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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 -conotoxin GVIA) and P-type
Ca2+ channels (with 40 nM
-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 M) 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). -Conotoxin GVIA was
obtained from Auspep (Parkville, Victoria, Australia) and iberiotoxin
from Alomone Laboratories (Jerusalem, Israel).
-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.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 M
; input time
constant,
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).
|
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).
|
|
EFFECT OF IBTX.
Addition of 20 nM IbTx to the perfusing solution increased cell input
resistance (Rin) by 44 ± 16%
(control, 97 ± 10 M; IbTx, 137 ± 18 M
,
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).
|
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.
|
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).
|
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 -CONOTOXIN GVIA.
Addition of the N-type Ca2+ channel blocker,
-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.
|
EFFECTS OF -AGATOXIN IVA.
Addition of the P-type Ca2+ channel blocker,
-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).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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).
|
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
).
|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|