ca2+-activated k+ currents underlying the afterhyperpolarization in guinea pig vagal neurons: a role...

Neuron, Vol. 7, 257-264, August, 1991, Copyright 0 1991 by Cell Press

Ca*+-Activated K+ Currents Underlying the Afterhyperpolarization in Guinea Pig Vagal Neurons: A Role for Ca*+-Activated Ca*+ Release

Pankaj Sah and Elspeth M. Mclachlan Department of Physiology and Pharmacology University of Queensland Queensland 4072 Australia

Summary

We examined the possibility that Ca*+ released from intracellular stores could activate K+ currents underlying the afterhyperpolarization (AHP) in neurons. In neurons of the dorsal motor nucleus of the vagus, the current underlying the AHP had two components: a rapidly decaying component that was maximal following the action potential (Ckca,,) and a slower component that had a distinct rising phase &kc&. Both components required influx of extracellular Ca*+ for their activation, and neither was blocked by extracellular TEA (10 mM). Ckc,,, was selectively blocked by apamin, whereas Ckca,* was selectively reduced by noradrenaline. The time course of Gkca,* was markedly temperature sensitive. Ckc,, was selectively blocked by application of ryanodine or sodium dantrolene, or by loading cells with ruthenium red. These results suggest that influx of Ca*+ directly gates one class of K+ channels and leads to release of Ca*+ from intracellular stores, which activates a different class of K+ channel.

Introduction

Influx of Ca*+ via voltage-gated Ca*+ channels leads to activation of K+ currents in many types of cells. From single-channel recordings, it is now clear that Ca*+- activated K+ channels can be divided into two types, so-called BK channels and SK channels. BK channels are voltage sensitive, have a large single-channel conductance (200-300 pS), and are blocked by submilli- molar concentrations of tetraethylammonium (TEA) ions. SKchannels arevoltage insensitive, havea lower single-channel conductance (IO-20 pS), and are insensitive to TEA. SK channels are potently blocked by the toxin apamin (Blatz and Magelby, 1986, 1987; Marty, 1989; Langand Ritchie,lYYO). In neurons, influxofCa*+ via voltage-activated Ca*+ channels has been shown to activate both types of Ca*+-activated K+ channels (Lancaster and Nicoll, 1987; Storm, 1987). In many neurons, activation of BK channels contributes to action potential repolarization (Lang and Ritchie, 1987; Lan- caster and Nicoll, 1987; Cola et al., 1990).

Although the SK channel has been suggested to underlie the current generating the afterhyperpolarization (AHP) (Lang and Ritchie, 1987; Lancaster and Nicoll, 1987), both apamin-sensitive (Pennefather et al., 1985; Kawai and Watanabe, 1986; Bourque and Brown, 1987; Schwindt et al., 1988) and apamin- insensitive AHPs (Lancaster and Nicoll, 1987; Con-

stanti and Sim, 1987; Morita and Katayama, 1989; Schwindt et al., 1988) have been reported in a variety of neurons. It therefore seems likely that there is a second type of voltage- and TEA-insensitive Ca*+- activated K+ channel (distinct from the SK channel) that is not blocked by apamin. Such channels have been recently described in excised patches from cultured hippocampal neurons (Lancaster et al., 1991). While it has not been demonstrated that this channel underlies the apamin-insensitive AHP in these neurons, it seems the most likely candidate.

AHPs in neurons range from several hundred milliseconds to several seconds in duration. Voltage- clamp studies have shown that the K+ current that underlies the AHP can be divided into two types. In some neurons (Pennefather et al., 1985), the AHP current (I& is maximal following an action potential and then decays exponentially with a time constant of several hundred milliseconds. In other cells (Hirst et al., 1985; Lancaster and Adams, 1986), the K+ current has a rising phase following the action potential and then decays over several seconds. This latter type of current has also been called IAHP (Lancaster and Adams, 1986; Constanti and Sim, 1987). In some neurons the two types of currents coexist (Cassell and McLachlan, 1987; Schwindt et al., 1988). In mammalian sympathetic ganglion cells, these two currents have been called Gkca,, and Gkc,,*, respectively (Cassell and McLachlan, 1987). This is the terminology we will use here.

