Diversity of Channels Involved in Ca2+ Activation of K+ Channels During the Prolonged AHP in Guinea-Pig Sympathetic Neurons

Juan Martínez-Pinna,2 Philip J. Davies,1 and Elspeth M. McLachlan1

 1Prince of Wales Medical Research Institute (affiliated with the University of New South Wales), Randwick, Sydney, NSW 2031, Australia; and  2Instituto de Neurociencias, Universidad Miguel Hernández, 03550 Alicante, Spain


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Martínez-Pinna, Juan, Philip J. Davies, and Elspeth M. McLachlan. Diversity of Channels Involved in Ca2+ Activation of K+ Channels During the Prolonged AHP in Guinea-Pig Sympathetic Neurons. J. Neurophysiol. 84: 1346-1354, 2000. The types of Ca2+-dependent K+ channel involved in the prolonged afterhyperpolarization (AHP) in a subgroup of sympathetic neurons have been investigated in guinea pig celiac ganglia in vitro. The conductance underlying the prolonged AHP (gKCa2) was reduced to a variable extent in 100 nM apamin, an antagonist of SK-type Ca2+-dependent K+ channels, and by about 55% in 20 nM iberiotoxin, an antagonist of BK-type Ca2+-dependent K+ channels. The reductions in gKCa2 amplitude by apamin and iberiotoxin were not additive, and a resistant component with an amplitude of nearly 50% of control remained. These data imply that, as well as apamin- and iberiotoxin-sensitive channels, other unknown Ca2+-dependent K+ channels participate in gKCa2. The resistant component of gKCa2 was not abolished by 0.5-10 mM tetraethylammonium, 1 mM 4-aminopyridine, or 5 mM glibenclamide. We also investigated which voltage-gated channels admitted Ca2+ for the generation of gKCa2. Blockade of Ca2+ entry through L-type Ca2+ channels has previously been shown to reduce gKCa2 by about 40%. Blockade of N-type Ca2+ channels (with 100 nM omega -conotoxin GVIA) and P-type Ca2+ channels (with 40 nM omega -agatoxin IVA) each reduced the amplitude of gKCa2 by about 35%. Thus Ca2+ influx through multiple types of voltage-gated Ca2+ channel can activate the intracellular mechanisms that generate gKCa2. The slow time course of gKCa2 may be explained if activation of multiple K+ channels results from Ca2+ influx triggering a kinetically invariant release of Ca2+ from intracellular stores located close to the membrane.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In many neurons, the activation of voltage-gated Ca2+ channels during the action potential (AP) leads to a prolonged afterhyperpolarization (AHP) lasting several seconds. The AHP causes spike frequency adaptation and is thus a major determinant of cell excitability. In one class of sympathetic neuron in the guinea pig celiac ganglion (long-afterhyperpolarizing or LAH neurons), entry of Ca2+ during a single AP activates two distinct K+ conductances. One conductance change (gKCa1) has fast kinetics of activation and decays exponentially with a time constant of ~100 ms. The second conductance change in LAH neurons (gKCa2) has a slower rising phase and decays exponentially with a time constant of ~1.4 s at 35°C (Cassell and McLachlan 1987). In some peripheral neurons (myenteric, Hirst et al. 1985; nodose, see Cordoba-Rodriguez et al. 1999), the slower current alone can be detected after a single action potential, whereas both gKCa1 and gKCa2 are present in preganglionic neurons of the guinea pig vagal dorsal motor nucleus (Sah and McLachlan 1991) and the rat intermediolateral column (Sah and McLachlan 1995). In other CNS neurons (e.g., Constanti and Sim 1987; Lancaster and Nicoll 1987; Schwindt et al. 1988; Williams et al. 1997), a prolonged AHP of similar time course appears after a high-frequency discharge or a prolonged depolarization, and the underlying current has been variously referred to as IAHP or sIAHP (Lancaster and Adams 1986; Pennefather et al. 1985; Sah and Bekkers 1996). Like cultured CA3 (Tanabe et al. 1998) but not CA1 hippocampal neurons (Zhang et al. 1995), gKCa2 in guinea pig vagal (Sah and McLachlan 1991) and celiac (Jobling et al. 1993) neurons is markedly reduced by blockers of Ca2+ release from intracellular stores, such as ryanodine.

The K+ channels activated during these two Ca2+-dependent K+ conductances in LAH neurons appear to be different. gKCa1 is substantially blocked by apamin, a blocker of most small conductance or SK-type Ca2+-activated K+ channels (Jobling et al. 1993; Sah and McLachlan 1991). In guinea pig myenteric neurons, which lack gKCa1, the slow AHP is unaffected by apamin, but blockers of large conductance or BK-type Ca2+-activated K+ channels reduce its amplitude (Kunze et al. 1994). Similarly, apamin has no effect on gKCa2 in guinea pig vagal neurons (Sah and McLachlan 1991) or the sIAHP generated following a train of action potentials in hippocampal pyramidal neurons (Lancaster and Adams 1986; Stocker et al. 1999). Thus it appears that the slow conductance change is commonly mediated by Ca2+-activated K+ channels that are resistant to blockade by apamin.

