Apamin-Sensitive Conductance Mediates the K+ Current Response During Chemical Ischemia in CA3 Pyramidal Cells

Mitsuo Tanabe, Masahiro Mori, Beat H. Gähwiler, and Urs Gerber

Brain Research Institute, University of Zurich, CH-8057 Zurich, Switzerland


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Tanabe, Mitsuo, Masahiro Mori, Beat H. Gähwiler, and Urs Gerber. Apamin-Sensitive Conductance Mediates the K+ Current Response During Chemical Ischemia in CA3 Pyramidal Cells. J. Neurophysiol. 82: 2876-2882, 1999. Pyramidal cells typically respond to ischemia with initial transient hyperpolarization, which may represent a neuroprotective response. To identify the conductance underlying this hyperpolarization in CA3 pyramidal neurons of rat hippocampal organotypic slice cultures, recordings were obtained using the single-electrode voltage-clamp technique. Brief chemical ischemia (2 mM 2-deoxyglucose and 3 mM NaN3, for 4 min) induced a response mediated by an increase in K+ conductance. This current was blocked by intracellular application of the Ca2+ chelator, bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), reduced with low external [Ca2+], and inhibited by a selective L-type Ca2+ channel inhibitor, isradipine, consistent with the activation of a Ca2+-dependent K+ conductance. Experiments with charybdotoxin (10 nM) and tetraethylammonium (TEA; 1 mM), or with the protein kinase C activator, phorbol 12,13-diacetate (PDAc; 3 µM), ruled out an involvement of a large conductance-type or an apamin-insensitive small conductance, respectively. In the presence of apamin (1 µM), however, the outward current was significantly reduced. These results demonstrate that in rat hippocampal CA3 pyramidal neurons an apamin-sensitive Ca2+-dependent K+ conductance is activated in response to brief ischemia generating a pronounced outward current.


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Considerable variation in the susceptibility to ischemic damage is observed among neuronal cell types and regions in the CNS (Pulsinelli 1985). The elucidation of the mechanisms conferring partial resistance to specific classes of neurons during energy deprived states would advance our understanding of cellular homeostatic processes and may provide new leads for drug development. A majority of studies addressing these questions have focused on the hippocampus, which exhibits a characteristic pattern of neuronal damage. CA1 pyramidal cells show the greatest sensitivity to ischemic insults, whereas CA3 pyramidal cells remain relatively resistant. This pattern is observed both in the hippocampus of stroke patients as well as in hippocampal in vitro models of ischemia (Schmidt-Kastner and Freund 1991).

Differences in functional properties between CA1 and CA3 pyramidal cells after ischemia have also been noted. Thus CA1 cells exhibit a decrease in excitability during hypoxia (Fujiwara et al. 1987; Hansen et al. 1982; Leblond and Krnjevic' 1989; Nowicky and Duchen 1998; Urban et al. 1989), which is not observed in CA3 neurons (Howard et al. 1998), long-term potentiation (LTP) can no longer be induced in CA1 versus CA3 cells after ischemia (Kirino et al. 1992), and CA1 cells are more prone to hypoxic seizures than CA3 cells (Kawasaki et al. 1990). The decreased excitability of CA1 cells is largely due to a transient hyperpolarization mediated by an increase in potassium conductance (Erdemli et al. 1998; Fujiwara et al. 1987; Hansen et al. 1982; Leblond and Krnjevic' 1989). Recent studies have shown that this conductance corresponds to the calcium-dependent and charybdotoxin-sensitive K+ current IC, conducted by large conductance (BK) channels (Harata et al. 1997; Nowicky and Duchen 1998). A brief period of ischemia similarly induces hyperpolarization or a transient outward current in CA3 pyramidal cells. (Ben-Ari 1990; Takata and Okada 1995; Tanabe et al. 1998). The aim of the present investigation was to characterize this current and to determine whether its properties differ from the ischemic current described in CA1 neurons.