Kinetic studies in invertebrate neurons (Barish and Thompson, 1983) have suggested that the time course of some Ca*+-activated K+ currents reflects the time course of the change in intracellular Ca*+ concentration. Direct measurement of the kinetics of intracellular Ca2+ concentration using Ca*+ indicators supports this hypothesis (Barish and Thompson, 1983; Smith et al., 1983; Stockbridge and Ross, 1984; Hernandez-Cruz et al., 1990). It is thought that the source of the Ca*+ that activates the Ca*+-sensitive K+ currents is influx from the extracellular space via voltage-gated Ca*+ channels.

It is clear that, following a brief period of influx, Ca*+ concentration near the intracellular face of the plasmalemmawill bemaximal immediatelyafterentry and will then decline as Ca*+ diffuses away from the membrane and is buffered within the cell (Yamada et al., 1989; Sala and Hernandez-Cruz, 1990). If the kinetics of the Ca2+-activated K+ current reflects Ca*+ concentration, it is puzzling that the two currents can have such different time courses. In particular, the mechanism for the slow-rising phase of Gkc,,? is unknown (Hirst et al., 1985; Constanti and Sim, 1987).

The preganglionic neurons in the dorsal motor nucleus of the vagus (DMV) play an important role in the efferent control of the cardiovascular and visceral systems. integration of diverse synaptic inputs from

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Figure 1. Two Components of the Current Underlying the AHP

(A) The AHP folowing a single action potential elicited at the resting potential of a guinea pig DMV neuron (-60 mV). (6) InvoltageclampthecurrentunderlyingtheAHP(uppertrace) mirrors the potential and has a rapidly decaying component followed by a slower component. The membrane potential during the voltage clamp is shown in the lower trace. Action potential peaks have been truncated. Details of the voltage clamp procedure are described in Experimental Procedures. The rapidly decaying component has been called Ckc,,,, and the slower component has been called Gkc,,*.

many brain stem and higher centers is responsible for the discharge of these neurons (Feldman and Ellen- berger, 1988), and the actions of these inputs in modi- fying firing properties are not understood. We have

undertaken an initial study of the characteristics of the conductances determining repetitive firing in these neurons. We found these neurons to be typical small neuronswith high input resistances (see Experimental Procedures), characteristics that made them attractive candidates for carrying out kinetic studies of currents under voltage clamp. In this study, we describe the properties of two Ca2+-activated K+ currents underlying the AHP in neurons of the DMV in guinea pigs. Our findings suggest that the time course of Gkca,2 can be explained by a mechanism that involves Ca2+- activated Ca2+ release from intracellular stores.

Results

Intracellular recordings were made from 135 neurons in the guinea pig DMV. As shown in Figure IA, a single action potential in these neurons was followed by a prolonged AHP that lasted many seconds. These cells were confirmed to be DMV neurons by the presence of an antidromic action potential. The currents under- IyingtheAHP in these neurons have been shown to be Ca*+-activated K+ currents (Yarom et al., 1985). Under voltage clamp, the kinetics of these currents were ex-

Figure 2. Some DMV Neurons Have Only One of the Two Com- ponents of the AHP

(A) A neuron that had only the rapidly decaying component of the AHP, shown in current clamp (upper trace) and in voltage clamp (lower trace). (B) Another neuron in the DMV that had only the slow component of the AHP.

amined. The outward tail current underlying the AHP hastwo phases(FigurelB, uppertrace).An initial component (henceforth called Gkca,J was maximal following the action potential and then decayed, after which another component had slow-rising and decay phases (called Gkc,,?). The relative sizes of the two components varied between cells. It was also possible to find cells in which one or the other component could not be detected (Figures 2A and 2B). In 10% of the cells Gkc,,2 was undetectable, and in 6% of the cells Gkca,, was undetectable. These observations suggest that the two currents are independent of each other.