We recently found that entry of Ca2+ through L-type Ca2+ channels contributes to the activation of gKCa2 in LAH neurons; the amplitude of the prolonged tail current was reduced by about 40% on average in 10 µM nifedipine (Davies et al. 1999). In nodose neurons, however, the slow AHP is abolished when N-type channels are blocked (Cordoba-Rodriguez et al. 1999). The slow AHP following a burst of action potentials in hippocampal CA3 pyramidal neurons is partly mediated by Ca2+ derived from L-type Ca2+ channels but not from N-type or P/Q-type Ca2+ channels (Tanabe et al. 1998). In contrast, activation of the slow AHP in cholinergic nucleus basalis neurons depends on Ca2+ flowing through N- and P-type channels (Williams et al. 1997). Therefore the source of Ca2+ for the activation of the slow conductance change by APs seems to vary between different types of neuron.

In this study, we have investigated the involvement of different Ca2+-activated K+ channels in the generation of gKCa2 in LAH neurons in sympathetic ganglia. For this purpose, we have used apamin (100 nM) and iberiotoxin (IbTx, 20 nM) as selective blockers of SK- and BK-type channels, respectively (Candia et al. 1992; Castle et al. 1989). These concentrations are well above the IC50 (apamin: 480 pM to 1.3 nM, Bourque and Brown 1987; Stocker et al. 1999; IbTx: 250 pM, Galvez et al. 1990), so that SK and BK channels, respectively, should be completely blocked. We appreciate that some cloned SK-type channels are not blocked by apamin (Köhler et al. 1996), but, for the purpose of this study, we have referred to channels blocked by apamin as being of the SK-type. In addition, we have investigated the effects of blockade of different types of Ca2+ channel to determine which source(s) of Ca2+ other than L-type channels activate(s) the slow conductance change in LAH neurons. All of the experiments depend on the specificity of the antagonists, which has not been independently verified for these neurons, given that isolation of cells for direct study of membrane channels would limit the study to somatic channels and can probably modify activation mechanisms. Rather, we have assumed the pharmacological selectivity of the antagonists for particular types of K+ and Ca2+ channels, as has been done previously for sympathetic (Davies et al. 1996; Ireland et al. 1998, 1999) and central neurons (Boland et al. 1994; Mintz et al. 1992; Olivera et al. 1994). Consideration of the data obtained for gKCa2, as well as that for gKCa1, leads to the conclusion that distinct intracellular mechanisms exist for activation of several types of Ca2+-activated K+ channels underlying the AHP.


    METHODS
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METHODS
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Guinea pigs (150-300 g of either sex) were deeply anesthetized with pentobarbitone (80 mg/kg ip) and exsanguinated by perfusion through the descending thoracic aorta with oxygenated physiological salt solution. The celiac ganglion and attached splanchnic and celiac nerve branches were dissected free. In two experiments, data were obtained from the relatively rare LAH cells present in the superior cervical ganglion (see Christian and Weinreich 1988). Ganglia were pinned out in vitro and superfused with oxygenated physiological salt solution at 35°C (composition, in mM: 151 Na+, 4.7 K+, 2.0 Ca2+, 1.2 Mg2+, 144.5 Cl-, 1.3 H2PO4-, 16.3 HCO3-, and 7.8 glucose; pH 7.2-7.4). These procedures were approved by the Animal Care and Ethics Committee of the University of New South Wales.

Intracellular recordings were made using microelectrodes filled with 0.5 M KCl (resistance 70-120 MOmega ) and records taken in bridge mode, single-electrode current clamp, and single-electrode voltage clamp as described in detail previously (Cassell et al. 1986; Davies et al. 1996). Recordings were taken only from cells classified as LAH by the presence of the slow conductance, gKCa2 (Cassell et al. 1986; McLachlan and Meckler 1989). Passive electrical properties and RMP of each neuron were routinely measured at intervals throughout every impalement. Cell input resistance, cell time constant, and cell input capacitance were determined from the voltage response to a small amplitude hyperpolarizing current step (250 ms long) at a holding potential between -60 and -65 mV, where the current-voltage relationship was linear. APs were generated after a brief (10 ms) depolarizing current step from resting membrane potential (RMP) and analyzed as described previously (Davies et al. 1996). The peak of the AHP was measured as the most negative value at the end of the falling phase of the action potential. Outward tail currents were generated after a voltage command step (50 ms) that elicited only one "action current" (i.e., an uncontrolled action potential, see Cassell and McLachlan 1987). Neurons with relatively large gKCa2 were selected for study to permit quantification of the reductions produced by the antagonists. Normally at least 10 current responses were averaged. The time course of gKCa1 in LAH neurons was estimated by subtracting a function describing the sum of two exponentials fitted to the peak and decay phase of gKCa2, from the overall tail current (for details see Cassell and McLachlan 1987). The amplitude of gKCa2 was measured at about 500 ms after the end of the voltage step; this corresponded to its peak when this could be detected and, in cases where its rising phase was obscured by gKCa1, to the time when only a small component of gKCa1 remained (see Cassell and McLachlan 1987). RMP was measured as the difference between potentials immediately prior to and following withdrawal of the microelectrode from the cell.