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Experiments were performed with organotypic hippocampal slice cultures. Hippocampi were removed aseptically from 6-day-old Wistar rats that had been killed by cervical dislocation. Tissue slices of 400-µm thickness were prepared and cultured by means of the roller tube technique as described previously (Gähwiler 1981). After 15-30 days in vitro, the cultures were transferred to a recording chamber mounted onto the stage of an inverted microscope (Axiovert 35 M, Zeiss, Jena, Germany), and superfused with an external solution (at 32°C, pH 7.4) containing (in mM) 148.9 Na+, 2.7 K+, 146.2 Cl-, 2.8 Ca2+, 0.5 Mg2+, 11.6 HCO3-, 0.4 H2PO4-, and 5.6 D-glucose. Single-electrode voltage-clamp recordings were made from CA3 pyramidal neurons (Axoclamp 2 amplifier, Axon Instruments, Foster City, CA) with microelectrodes having tip resistances of 50-80 MOmega and filled with 2 M potassium methylsulphate. Most experiments were performed in the presence of tetrodotoxin (TTX, 0.5 µM) at a holding potential (VH) between -50 and -60 mV. Slow afterhyperpolarization currents (sIAHP) were evoked with a brief depolarizing voltage jump (to 0 mV for 100-200 ms, 0.04 Hz). Input resistance was assessed with 500-ms hyperpolarizing voltage commands of -10 mV. Chemical ischemia was induced by bath application of sodium azide (NaN3, 3 mM) and 2-deoxyglucose (2-DG, 2 mM) to block the cytochrome oxidase system and glycolysis, respectively. The duration of chemical ischemia is indicated by a horizontal bar in the figures (normally 4 min). In experiments to assess the role of extracellular Ca2+, the normal superfusing solution was replaced with one containing low Ca2+ (0.5 mM) high Mg2+ (10 mM). Completely removing extracellular Ca2+ resulted in unstable recordings. When working with large polypeptides such as apamin, variability in responses can arise, which may reflect problems with tissue penetration (Lancaster and Adams 1986). For these experiments, we therefore preincubated tissue with apamin (1 µM) for 15 min.

NaN3, 2-DG, tolbutamide, and tetraethylammonium chloride (TEA) were purchased from Sigma (St. Louis, MO). Bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) was from Molecular Probes (Eugene, OR). Phorbol 12,13-diacetate (PDAc) was from LC Laboratories Europe (Läufelfingen, Switzerland). TTX was from Sankyo (Tokyo). Isradipine was a gift from Novartis (Basel). All drugs except BAPTA were directly applied via the superfusing solution.

Numerical data are expressed as means ± SE. Student's t-test was used to compare means when appropriate. P < 0.05 was considered significant.


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Brief chemical ischemia induces outward current in CA3 cells

Transient chemical ischemia (2 mM 2-DG and 3 mM NaN3 for 4 min) induced an outward current (348 ± 23 pA, mean ± SE, n = 64) associated with a conductance increase (28 ± 3%, n = 64) in CA3 hippocampal pyramidal cells in the presence of TTX (Fig. 1A). Reoxygenation often induced an additional outward current, reflecting reactivation of the electrogenic membrane Na+-K+ pump (Fujiwara et al. 1987). This transporter response was not further examined in this study. The current-voltage relationship of the outward current induced by ischemia was determined in the range from -130 to -50 mV using step pulse commands (500 ms duration) with 10-mV increments from a VH of -50 mV. The reversal potential was -96 ± 6 mV (n = 4), which is close to the theoretical equilibrium potential for K+ (-100 mV) with 2.7 mM K+ in the external solution, assuming an intracellular K+ concentration of 120 mM for CA3 pyramidal cells (Lüthi et al. 1996) (Fig. 1B). With 8.0 mM K+ in the external solution the reversal potential shifted to -70 ± 4 mV, which is close to the predicted value of -72 mV according to the Nernst equation (n = 4; Fig. 1C).