Bothcomponentsoftheoutwardcurrentwereabol- ished either by addition of divalent cations that block Ca2+ channels (Figure 3A, n = 2), or by removing the Ca2+ from the perfusing medium (n = 2; see also Yarom et al., 1985). This indicates that influx of extracellular Ca*+ is necessary for activation of both currents. However, the two components of the K+current could be separated pharmacologically. Apamin, a polypeptide isolated from bee venom, has been shown to block the SK type of Ca*+-activated K’channel (Castle et al., 1989). Apamin (20-100 nM) blocked the fast component of the K+ current (Gkc,,l) but had no effect on the slowly decaying component (Figure 3B, n = 6). In hippocampal pyramidal neurons, the current underlying the AHP resembles Ckca,* (see be- low and Discussion). In these neurons the AHP and the underlying current are blocked by application of noradrenaline (Madison and Nicoll, 1986; Lancaster and Nicoll, 1987). We therefore examined the effects of noradrenaline on the Ca2+-activated currents in DMV neurons. Noradrenaline (Figure 3C, IO-100 PM) blocked the slow component of the K+current (Gk& with little effect on the fast component (n = 3).

The kinetic properties of the two currents are analyzed in Figure4. Gkc,,, (Figure4A, lefttrace)was maximal immediately following the action potential and decayed exponentially back to the baseline. This current reached its peak amplitude within 5 ms following an action potential (data not shown). The time con-

AHP and Intracellular CaZ+ Release 259

I cobalt

Figure 3. The Two Components of the AHP Tail Current Can Be Separated Pharmacologically

(A)The tail current underlying the AHP in control solution (4 mM Ca2’) and in a solution containing 3.5 mM Co*+ and 0.5 mM Ca2+. (8) The tail current underlying the AHP in control solution and in a solution containing 100 nM apamin (different neuron). (C) The tail current underlying the AHP in control solution and in a solution containing 50 PM noradrenaline (different neuron).

stant of decay of this current, which was voltage independent, was 152 * 7 ms (mean f SEM; n = 21, includ- ing eight ceils in which Gkca,* was pharmacologically blocked with either noradrenaline [three] or caffeine [five]). In contrast, Gkca,2 (Figure 4A, right trace) has a distinct rising phase and slow decay. The time course could be described with a double exponential function of the form A x (1 - e-““) x emu’*. The time constant for the rising phase (~1) was 555 + 24 ms and for the decay phase (~2) was 1422 f 45 ms (n = 28). The kinetics of Ckca,2 was also voltage independent.

Neither Gkc,, nor Gkca,z was blocked by addition of

“‘_h_‘ 200ms

& lsec

JCOms

lO@A I

JcY??a-

lsec

Figure 4. Kinetic Properties of Ck,,,, and Gkc.,z

Shown in (A) are the tail current in a cell that had only Gkc,,, (a) and the tail current in a cell that only had Ckc,,* (b). A line representing a single exponential function with a time constant of 130 ms has been drawn through the data in (a). Drawn through the data in (b) is a line representing a function of the form A x (1 - exp[-tlrl]) x exp(-t/12) with ~1 = 500 ms and r2 = 1655 ms. TEA (10 mM) applied extracellularly does not block either Gkc,,, (6) or Gkc,,? (C).

32oc

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l-

: 0.8 -

z 0.6 - F 6 0.4 - z

0.2 -

0 I I -500 2250 5000

Time (ms)

Figure 5. Effect of Temperature on Ckc+z

(A) The tail current underlying the AHP in one cell recorded at 32%, 26”C, and then back at 32OC. (B) The same currents shown normalized and superimposed.

IO mM TEA (Figures 4B and 4C, n = 6), indic:ating that BKchannels are not involved in either current. In fact, both Gkc,,, and Gkca,Zwere somewhat larger following addition of TEA presumably because of increased Ca2+ influx due to blockade of BK channels and action potential widening in the presence of TEA (Lancaster and Nicoll, 1987). These observations demonstrate that in guinea pig DMV neurons, influx of Ca2+ activates two different types of K+ currents that can be separated on kinetic and pharmacological grounds.