Drugs used

Apamin, tetraethylammonium, 4-aminopyridine, and glibenclamide were all purchased from Sigma (St Louis, MO). omega -Conotoxin GVIA was obtained from Auspep (Parkville, Victoria, Australia) and iberiotoxin from Alomone Laboratories (Jerusalem, Israel). omega -Agatoxin IVA was obtained from Peptide Institute (Osaka, Japan) and was dissolved initially in distilled water containing 1 mg/ml cytochrome C (Sigma). All drugs were dissolved in distilled water, aliquotted as stock solutions, and kept frozen until immediately before use when they were diluted in physiological solution to reach the final working concentration. Drugs were added to the bath by transferring the inlet of the perfusion system to a solution containing the stated concentration. Effects of all drugs were recorded at least 20-30 min later when a steady state of block had been achieved (Davies et al. 1996). The effects of the toxins could not be reversed by washing for >2 h.

Statistical analysis

All values are expressed as means ± SE. Differences between properties in control and drug solutions were tested using a paired t-test, or a Wilcoxon signed-rank test if n >=  6 and variances were not equal for control and drug data. All reported significant differences had P < 0.05.


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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

General properties

RMP of LAH neurons selected for this study was -55 ± 1 mV (mean ± SE, n = 29). Passive membrane properties determined between -60 and -70 mV (cell input resistance, Rin, 104 ± 7 MOmega ; input time constant, tau in, 17 ± 1 ms; input capacitance, Cin, 166 ± 11 pF, n = 28) were similar to those previously reported (Cassell and McLachlan 1987). A single AP was followed by an AHP of 11 ± 1 mV peak amplitude lasting 2.28 ± 0.13 s (n = 26; Fig. 1A).



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Fig. 1. Properties of the slow afterhyperpolarization (AHP) and outward tail current recorded in a long-afterhyperpolarizing (LAH) neuron in the guinea pig celiac ganglion. A: the AHP after a single action potential (AP) evoked at -58 mV. This trace is an average of 5 trials. In this and subsequent traces of AHPs, the peak of the AP has been truncated. The bottom trace shows the brief current step used to trigger the AP. B: outward tail current (top trace) recorded in voltage clamp at a holding potential of -50 mV following a brief depolarizing voltage step (bottom trace) that initiated a single active response. These traces are averages of 10 trials. The slow component of the outward current (gKCa2) has been fitted by the sum of 2 exponentials (solid line) with tau on = 285 ms and tau off = 1.3 s (see Cassell and McLachlan 1987). C: subtraction of the fit to the slow component reveals the initial faster component of the outward current (gKCa1) that decayed as a single exponential (solid line) with tau  = 130 ms. Current scale in B also applies in C. Note time scale in A and B differs from that in C.

In voltage clamp, a depolarizing step (50 ms) sufficient to initiate a single action current was followed by an outward current with two components: a transient fast decaying component and another component that rose to a peak at 500-800 ms and then decayed slowly (Fig. 1B). As these currents are measures of the underlying conductances, they are referred to here as gKCa1 and gKCa2, respectively. gKCa1 (measured following subtraction of gKCa2) had an amplitude of 121 ± 10 pA and a decay time constant of 98 ± 4 ms (n = 29; Fig. 1C). The peak amplitude of gKCa2 was 56 ± 7 pA, and its rise and decay could be fitted with exponential functions with time constants of 253 ± 10 ms and 1.24 ± 0.05 s, respectively.

Effect of blockade of K+ channels

EFFECT OF APAMIN. Addition of 100 nM apamin had no significant effects on RMP, passive properties or the AP (n = 7; Fig. 2A) (see also Davies et al. 1996; Ireland et al. 1998). The early peak amplitude of the AHP was not significantly changed. However, the derived amplitude of gKCa1 was reduced (by 64 ± 5%, Table 1; Fig. 2C, inset), and the early part of the slow AHP was correspondingly attenuated (Fig. 2B, inset). The reduction in the peak amplitude of gKCa2 was highly variable between cells (range +0 to -83%) with an overall reduction of 35 ± 10%; however, its time course was not affected (Table 1; Fig. 2C).