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Fig. 1. Chemical ischemia activates a K+ conductance in hippocampal CA3 pyramidal cells. Chemical ischemia was induced with a solution containing NaN3 (3 mM), and 2-deoxyglucose (2 mM) in place of glucose. A: typical recording from a hippocampal CA3 cell at a holding potential (VH) of -55 mV. In this and subsequent figures, downward deflections represent current responses to 10-mV hyperpolarizing voltage steps of 500 ms duration applied at 0.1 Hz to assess changes in membrane conductance. B: steady-state current-voltage relationships from -130 to -50 mV recorded before () or during (open circle ) ischemia from the cell depicted in A. C: current-voltage relationships obtained by subtracting the current before ischemia from that during ischemia. The reversal potentials are -96 ± 6 mV (n = 4) with 2.7 K+ () and -70 ± 4 mV (n = 4) with 8 K+ (open circle ) in the extracellular solution.

In subsequent experiments, the nature of the K+ conductance underlying the outward current was investigated. During transient energy deprivation the intracellular [Ca2+] in neurons increases (Szatkowski and Attwell 1994). To determine whether the K+ conductance activated during ischemia is sensitive to changes in intracellular [Ca2+], responses were examined in cells filled with BAPTA, a Ca2+ chelator. BAPTA (150 mM in the microelectrode), applied by ionophoretic injection for 20 min, effectively chelated intracellular Ca2+, as shown by the complete blockade of the Ca2+-dependent IAHP (Fig. 2A2). In BAPTA-loaded neurons, the outward K+ current was no longer observed during ischemia, but rather an inward current became apparent (278 ± 138 pA, n = 5; Fig. 2A1). A similar inward current also occurred when K+ conductance was blocked with 2 M CsCl in the electrode and 10 mM TEA in the extracellular solution (185 ± 62 pA, n = 5, not shown).



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Fig. 2. The K+ conductance responsible for the ischemic current is Ca2+-dependent. A1: with a bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA)-filled microelectrode (150 mM), ischemia failed to induce an outward current, but rather revealed an inward current. A2: blockade of the sIAHP after 20 min of BAPTA diffusion confirms the chelation of intracellular Ca2+. sIAHP was elicited with a voltage pulse (200 ms) to 0 mV from a VH of -55 mV. B: no change in the ischemic outward current is observed in the presence of tolbutamide (500 µM), a blocker of ATP-sensitive K+ channels .

Previous studies have shown that an ATP-sensitive K+ conductance (Fujimura et al. 1997; Fujiwara et al. 1987; Jiang et al. 1994) contributes to hyperpolarization observed in response to hypoxia. In CA3 cells, however, tolbutamide (500 µM), a sulfonylurea that specifically blocks ATP-sensitive K+ conductance (Ashcroft 1988), did not significantly change the amplitude of the ischemic K+ current (control 473 ± 123 pA vs. tolbutamide 603 ± 153 pA, n = 5; Fig. 2B). These results indicate that in CA3 pyramidal cells the outward current induced by ischemia is primarily due to the activation of a Ca2+-activated K+ conductance.

Activation of the ischemic K+ current depends on Ca2+ influx

The amplitude of the ischemic K+ current was reduced with low Ca2+ (0.5 mM) high Mg2+ (10 mM) in the external solution (control 338 ± 65 pA vs. low Ca2+ 71 ± 8 pA, P < 0.05, n = 5, Fig. 3A). This suggests that the activation of ischemic K+ current is mediated by Ca2+ influx from the extracellular compartment. A recent report has shown that in CA1 pyramidal cells, Ca2+ influx through L-type Ca2+ channels is required to induce the outward K+ current in response to chemical ischemia (Nowicky and Duchen 1998). To examine whether a similar mechanism operates in CA3 pyramidal cells, the ischemic K+ current was recorded in the presence of a dihydropyridine derivative, isradipine. For these experiments, cells were clamped at a depolarized holding potential of -40 mV, because the blockade of L-type Ca2+ current by dihydropyridines is voltage and use dependent (Gähwiler and Brown 1987). In cells exposed to isradipine (2 µM for 7 min) the ischemic K+ current was strongly attenuated (control 338 ± 58 pA, isradipine 58 ± 16 pA, n = 5; P < 0.05, Fig. 3B).