The time course of Ca2+-activated K+ currents is thought to reflect the time course of intracellular Ca2+ concentration. It can be seen in Figure 1 that Gkca,2 increased while Gkca,l was decaying. If extracellular Ca2+ is the source of the Ca2+ activating both of these currents, how does one account for their different time courses? Since the Cal+ concentration at the intracellular side of the membrane will be maximal immediately following influx (i.e., during the action potential), it seems likely that this Ca2+ directly activates apamin-sensitive (Gkca,,) channels close to the site of entry. The time course of Gkca,2 might then be due either to diffusion of Ca2+ to K+ channels located at some distance from the site of Ca2+ influx, to a slow openingoftheK+channels,ortoinfluxof Ca2+leading to generation of a second messenger that activates the K+ channels.

If the slow-rising phase of Gkca,Zwere due to simple diffusion of Ca*+ to a distant site then one would ex- pect that the time course of Gkc,,z would be relatively insensitive to temperature. To examine this question werecordedAHPtaiIcurrentattwodifferenttempera- tures. Data from one cell are illustrated in Figure 5. It can be seen that as the temperature was lowered from 32OC to 26%, both the rising phase and the decay

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control ryanodine

Jr. . . ..- - p I

Figure 6. Inhibition of Intracellular Ca*+ Release Blocks the Slow Component of the AHP

(A) AHP recorded in current clamp in a cell in control Ringer’s solution (left) and following addition of 10 PM ryanodine (right). (B) Recordings from a different cell in voltage clamp. Ryanodine (IO pM) selectively blocked the slow component of the AHP current. (C) Sodium dantrolene (25 PM) selectively reduced the slow component of the AHP current.

of the slow K+ current were markedly slowed. The average temperature coefficient (QIo) of the rising phase of Gkc,,* was 4.6 + 0.4, and that of the decay phase was 2.9 + 0.4 (n = 3; cf. Cassell and McLachlan, 1987). These observations suggest that the slow rise and decay of Gkea,l cannot be explained by simple diffusion of Ca*+ ions.

Apamin-andTEA-insensitive Ca2’-activated K+channels recorded in excised patches from hippocampal neurons show no appreciable delay between the application of Ca2+ and the opening of K+ channels (Lan- caster et al., 1991). Thus it seems unlikely that the rising phase of Gkca,? is due to channels that open slowly. In addition, since Ca2+ alone can open these channels in inside/out patches this implies that second messengers are not needed for activation of these channels.

Neurons are known to have intracellular stores of Ca*+ that can be released by caffeine or following a rise in intracellular Ca2* concentration (Neering and McBurney, 1984; Lipscombe et al., 1988). Thus an alternative explanation might be that the time course of Gkca,2 reflects the arrival of a wave of Ca2+ secondary to its release from an intracellular store. We tested this idea by applying substances known to inhibit the release of Ca*+ from intracellular stores in skeletal muscle, ryanodine (Sutko et al., 1985; Meissner, 1986) and sodium dantrolene (Van Winkle, 1976).

The effects of ryanodine (IO PM) and sodium dantrolene (25 PM) in the bath solution on the two Ca2+- activated K+ currents in DMV neurons are illustrated in Figure 6. It can be seen that ryanodine blocked the slow component of the AHP without affecting the fast

A I Zmin I 15min

Figure 7. Intracellular Loading with Ruthenium Red Reduces Gkcas

(A) AHP recorded in a cell impaled with an electrode containing 5 mM ruthenium red. The trace on the left shows the AHP 2 min following impalement, and the trace on the right shows the AHP 15 min after impalement. (B) Another cell in voltage clamp. The tail current underlying the AHP is shown 2 min and 15 min after impalement. Electrodes were loaded with ruthenium red as described in Experimental Procedures.

component (Figure 6A). In voltage clamp, both ryanodine (Figure 6B, n = 6) and dantrolene (Figure 6C, n = 4) substantially reduced Gkc,,* but had little effect on Gkc,,,. The effect of ryanodine was slow in onset and required 30-40 min to develop fully; it was not possible to reverse the effect despite washing for up to 1 hr. The effects of dantrolene were faster in onset but also irreversible. The fact that these compounds did not affect Gkc,, demonstrates that they did not affect the influx of Ca2+. In support of this we also found that ryanodine had no effect on the Ca2+ action potential (see also Kawai and Watanabe, 1989).

