used relatively high concentrations of the inhibitors compared with the reported K_i values (Honkanen 1993; Honkanen et al. 1990; Ishihara et al. 1989; MacKintosh and MacKintosh 1994) because lower concentrations produced only a partial inhibition of sI_AHP probably due to limited diffusion (Pedarzani and Storm 1993). Consequently, we could not rely on the different specificity of microcystin, calyculin A, and cantharidic acid for PP-1, PP-2A, and PP-2B (PP-2C could be excluded because it does not seem to be inhibited by microcystin) to deduce which phosphatase is mainly responsible for the basal modulation of sI_AHP. However, our results with selective inhibitors of PP-1 and PP-2B do not support an involvement of these enzymes in the basal modulation of sI_AHP. Because PP-2B is calmodulin dependent, this conclusion also is supported by our previous observation that intracellular application of a calmodulin-binding peptide did not significantly reduce sI_AHP (Pedarzani and Storm 1996). Nevertheless, we cannot categorically exclude that PP-1 or PP-2B contribute to the basal modulation of sI_AHP.

The results obtained by selectively inhibiting PKA with Rp-cAMPS indicate that this kinase is mainly responsible for the run-down of sI_AHP during phosphatase inhibition and therefore for the steady-state modulation of sI_AHP under basal conditions. This result, which adds to the function of PKA in the modulation of sI_AHP by monoamine transmitters (Blitzer et al. 1994; Pedarzani and Storm 1993, 1995; Torres et al. 1995), is consistent with the gradual increase in sI_AHP induced by bath application of the broad-spectrum kinase inhibitor staurosporine (Gerber et al. 1992; Sim et al. 1992) and of Rp-cAMPS in cultured CA3 neurons (Gerber and Gähwiler 1994).

Our data, however, do not provide any direct indication of the target molecule being phosphorylated or dephosphorylated, thereby leading to the basal modulation of sI_AHP. It recently has been proposed, for example, that serotonin suppresses sI_AHP by modulating intracellular calcium-induced calcium release (CICR) through the phosphorylation of CICR channels by PKA (Torres et al. 1996). Direct evidence to solve this issue can be provided only by biochemical and electrophysiological studies on AHP channels in isolation after purification or cloning.

Inhibition of adenylyl cyclase and of cAMP-phosphodiesterase type IV activity led to an increase or decrease in the sI_AHP amplitude, respectively, in agreement with results obtained with conventional microelectrode recordings on AHP (Madison and Nicoll 1986). These observations suggest that a tonic activity of adenylyl cyclase leads to the production of sufficient levels of cAMP to reduce sI_AHP under resting conditions. This cyclase activity does not seem to be due to the release of monoamine transmitters from presynaptic terminals because neither antagonists of various monoamine receptors, alone or in combination (Pedarzani and Storm, unpublished data), nor the G-protein inhibitor GDP--S (Krause and Pedarzani, unpublished data) enhanced sI_AHP. Furthermore, the hypothesis of a tonic adenylyl cyclase activity is supported by the enhancement of sI_AHP, sAHP, and spike frequency adaptation previously observed in response to adenosine (A₁) or -aminobutyric acid-B receptor stimulation (Gerber and Gähwiler 1994; Haas and Greene 1984).

In conclusion, our results support the idea of a continuous modulation of neuronal excitability through sI_AHP due to a reversible and balanced activity of phosphorylating and dephosphorylating systems constituting a cyclic cascade (Shacter et al. 1988).

	ACKNOWLEDGEMENTS

  We thank Dr. M. Stocker for discussion and useful comments on the manuscript, Dr. G. L. Busch for critical reading of the manuscript, Dr. R. Andrade for suggesting the experiment with ATP--S, Dr. O. Paulsen for help with Igor analysis routines, Dr. A. Nairn (Rockefeller University, NY) and Fujisawa GmbH (Munich, Germany) for the generous gifts of PP-1 peptide inhibitor and FK-506, respectively, and S. Voigt and R. Schliephacke for excellent technical assistance.

  This work was supported by a Human Frontier Science Program Fellowship to P. Pedarzani.

	FOOTNOTES

  Address for reprint requests: P. Pedarzani, Dept. of Molecular Biology of Neuronal Signals, Max-Planck-Institute for Experimental Medicine, Hermann-Rein-Str. 3, 37075 Göttingen, Germany.

  Received 31 December 1997; accepted in final form 3 February 1998.

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