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Fig. 3. Influx of extracellular Ca2+ is required to induce the outward K+ current. A: the K+ current is reduced when external [Ca2+] is lowered from 2.8 mM (top trace) to 0.5 mM ([Mg2+]o raised to 10 mM). VH was -50 mV. B: isradipine (2 µM), an inhibitor of L-type Ca2+ current, blocks the ischemic K+ current. VH was -40 mV.

Ca2+-dependent K+ conductance mediating the outward current is apamin sensitive

Three types of Ca2+-dependant K+ currents have been characterized in hippocampal pyramidal neurons, the fast afterhyperpolarization current IC, and two types of slow afterhyperpolarization currents IAHP and sIAHP (Sah and Clements 1999; Storm 1993). IC is a large conductance Ca2+-dependent K+ current that underlies the fast components of the AHP. Submillimolar concentrations of extracellular TEA block IC, thereby prolonging action potential duration (Lancaster and Nicoll 1987; Storm 1989). Experiments were performed to evaluate the possible contribution of IC to the ischemic outward current. Although TEA (1 mM) increased the duration of action potentials, the ischemic response was not modified (Fig. 4A, n = 3). IC is conducted by BK channels, which are selectively blocked by charybdotoxin (Miller et al. 1985). To confirm the finding with TEA, the effects of charybdotoxin on the outward current were examined. Slice cultures were preincubated with charybdotoxin (10 nM) for 15 min to prevent activation of BK channels. As previously shown (Lancaster and Nicoll 1987; Storm 1989), charybdotoxin under control conditions prolonged the repolarization phase of the action potential (Fig. 4B2, n = 4). The ischemic K+ current response was, however, not affected (Fig. 4B1, n = 4). Even at a higher concentration of charybdotoxin (300 nM), the ischemic K+ current was not inhibited (n = 5, data not shown), ruling out an involvement of IC.



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Fig. 4. BK channels do not mediate the ischemic K+ current. The outward current is not reduced by TEA at a concentration of 1 mM (A1), which inhibits BK channels as reflected by the prolongation in the repolarization phase of evoked action potentials (A2). VH was -55 mV for TEA experiments, and action potentials were evoked with current injection at a membrane potential of -60 mV. B: the outward ischemic current persisted in the presence of charybdotoxin (10 nM; B1). In the same cell, the duration of evoked action potentials was prolonged after a 15-min application of charybdotoxin (B2). The action potentials evoked before (- - -) and 15 min after application of charybdotoxin (---) are shown superimposed.

The possibility that sIAHP corresponds to the ischemic K+ current was then tested. This current is insensitive to apamin (Lancaster and Nicoll 1987), but is blocked by protein kinase C activators (Baraban et al. 1985). sIAHP was activated by clamping cells at potentials between -50 and -60 mV and applying voltage step commands to 0 mV for 200 ms every 25 s. Although sIAHP was completely suppressed by PDAc (3 µM), an activator of protein kinase C, this treatment did not reduce the ischemic K+ current (Fig. 5A).



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Fig. 5. An apamin-sensitive Ca2+-dependent K+ current mediates the ischemic response. Blockade of the sIAHP with an activator of protein kinase C, phorbol 12,13-diacetate (PDAc; 3 µM; A2) does not inhibit the outward current response (A1). B: apamin (1 µM for 15 min) blocks the ischemic K+ current. Depolarizing voltage pulses to 0 mV for 100 ms were applied to this cell held at -60 mV.