In muscle fibers, the Ca2+-activated Ca*+ release is also blocked bythe dye ruthenium red (Lai et al., 1988). To test further the idea that Ca2+-induced Ca*+ release is involved in the generation of Gkca,2, we loaded cells with ruthenium red. As illustrated in Figure 7, ruthenium red produced aclear reduction in the amplitude of Gkc,z while having no effect on Gkca,<. There was no obvious difference in resting potential or input resistance in cells loaded with ruthenium red as com- pared with control neurons. A total of 20 cells were recorded loaded with ruthenium red. In eight of these cells (40%) there was no evidence for a slow Gke,,? current following single action potentials. This com- pares with 10% in the control neurons. In seven cells recorded under current clamp there was a selective reduction in the slow component of the AHP by 47% + 9% 15 min following impalement with a ruthenium red-containing electrode. In four cells under voltage clamp in which Gkea,Zwas detected, it was reduced by 51% rfr 5% 20 min after impalement. In one cell there was no effect of ruthenium red. In electrodes not loaded with ruthenium red both Gkc,, and Gkc=,*were stable in amplitude for several hours.

Caffeine is an agonist at the Ca2+-activated Ca2+ release channel (Sitsapesan and Williams, 1990). We

AHP and Intracellular Caz+ Release 261

thus expected that application of caffeine might in- crease the size of Gkca,z (Kuba, 1980). Instead, application of caffeine (I-5 mM) caused a reduction of Gkca,2 amplitude (n = 5). However, caffeine is known also to block a phosphodiesterase that may cause a rise in intracellular cyclic nucleotide levels (Rail, 1982). The reduction of Gkca,2 might therefore be explained by this action (Madison and Nicoll, 1986). In support of this idea application of the phosphodiesterase inhibitor IBMX (3-isobutyl-I-methyl-xanthine; 100 PM) also blocked Gkca,P (n = 4).

Our experiments suggest that the rising phase of Gkca,z is due to delivery of Ca2+ via Ca2+-induced Ca*+ release. What determines the decay of this current? As mentioned earlier the current underlying the AHP in hippocampal pyramidal neurons shares many of the kinetic and pharmacological features of Gkca,?. Single-channel studies of apamin- and voltage-insensitive Ca2+-activated K+channels from cultured hippocampal neurons have shown them to have an open time of 8 ms, and they have been suggested to underlie the AHP in these cells (Lancaster et al., 1991). If these channels also underlie Gkca,2, the decay of the current is unlikely to be determined by the open time of the channel. The high Qlo of the decay phase suggests that simple diffusion of Ca2+ away from the K+ channelsdoesnotdeterminethetimecourseofdecay of this current. One possibility is that the decay of this current reflects removal of Ca*+ by a Ca2+-Mg2+- ATPase. Caimodulin has been shown to regulate the plasma membrane Ca2+-Mg2+-ATPase in erythrocytes (Carafoli and Zurini, 1982). Superfusion of thecalmod- ulin antagonists calmidazolium (10 uM, n = 3) or trifluperazine (300 uM, n = 2) was without effect on the time course of GkCa,2. We also applied the nonspe- cific ATPase inhibitor sodium orthovanadate (Carafoli and Zurini, 1982) externally (1 mM, n = 3) or in the recording electrode (400 PM, n = 8); both were without effect on Gkca,2.

Two other possible mechanisms for the removal of Ca2+ are efflux via the Na+/Ca*+exchanger and uptake into mitochondria (Blaustein, 1988). We examined the role of the Na+/Ca2+ exchanger by reducing the extracellular Na+ (n = 8; NaCl in the Ringer’s solution was replaced by LiCI). The role of mitochondrial uptake was examined by adding the mitichondrial uncou- pling agent CCCP (carbonyl cyanide m-chlorophenyl- hydrazone; 5 PM, n = 5). Both manipulations led to a rapid and marked reduction of membrane potential and input resistance presumably because of a rise in intracellular Ca2+ concentration. Thus the possible contribution of these mechanisms to the removal of Ca2+ in these neurons remains unknown.