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Fig. 2. Blockade of SK-type channels in an LAH neuron. Records show APs (A), AHPs (B), and outward tail currents (C). In this and subsequent figures, control and drug responses are shown superimposed with those in the presence of the drug as thicker lines (arrow). A: addition of 100 nM apamin had little effect on the amplitude or time course of the AP. B: apamin reduced both components of the AHP (top traces). C: both gKCa1 and gKCa2 were reduced in amplitude by apamin (top traces) in voltage clamp at a holding potential of -50 mV. Insets in B and C and in following figures show expanded traces (in this case 200 ms) of the early part of the response scaled in amplitude by 50%. Resting membrane potential (RMP) in A and B was -56 mV. Time scale in B also applies in C.


                              
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Table 1. Reduction in gKCa1 and gKCa2 by antagonists of K+ and Ca2+ channels

EFFECT OF IBTX. Addition of 20 nM IbTx to the perfusing solution increased cell input resistance (Rin) by 44 ± 16% (control, 97 ± 10 MOmega ; IbTx, 137 ± 18 MOmega , n = 9, P = 0.03), without changing cell capacitance (control, 162 ± 16 pF; IbTx, 159 ± 20 pF; n = 9). This may have resulted from blockade of a Ca2+-dependent K+ conductance active at RMP, possibly gKCa2 (see North and Tokimasa 1987). IbTx had no effect on the amplitude of the AP but it increased its half-width by 7% (control, 1.33 ± 0.08 ms; IbTx, 1.45 ± 0.09 ms, n = 8, P < 0.05; Fig. 3A).



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Fig. 3. Effect of blockade of BK-type channels in an LAH neuron. Records show APs (A), AHPs (B), and outward tail currents at a holding potential of -50 mV (D). A: addition of 20 nM iberiotoxin (IbTx) slowed AP repolarization. B: both components of the AHP were reduced by IbTx. RMP in A and B was -56 mV. C: plot showing the time course of blockade of gKCa2 by IbTx. Thick bar indicates period during which IbTx was present. D: IbTx reduced the amplitude of both components of the outward tail current. Insets in B and D show expanded the 1st 400 ms of the traces. Time scale bar in B also applies in D.

After 20 min in IbTx, the peak amplitude of the AHP was generally slightly smaller (control, 12 ± 1 mV; IbTx, 10 ± 1 mV, n = 8, P = 0.22), and the peak amplitude of gKCa1 was reduced on average by 27 ± 11% (range +17 to -61%, Table 1; Fig. 3D, inset). This suggests that gKCa1 has components both sensitive and resistant to IbTx as well as those blocked by apamin. However, the most prominent effect of IbTx was a reduction in the peak amplitude of gKCa2 (Fig. 3, C and D), which was 55 ± 7% (range 30-85%) smaller than that in control solution (Table 1). IbTx had no effect on the time course of gKCa2.

We also tested the effects of blocking BK-type channels by addition of a low concentration (0.4-0.5 mM) of tetraethylammonium (TEA). Addition of TEA increased AP half-width by 6% (control, 1.28 ± 0.04 ms; TEA, 1.35 ± 0.05 ms, n = 4). However, neither the amplitude of gKCa1 (control, 93 ± 13 pA; TEA, 110 ± 20 pA, n = 4; P = 0.1) nor that of gKCa2 (control, 40 ± 6 pA; TEA, 48 ± 14 pA, n = 4; P = 0.44) was reduced in the presence of this concentration of TEA.

In three experiments, the effects of subsequent addition of 0.5 mM TEA was tested in the presence of IbTx and apamin. Following the addition of TEA, there was no further reduction in the amplitude of gKCa2 (apamin + IbTx, 30 ± 0 pA; TEA, 34 ± 3 pA, P = 0.32). However, AP half-width tended to increase (apamin + IbTx, 1.27 ± 0.03 ms; TEA, 1.35 ± 0.04 ms, P = 0.1).

OUTWARD TAIL CURRENT RESISTANT TO APAMIN AND IBTX. When apamin and IbTx were added sequentially, the addition of the second drug always produced a smaller degree of blockade than the first. This occurred independently of the order of application. Apamin added after IbTx reduced the remaining slow current by 0, 15, and 17%, whereas IbTx added after apamin reduced it by 17 and 25%.

In the presence of both apamin and IbTx, a component of gKCa2 remained, which ranged from 15 to 70% (mean 45 ± 11%) of the original gKCa2 amplitude in different cells (amplitude control, 85 ± 29 pA; apamin + IbTx, 28 ± 4 pA, n = 5). This current had the same decay time constant as in control solution (control, 1.13 ± 0.09 s; apamin + IbTx, 1.13 ± 0.17 s, n = 5; Fig. 4). In the same cells, the resistant component of gKCa1 was 15.7 ± 6.7% of the amplitude in control solution. The resistant component remained in one cell after the concentration of apamin was increased to 200 nM.