Hippocampal CA1 pyramidal cells exhibit an additional calcium-dependent IAHP mediated by apamin-sensitive small conductance (SK) channels (Sah and Clements 1999; Stocker et al. 1999). Furthermore, autoradiographic studies provide evidence for the expression of apamin binding sites in the hippocampal pyramidal cell layer (Gehlert and Gackenheimer 1993; Mourre et al. 1986). We therefore tested whether an apamin-sensitive Ca2+-dependent K+ current mediates the ischemic current in CA3 cells. Slice cultures were preincubated with apamin (1 µM, 15 min). At this concentration, apamin did not modify the resting membrane potential. The outward K+ current induced by ischemia was, however, significantly decreased in the presence of apamin (36 ± 44 pA, n = 7, P < 0.05; Fig. 5B).


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Our results show that the transient outward current observed in CA3 pyramidal cells in response to a brief period of energy deprivation is mediated by a Ca2+-dependent K+ conductance. As has been demonstrated in CA1 pyramidal cells (Nowicky and Duchen 1998), this response is blocked by inhibitors of voltage-gated L-type Ca2+ channels, indicating that an influx of extracellular Ca2+ triggers the outward K+ current. In CA1 cells, mainly BK channels appear to conduct this Ca2+-dependent K+ current (Harata et al. 1997; Nowicky and Duchen 1998). In contrast, our experiments demonstrate that in CA3 cells activation of apamin-sensitive SK channels underlies the K+ current.

Ischemic outward current

Many types of central neurons respond to energy deprivation with an initial transient hyperpolarization resulting from the activation of a membrane K+ conductance (see Luhmann 1996, for review). Two main mechanisms have been put forward to explain this increase in conductance. The first is the activation of ATP-sensitive K+ channels that open in response to decreased levels of cytosolic ATP under ischemic conditions (Ashcroft 1988). In the hippocampus, however, experiments in which ATP-sensitive K+ channels were blocked with sulfonylureas have provided contradictory results. Application of the sulfonylurea tolbutamide significantly reduced hypoxic or ischemic responses in CA1 cells (Fujimura et al. 1997; Godfraind and Krnjevic' 1993; Grigg and Anderson 1989), but was reported to have no effect in other studies (Harata et al. 1997; Nowicky and Duchen 1998). Our results indicate that in CA3 cells ATP-sensitive K+ channels do not contribute to the ischemic outward current, as previously reported (Ben-Ari 1990).

Our results are consistent with the second proposed mechanism involving the activation of a K+ conductance sensitive to a rise in intracellular [Ca2+]. The dependency of the K+ conductance on an increase in cytosolic [Ca2+] was shown in our experiments where the ischemic outward current was prevented by chelation of intracellular Ca2+ with BAPTA, as previously reported in CA1 cells (Erdemli et al. 1998; Yamamoto et al. 1997). Moreover, the ischemic hyperpolarization or outward current occurs during the initial phase of ischemia, which is associated with an increase in cytosolic [Ca2+] in hippocampal neurons (Dubinsky and Rothman 1991; Duchen et al. 1990; Kass and Lipton 1986; Mitani et al. 1993; Nowicky and Duchen 1998).

Ca2+ dependence of the current

The ischemic outward current was reduced by decreasing extracellular [Ca2+] and abolished in the presence of isradipine, a selective L-type Ca2+ channel antagonist, indicating that Ca2+ influx is involved in generating the response. A similar observation was reported in CA1 cells, where Ca2+ influx through L-type channels appears to account for most of the increase in intracellular Ca2+ concentration (Nowicky and Duchen 1998). Furthermore, L-type channel blockers have been shown to prevent the Ca2+ rise in cortical cells during ischemia (Pisani et al. 1998). The mechanism through which voltage-gated Ca2+ channels are activated during the ischemic hyperpolarization remains unresolved. One possibility might be that energy deprivation causes a dysfunction in the voltage-sensing elements of the channel proteins, which could shift the voltage sensitivity to a more hyperpolarized level. In addition, even a small influx of Ca2+ may suffice to induce a large intracellular signal through a process of amplification involving Ca2+ release from intracellular stores (Belousov et al. 1995; Yamamoto et al. 1997).