Discussion

In this paperwe have demonstrated that in guinea pig DMV neurons, single action potentials are followed by a prolonged AHP. This AHP has two components: a rapidly decaying component followed by a slower

component. In voltage clamp, the currents underlying these two components can be shown to be distinct both kinetically and pharmacologically. Both are Ca2+-activated K+ currents, which have been called Gkca,, and Gkca,2. It seems likely that the time course of Gkca,, reflectsthetimecourseof Ca2+concentration near its point of entry. Our data suggest that influx of Ca2+ during the action potential also leads to release of intracellular Ca*+, which activates Gkca,2.

Both the currents reported here are voltage insensitive and are not blocked by TEA (Figure 3), making it unlikely that either one is mediated by BK channels. Because apamin blocks the fast current (Gkca,,), it probably reflects activation of SK channels. This current is maximal following the action potential, during which Ca2+ enters the cell and then decays with a time constant of about 150 ms (at 30%). In contrast, the apamin-insensitive K+current (Gkca,2) has a slow-rising phase and then decays with a time constant of 1.4 s. Similar differences in time course between apamin- sensitive and apamin-insensitive AHPs have been described in other neurons (Constanti and Sim, 1987; Schwindt et al., 1988).

Influx of Ca2’ via voltage-gated channels is necessary for activation of both the Ca2+-activated currents described here, because substitution of Co*+ for Ca2+ abolishes both currents. Single-channel studies have shown that apamin-sensitive, Ca*+-activated K+ channels (SK channels) can be recorded in excised membrane patches;‘these channels are directly gated by Ca*+ with a Kd of IO-’ to 10v6 M (Blatz and Magleby, 1986; Capiod and Ogden, 1989). Our experiments have notaddressed thetimecourseof theapamin-sensitive current, but since the burst length of the underlying channels is 35-110 ms (Capiod and Ogden, 1989) its time course seems likely to reflect local buffering and diffusion of Ca2+ away from the site of entry (see also Hernandez-Cruz et al., 1990).

Three mechanisms can be considered that might account for the slow time course of the apamin- insensitive current. The first possibility is that its kinetics simply reflects the time course of changes in Ca2+ concentration at some site distant from the point of Ca2+ entry. Second, Ca2+ might enter the cell and be rapidly buffered locally so that the time course of the current is entirely determined by channel kinetics. The third possibility is that Ca2+ influx leads to generation of a second messenger(s) (e.g., via phospholipase C) or release of Ca*+ from intracellular stores, ulti- mately leading to activation of the K+ channel.

The high temperature sensitivity of thecurrent indi- catesthatfreediffusion of Ca2+is unlikelytoexplain its slow time course. In addition, given that the diffusion coefficient of Ca2+ in cytoplasm is likely to be around 10m6 cm2/s (Hodgkin and Keynes, 1957), a time constant of 500 ms for the rising phase suggests that the K+ channels would have to be located about 20 urn away from the entry point of Ca2+. However, simulations of intracellular Caz+ concentration following a brief period of influx suggest that at a distance of 20 pm

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from the point of entry, the concentraton of Ca2+ would be too low to activate these channels (Yamada et al., 1989; P. S., unpublished data).

In hippocampal pyramidal neurons, the K+ current underlying the AHP is very similar to Gkca,z described here. Thus it is voltage insensitive, has slow-rising and decay phases, is apamin insensitive, and is blocked by application of noradrenaline (Lancaster and Adams, 1986; Lancaster and Nicoll, 1987). Apamin- and TEA- insensitve Ca2+-activated K+ channels have recently been recorded in excised patches of hippocampal neurons and have been suggested to underlie the AHP in these neurons (Lancaster et al., 1991). These experiments showed no appreciable delay between application of Ca2+ and opening of K+channels. Given the similarities between the two currents in hippocampal neurons and DMV neurons, these observations make it unlikely that the rising phase of Gkca,? is due to a slow opening of channels. Furthermore, in hippocampal neurons it is clear that a rise in intracellular Ca2+ is necessary for activation of this current (Lancaster and Nicoll, 1987). We have confirmed that in DMV neurons injection of the Ca2’ chelator EGTA blocks activation of Gkca,2 (P. S., unpublished data). In addition (as mentioned above) apamin-insensitive channels are directly gated by Ca*+ in excised membrane patches. These observations provide strong evidence that Ca2+ is likely to be the agonist that gates Gkc,,2 channels.