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Fig. 4. Effect of sequential application of iberiotoxin (IbTx) and apamin in an LAH neuron. Records show AHP at -55 mV (a) and outward tail currents at a holding potenital of -50 mV (b) in control solution (A) and 30 min following addition of 20 nM IbTx (B) and then 28 min after adding 100 nM apamin (C). The slow components of the AHP and the outward tail current were reduced in the presence of IbTx (B). Subsequent application of apamin (C) reduced the faster component of the AHP and abolished the early peak in the tail current (gKCa1). It is clear that a component of gKCa2 is resistant to blockade by both drugs. Scales in C apply throughout.

In five experiments, increasing the number of action currents generated during the voltage step increased the amplitude of the residual component (1 action current, 35 ± 4 pA; 2 action currents, 68 ± 7 pA, P = 0.012) but did not alter its time course (1 action current, 1.25 ± 0.19 s; 2 action currents, 1.12 ± 0.11 s), as occurs in control solution (Cassell and McLachlan 1987).

gKCa2 is abolished by addition of 50 µM noradrenaline (see Jobling et al. 1993), and this effect was confirmed in one experiment for the residual current remaining in apamin and IbTx.

EFFECTS OF OTHER K+ CHANNEL BLOCKERS. The sensitivity of the residual component of slow outward current present in apamin and IbTx to other K+ channel antagonists was examined (Fig. 5). Addition of 10 mM TEA more than doubled AP half-width (control, 1.30 ± 0.02 ms; TEA, 2.84 ± 0.08 ms, n = 3, P = 0.004; Fig. 5, Aa and Ba) but did not change the amplitude of either gKCa1 (control, 40 ± 10 pA; TEA, 40 ± 12 pA, n = 3) or gKCa2 (control, 37 ± 7 pA; TEA, 37 ± 9 pA, n = 3), or the time constants of decay of either current (Fig. 5Bb) (see also Hirst et al. 1985 for myenteric neurons).



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Fig. 5. Effect of blockade of K+ channels with tetraethylammonium (TEA) and 4-aminopyridine (4-AP) on the resistant component of gKCa2. Records show APs at -50 mV (a) and outward tail currents at a holding potential of -50 mV (b) recorded in the presence of apamin and IbTx (A) and after addition of 10 mM TEA (B) and subsequent application of 1 mM 4-AP (C). Scales in A apply throughout.

The subsequent addition of 1 mM 4-AP markedly increased Rin and almost doubled the membrane time constant (P = 0.014), implying blockade of a resting K+ conductance. The AP was further widened in all cases (TEA, 2.84 ± 0.08 ms; 4-AP, 6.26 ± 0.93 ms, n = 3, P = 0.06; Fig. 5Ca) and the cells became highly excitable. The amplitude and duration of the residual outward tail current was increased in two cells, by 20% (Fig. 5Cb) and 75%, respectively, but gKCa2 was abolished in the third (see also Hirst et al. 1985). Because of the presumably large increase on Ca2+ influx during the widened AP, these data suggest that 4-AP blocks some (but not all) of the Ca2+-dependent K+ channels involved in gKCa2.

The addition of glibenclamide (5 mM), which blocks ATP-dependent K+ channels, did not affect passive properties or the AP, although it increased the amplitude of gKCa1 by 15% (P = 0.038). The drug had no effect on gKCa2 (amplitude: control, 46 ± 10 pA; glibenclamide, 53 ± 10 pA; decay time constant: control, 1.08 ± 0.01 s; glibenclamide, 1.29 ± 0.15 s, n = 3, P > 0.05).

Effect of blockade of voltage-activated Ca2+ channels

Blockade of L-type Ca2+ channels with 10 µM nifedipine has been shown to reduce the amplitude of gKCa2 by about 40% (Davies et al. 1999). We tested the effects on gKCa2 of blocking other subtypes of Ca2+ channel.

EFFECTS OF omega -CONOTOXIN GVIA. Addition of the N-type Ca2+ channel blocker, omega -conotoxin GVIA (CgTx, 100 nM), had no effect on passive membrane properties (n = 7). CgTx did not affect the amplitude of the AP but reduced its half-width by 5% (control, 1.24 ± 0.08 ms; CgTx, 1.18 ± 0.07 ms, n = 6, P = 0.02; Fig. 6A) (see also Ireland et al. 1998). The early part of the AHP was reduced in CgTx (peak amplitude, control, 10 ± 2 mV, CgTx 6 ± 1 mV, P = 0.02; Fig. 6B). The amplitude of gKCa1 was in all cases reduced (Fig. 6C) (see Davies et al. 1999; Ireland et al. 1998), whether CgTx was added to control solution (Table 1) or in the presence of apamin (n = 3, P = 0.07). The amplitude of gKCa2 was reduced by 34 ± 6% in CgTx (see Table 1, Fig. 6C). However, the time course of neither current was affected.