Apamin-sensitive Ca2+-dependent K+ conductance is responsible for the ischemic K+ current

Three types of Ca2+-dependent K+ conductances have been characterized in central neurons, a charybdotoxin-sensitive BK conductance, an apamin-insensitive SK conductance, and an apamin-sensitive SK conductance (Sah 1996). In CA1 cells the increase in intracellular Ca2+ during energy deprivation activates a Ca2+-dependent K+ current that is blocked by charybdotoxin (Harata et al. 1997; Nowicky and Duchen 1998). The sensitivity of the response to charybdotoxin identifies the underlying current as IC, which is carried by BK channels (Miller et al. 1985). In CA3 cells, however, blockade of IC by TEA or charybdotoxin failed to inhibit the ischemic K+ current. The Ca2+-dependent K+ current, sIAHP, underlying the slow afterhyperpolarization, may also contribute to the hypoxic response in CA1 cells, as muscarinic and beta -adrenergic agonists suppress both the hypoxic hyperpolarization as well as the sIAHP (Erdemli et al. 1998; Krnjevic' and Xu 1990). Again, in CA3 cells under conditions where sIAHP was blocked, we observed no change in the outward K+ current response. The third type of Ca2+-dependent K+ current, which is sensitive to apamin and is mediated by SK channels, has been described in hippocampal interneurons (Zhang and McBain 1995) and CA1 pyramidal cells (Sah and Clements 1999; Stocker et al. 1999), but not in CA3 pyramidal cells. In our experiments, apamin significantly inhibited the Ca2+-dependent K+ current induced by ischemia. Thus CA3 pyramidal cells also express an apamin-sensitive K+ conductance. Furthermore, although our results show that CA3 cells possess all three types of known Ca2+-dependent K+ conductances, the increase in intracellular Ca2+ occurring during ischemia appears to preferentially activate the apamin-sensitive channels. It is unclear why the other Ca2+-activated K+ currents are not activated by the increase in intracellular Ca2+. The sIAHP is blocked during ischemia (Tanabe and Gerber, unpublished results), suggesting that an energy-dependent process is involved in the activation of these channels. Alternatively, sIAHP, which is strongly modulated by various neurotransmitters through second-messenger systems (Nicoll 1988), may be inhibited as a result of an intracellular disequilibrium during energy deprivation. In the case of the fast IAHP mediated by BK channels, further experiments will be required to explain the difference in the response between CA1 and CA3 pyramidal cells.

In conclusion, we have identified a difference in the mechanism underlying the transient hyperpolarization in response to energy deprivation in two main types of hippocampal pyramidal cells. During hypoxia or ischemia, an increase in extracellular glutamate depolarizes neurons, a process that can ultimately end in excitotoxic cell death through Ca2+ overloading (Szatkowski and Attwell 1994). Hyperpolarization of CA3 pyramidal cells through activation of the apamin-sensitive K+ conductance would tend to prevent the membrane potential from attaining the threshold for voltage-dependent Ca2+ influx, thus protecting cells. Future work will aim to determine whether this can account for the contrasting responses and susceptibility of CA1 versus CA3 pyramidal cells to metabolic stress.


    ACKNOWLEDGMENTS

We thank Dr. Y. Fischer for valuable discussions and advice and Dr. R. Dürr, L. Heeb, R. Kägi, H. Kasper, L. Rietschin, and R. Schöb for excellent technical assistance.

This work was supported by Sankyo Ltd., the Ministry of Education, Science, and Culture of Japan, the Prof. Dr. Max Cloëtta Foundation, and the Swiss National Science Foundation (31-45547.95).


    FOOTNOTES

Address for reprint requests: U. Gerber, Brain Research Institute, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 26 April 1999; accepted in final form 7 July 1999.


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