Neurons are known to contain intracellular stores of Ca2+ that can be released by inositol trisphosphate (IPs; Ferris et al., 1989), by caffeine, or by a rise in Ca2+ concentration (Neering and McBurney, 1984; Lips- combe et al., 1988; Thayer and Miller, 1990). It is thought that the IP3-sensitive store is physiologically distinct from the store that can be released by caffeine or a rise in Ca*+ (see Pietrobon et al., 1990). Both ryanodine and ruthenium red interfere with Ca2+-activated Ca2+ release and do not affect IP3-sensitive Ca2+ release (see Petersen and Wakui, 1990). Our finding that ryanodine, dantrolene, and ruthenium red can selectively reduce Gkc,,? supports the notion that Ca2+ is delivered to these K+ channels by a mechanism in- volving Ca*+-activated Ca2+ release (see also Kuba, 1980; Kawai and Watanabe, 1989). The release of Ca2+ from Cal+-sensitive stores is thought to occur via a channel, the ryanodine receptor (Meissner, 1986; Pie- trobon et al., 1990), the primary structure of which has recently been described (Takeshima et al., 1989). Ryanodine receptors have been demonstrated in the mammalian central nervous system (Ellisman et al., 1990; Nakai et al., 1990) and when incorporated into planar lipid bilayers have been shown to form functional Ca2+ channels that can be gated by Ca2+ (Ashley, 1989). It is thought that the Ca2+-sensitive intracellular Ca2+ pool is in the smooth endoplasmic reticulum (Pie- trobon et al., 1990). Both histological (Henkart et al., 1976) and electrophysiological (Osipchuk et al., 1990) data suggest that these pools are located close to the

plasma membrane, which would be appropriate for activation of K+ currents.

Our experiments show that TEA-insensitive Ca2+- activated KC currents can be subdivided into apamin- sensitive and apamin-insensitive types. Single-channel studies have shown that the two types of channels have a similar Ca2+ sensitivity (Blatz and Magleby, 1986; Lang and Ritchie, 1987; Lancaster et al., 1991). Since apamin has no effect on Gkca,2, SK channels cannot contribute to Gkc,,2. Furthermore, if Ca2+ gates both types of channels, this suggests that the Ca2+ that enters the neuron viavoltage-gated channels only has access to apamin-sensitive K+channels, whereas Ca2” released from intracellular stores only has access to the apamin-insensitive type.

One way to achieve such compartmentalization would be to separate the two types of channels physi- cally. For example, since Ca2+ channels are thought to be localized on dendrites (Llinas and Sugimori, 1980; Westenbroek et al., 1990) the apamin-sensitive K” channels may also be localized in the dendrites. The apamin-insensitive channels could then be on the so- matic membrane. If the spread of Ca2+ is limited by the the intrinsic buffering properties of the cytoplasm, Ca2+ influx or release from an intracellular or- ganelle could only act over a short distance. Such “compartments” have also been proposed for cardiac muscle(Ledereretal.,1990)and pancreaticacinarcells (Kasai and Augustine, 1990). In pancreatic cells, release of Cai+ near the cell membrane by IP3 leads to activation of two Ca2+-activated currents that have time courses similar to those of Gkca,, and Gkca,?. From direct imaging of intracellular Ca*+ the time course of the slower current can be explained by the arrival of a wave of Ca2+ secondary to its release from an intracellular store (Kasai and Augustine, 1990). This wave has been suggested to be mediated by repeated Ca2+-activated Ca2+ release. If the influx of Ca2+ through voltage-gated channels in neurons triggers the same sequence of events, such a mechanism could also explain the time course of the apamin- insensitive K+ current in neurons.

Experimental Procedures

All experiments were performed on transverse slices from guinea pig medulla maintained in vitro. Guinea pigs were anes- thetized with intraperitoneal pentobarbitone (50 mglkg) and de- capitated. The brain stem was exposed by scooping out the cere- bellum, and a section of it (obex & ~0.5 cm) was removed and placed in cold Ringer’s solution of the following composition: 115 mM NaCI, 5 mM KCI, 1.2 mM MgSO.,, 4 mM CaC12, 1.2 mM NaH2P0+ 25 mM NaHCOs, and 10 mM glucose (pH 7.2) when bubbled with 95% 0*-S% COz. Pransverse sections (400 pm thick) were then made on a Vibratome (TPI, St. Louis) starting approxi- mately 1 mm caudal to the obex. Slices were allowed to recover for at least 1 hr before recording was attempted. A single slice was then transferred to a recording chamber where it was held completely submerged between two nylon nets. The chamber was continuously perfused with oxygenated Ringer’s solution at 2WC-30°C.