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Fig. 6. Effect of blockade of N-type Ca2+ channels in an LAH neuron. Records show APs (A), AHPs (B), and outward tail currents at a holding potential of -50 mV (C). A: addition of 100 nM omega -conotoxin VIA (CgTx) accelerated repolarization of the AP. B: CgTx reduced the 1st component of the AHP more markedly than the 2nd. C: CgTx reduced gKCa1 much more than gKCa2. Insets in B and C show expanded the 1st 400 ms of the traces. RMP in A and B was -60 mV.

EFFECTS OF omega -AGATOXIN IVA. Addition of the P-type Ca2+ channel blocker, omega -agatoxin IVA (AgaTx, 40 nM), had no effect on passive membrane properties or the AP (n = 3; Fig. 7A). AgaTx had no effect on the early component of the AHP (peak amplitude, control, 9 ± 3 mV; AgaTx, 9 ± 3 mV, P = 0.83; Fig. 7B), or on gKCa1 (Table 1; Fig. 7C). However, the amplitude of the slow component of gKCa2 was reduced in all experiments (by 37 ± 11%, Table 1), without any change in its time course (Fig. 7C).



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Fig. 7. Effect of blockade of P-type Ca2+ channels in an LAH neuron. Records show APs (A), AHPs (B), and outward tail currents at a holding potential of -50 mV (C). A: addition of 40 nM omega -agatoxin GVIA (AgaTx) did not affect the AP. B: AgaTx reduced only the 2nd component of the slow AHP. C: AgaTx selectively reduced gKCa2 without affecting gKCa1. Insets in B and C show expanded the 1st 400 ms of the traces. RMP in A and B was -59 mV.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study we have used selective pharmacological blockade in an attempt to define the types of K+ channel activated to produce the prolonged AHP following a single AP in one class of guinea pig celiac neuron (LAH neurons). The data suggest that multiple types of K+ channel are involved in gKCa2, the slow conductance change underlying the slow component of the AHP. If the antagonists used are truly selective, both SK and BK channels, as well as a third type of K+ channel resistant to apamin, IbTx, 4-AP, TEA, and glibenclamide, must be activated during the prolonged conductance change. Using a similar approach to identify the sources of Ca2+ responsible for activation of these K+ channels, we conclude that Ca2+ (Cassell and McLachlan 1987) entering through at least three different Ca2+ channels (N-, L-, and P-type Ca2+ channels) is involved (see Fig. 8).



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Fig. 8. Relative contributions of specific Ca2+-activated K+ channels (A) and Ca2+ influx through specific Ca2+ channels (B) to gKCa2 amplitude in LAH neurons. Columns indicate the percentage block produced by addition of each of the antagonists relative to control. Error bars indicate standard error of the mean. The contribution from resistant K+ channels (R) is calculated from the component remaining following addition of apamin and iberiotoxin. The contribution of L-type Ca2+ channels was obtained from an earlier study (Davies et al. 1999).

Effects of apamin

We used apamin as a selective blocker of SK-type Ca2+-dependent K+ channels. Apamin has no reported blocking action on BK-type (Reinhart et al. 1989) nor most intermediate-conductance (IK)-type channels (e.g., Hay and Kunze 1994; Ishi et al. 1997) and, unlike IbTx, apamin did not affect electrical properties or widen the AP. The selective reduction in gKCa1 resembles the effects of apamin in other autonomic neurons (Callister et al. 1997; Ireland et al. 1998; Kawai and Watanabe 1986) and its blockade of the currents underlying medium duration AHPs in central neurons (see Sah 1996; Stocker et al. 1999).

Apamin reduced the amplitude of the slower conductance, gKCa2 in most but not all LAH neurons (see also Jobling et al. 1993; Vanner et al. 1993). In contrast, the prolonged AHP in many central neurons (e.g., Lancaster and Adams 1986; Sah and McLachlan 1991; Schwindt et al. 1988; Stocker et al. 1999) is resistant to blockade by apamin, as is the slow component of the AHP in nodose neurons (Jafri et al. 1997). Thus the involvement of apamin-sensitive channels in the prolonged AHP seems to be exclusive for LAH neurons in celiac ganglia.