Using a combination of trans- and epi-illumination, the DMV was visualized as a translucent area just dorsal to the hypoglossal

AHP and Intracellular Ca2+ Release 263

nucleus. In many slices the efferent fibers of the vagal neurons could also be seen coursing ventrolaterally. Bipolar steel stimu- lating electrodes (Fredrick Haer) were inserted into the region of these fibers for antidromic identification.

Intracellular recordings were obtained from neurons in the DMV that were stable for many hours. Intracellular electrodes were pulled from 1.2 mm o.d. glass (Clark Electromedical) on a Brown Flaming electrode puller and were filled with 0.5 M KCI (impedance 70-140 MO). When using KCI-filled electrodes, pic- rotoxin (100 PM) was included in the perfusing Ringer’s solution to block the reversed spontaneous inhibitory potentials that ap- peared. Cells had resting membrane potentials of -60 to -70 mV and input resistances of 100 to 600 MD.

Signals were recorded using an Axo-Clamp 2a (Axon Instru- ments), sampled at 0.5 to 5 kHz, and stored and analyzed on an IBM-compatible computer. For voltage-clamp experiments, electrode tips were coated with silicone oil in order to reduce the electrode capacitance. The headstage of the Ax&Clamp was continuously monitored to ensure complete settling of the voltage transient between samples. Switching frequencies of 2-3 kHz were used. Most of the cells described in this study were identified as vagal neurons by antidromic invasion from their efferent axons. In eight cases the cells were filled with biocytin and all were positively identified as being within the DMV.

Action potentials were elicited by injecting a brief (10 ms) depolarizing current pulse. To measure the currents underlying the AHP, the following procedure was used (see Cassell et al., 1986). The cell was voltage clamped at the resting membrane potential (-60 to -70 mV), and a brief (10 ms) depolarizing voltage (usually IO-15 mV) was delivered. The magnitude of this pulse was increased until a single active response was elicited. The Na+ current underlying the action potential cannot be controlled by the voltage clamp and results in an “action current.” Voltage control is poor during the action current; however, good voltage control is always obtained within 50 ms following the action current (Figure IB, lower trace; see also Cassell et al., 1986). Fol- lowing the action current, the tail current underlying the AHP can be measured. In some experiments a hybrid voltage clamp was used (Lancaster and Adams, 1986) to record tail currents underlying the AHP. There were no significant differences in the amplitude or time course of tail currents between the two methods. All traces shown represent the average of five to ten trials.

To load cells with dye, ruthenium red was dissolved in DMSO and then in 0.5 M KCI to give a final concentration of 5 mM (final concentration of DMSO was 0.06%). The tip of the electrode was filled with the ruthenium red solution bycapillaryaction, and the electrodewas then backfilled with 0.5 M KCI. Sodium dantrolene was dissolved in DMSO to make a stock of 10 mM; this was then added to the Ringer’s solution to make a final concentration of 25 PM (final DMSO concentration was 0.3%). DMSO on its own had no effect on membrane properties or Ca*+-activated currents. For intracellularstaining,theelectrodetipwas loadedwith biocytin (4% in 0.5 M KCI and 0.05 M Tris buffer [pH 7.41) and then backfilled with KCI. After the experiment, the tissue was briefly fixed in 4% buffered paraformaldehyde, rinsed, perme abilized by exposure to 0.5% Triton overnight, and reacted using the avidin-biotin procedure with diaminobenzidine (King et al., 1989). All drugs were purchased from Sigma.

Acknowledgments

We thank Bruce Gynther and David Hirst for comments on the manuscript and Mandy Bauer for doing the histology. This work was supported by grants from the National Health and Medical Research Council of Australia.

Received March 14, 1991; revised April 24, 1991.

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