Effects of iberiotoxin

We used IbTx in an attempt to block the BK-type Ca2+-dependent K+ channel more selectively than can be achieved with charybdotoxin (CbTx), which also blocks IK-type channels (Greffrath et al. 1998; Reinhart et al. 1989) as well as voltage-dependent inactivating K+ channels (Grissmer et al. 1994). Application of IbTx increased the duration of the AP, as it does in many peripheral and central neurons (Davies et al. 1996; Ireland et al. 1998; Lancaster and Nicoll 1987; Sah and McLachlan 1991), consistent with blockade of BK channels. It also tended to reduce the amplitude of gKCa1, which did not occur in guinea pig lumbar paravertebral (Ireland et al. 1998) or vagal neurons (Sah and McLachlan 1992).

In contrast, IbTx reduced gKCa2 in LAH neurons by an average of 55%. This effect parallels the reported reduction by CbTx of the prolonged AHP in myenteric neurons (Kunze et al. 1994) but contrasts with the lack of effect of CbTx in guinea pig vagal neurons (Sah and McLachlan 1991) and some central neurons (Lancaster and Nicoll 1987; Pineda et al. 1992; Stocker et al. 1999). IbTx does not block the CbTx- and low TEA-sensitive IK-type channels that underlie the slow AHP in supraoptic neurons (Greffrath et al. 1998). One explanation is that IbTx's actions are most likely to be by blockade of channels of the BK-type.

In the present experiments, 0.5 mM TEA (which also blocks BK channels) did not reduce gKCa2 (see also Vanner et al. 1993). This might be explained by a compensatory increase in Ca2+ entry due to the prolongation of AP half-width in TEA, although similar widening of the AP by IbTx was associated with a reduction in gKCa2. As addition of 0.5 mM TEA in the presence of IbTx further widened the AP, even this low concentration probably blocked part of the delayed rectifier.

Alternatively, it is possible that a novel IbTx-sensitive TEA-resistant channel exists in LAH neurons, in addition to the apamin-/IbTx-resistant channel. A reduction in gKCa2 by blockade of BK-type channels is hard to reconcile with the biophysical and pharmacological properties of these channels. BK-type channels are highly voltage dependent (see Gribkoff et al. 1997), whereas the time course of gKCa2 is voltage independent (Cassell and McLachlan 1987). The low affinity of BK channels for Ca2+ at potentials reached during the AHP would require prolonged high [Ca2+] on the cytosolic face of the neuronal membrane. Ca2+ transients have not been measured in sympathetic neurons that express gKCa2. In nodose neurons (Cohen et al. 1997; Moore et al. 1998) and hippocampal somata (Sah and Clements 1999), the cytoplasmic Ca2+ transient is faster than the slow outward current/AHP triggered by an AP. However, the relationship of the cytoplasmic concentration of Ca2+ to that adjacent to the sensitive K+ channels in the plasma membrane is unknown. If the channels blocked by IbTx are BK channels, Ca2+ delivery might be regulated very precisely so as to reach high enough local concentrations without more widespread effects. This scheme would be compatible with the IbTx-sensitive channels being of the BK-type.

Resistant K+ channels

When antagonists of SK and BK channels were applied sequentially, about 50% of the original slow outward current remained. If blockade of SK and BK channels had been purely additive, only about 10% of the original gKCa2 would have remained. Such occlusion might be explained if both toxins had nonspecific actions in blocking the same as-yet-unknown channels. Alternatively, gKCa2 might be mediated by a mixed population of BK channels, SK channels, channels that are blocked nonselectively by either apamin or IbTx and resistant channels.

The apamin/IbTx resistant component of gKCa2 was not abolished in high concentrations of TEA and (in 2/3 cases) 4-AP, confirming that voltage-dependent K+ channels such as A channels, M channels (Hille 1992), and Na+-activated K+ channels (Bader et al. 1985) are usually not involved in the generation of gKCa2. We also ruled out the participation of ATP-sensitive K+ channels using glibenclamide. Nevertheless, there is clearly considerable diversity in the types of K+ channel involved in gKCa2.

Ca2+-dependent K+ channels resistant to apamin and IbTx underlie the sIAHP in hippocampal pyramidal CA1 neurons. These channels are not blocked by TEA, CbTx, or apamin and are not voltage dependent. Noise analysis indicated that these channels have a low unitary conductance (2-5 pS) and open time of ~2.5 ms (Sah and Isaacson 1995). Their open probability is reduced by transmitters, such as noradrenaline, which inhibit the slow outward current via protein kinases (Pedarzani and Storm 1993). It has been suggested that the slow time course of the outward current in CA1 neurons reflects slow binding of Ca2+ to the channels (Sah and Clements 1999). However, gKCa2 in celiac neurons differs in several respects from the current in CA1 neurons. As well as part of gKCa2 being apamin-sensitive, the high temperature sensitivity of its kinetics is not compatible with a simple binding and diffusion model.

Ca2+ channel blockers

L-type channels contribute to the resting Ca2+ conductance that activates a K+ conductance involved in setting the RMP in LAH neurons (Davies et al. 1999). Here, blockade of Ca2+ entry with either CgTx or AgaTx did not affect RMP or Rin, but IbTx increased Rin. This suggests that L-type channels provide Ca2+ that activates IbTx-sensitive channels open at RMP.

The Ca2+ sources for the activation of gKCa2, like the K+ channels through which current flows, were found to be diverse. Both CgTx and AgaTx reduced gKCa2 by about 30-40%, as did nifedipine (Davies et al. 1999). Thus all of L-, N-, and P-type channels contribute Ca2+ for activation of gKCa2, apparently in a less than additive manner. The source of Ca2+ for triggering calcium-induced calcium release (CICR) is also nonspecific in parasympathetic cardiac neurons (Meriam et al. 1999), whereas, in nodose neurons, CICR is apparently triggered only via N- or L-type channels (Cordoba-Rodriguez et al. 1999). We have not tested whether resistant (R-type) channels also supply Ca2+ for gKCa2 in LAH neurons.

CgTx decreased the half-width of the AP indicating that the Ca2+ current through N-type channels may be large enough to affect to its duration, confirming that N-type channels play little role in providing Ca2+ to activate BK channels. Blockade of neither P-type nor L-type channels (Davies et al. 1999) affected the AP configuration. These results are consistent with the majority of whole cell Ca2+ current in dissociated sympathetic somata being carried through N-type channels (Toth and Miller 1995).

Possible mechanisms underlying gKCa2

To explain how various Ca2+-activated K+ conductances with very different kinetics of activation produce a prolonged current in LAH neurons, we assume that Ca2+ from several sources activates a process of Ca2+ release from a common pool with slow and characteristic kinetics (Fig. 9). Perhaps intracellular Ca2+ buffers associated with each type of K+ channel can deliver appropriate amounts of Ca2+ with different kinetics (see Velumian and Carlen 1999). An obvious candidate for involvement is the intracellular store in the endoplasmic reticulum (Berridge 1998). This store is thought to be located in subsurface cisternae lying only 20 nM from the plasma membrane (Fujimoto et al. 1980; Watanabe and Burnstock 1976), where the Ca2+ could be delivered directly to all nearby K+ channels, irrespective of type (see Fig. 9). From its time course, gKCa2 might result from a regenerative burst of spontaneous miniature outward currents (SMOCs; activated by the punctate release of Ca2+ from stores), as it resembles the currents generated by caffeine (Berridge 1998; Marrion and Adams 1992). The slow onset time course might be explained by the involvement of Ca2+ influx through voltage-dependent channels triggering phospholipase C and inositol trisphosphate-mediated Ca2+ release from stores (see Moore et al. 1998). This model would concur with the high temperature sensitivity of gKCa2 and its sensitivity to noradrenaline (Cassell and McLachlan 1987; Jobling et al. 1993).



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Fig. 9. Hypothetical mechanisms leading to activation of gKCa2 in LAH neurons. Ca2+ entering through at least 3 types of Ca2+ channels triggers CICR either directly or indirectly. RyR, ryanodine receptor channel; InsP3R, inositol trisphosphate receptor. Note that there is no significance in the location of the different types of Ca2+ channel. Ca2+ is released from subsurface cisternae to activate K+ channels in the adjacent membrane. The time course of the resulting conductance change, gKCa2, is determined by a kinetically invariant process that changes Ca2+ concentration in the vicinity of the channels. Adapted from Berridge (1998).

To summarize, we conclude that Ca2+ influx during the AP in LAH neurons opens SK channels, possibly BK channels, and at least one unknown type of Ca2+-dependent K+ channel. The population of native K+ channels activated may arise from various combinations of the cloned Ca2+-activated K+ channel subunits (Ishi et al. 1997; Jensen et al. 1998; Köhler et al. 1996; Reinhart et al. 1989). L-, N-, and P-channels all provide Ca2+ that triggers the slow change in K+ conductance. The time course of the current is not consistent with direct activation of the K+ channels by the Ca2+ entering through adjacent voltage-dependent channels, as described for the relatively faster conductance, gKCa1 (Davies et al. 1999). Rather, a regulated release process of fixed kinetics, probably from intracellular stores in submembrane cisternae, is postulated to underlie gKCa2 in LAH neurons.


    ACKNOWLEDGMENTS

We thank P. Lund for technical assistance.

This work was supported by National Health and Medical Research Council of Australia Grant 970852. J. Martínez-Pinna's travel was supported by funds from the Dirección General de Enseñanza Superior, Spain.


    FOOTNOTES

Address for reprint requests: P. J. Davies, c/o E. McLachlan, Prince of Wales Medical Research Institute, Barker St., Randwick, NSW 2031, Australia (E-mail: e.mclachlan{at}unsw.edu.au).

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 10 January 2000; accepted in final form 19 May 2000.


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