Centro de Investigaciones Biomédicas, Universidad de Colima, Colima 28000, Mexico
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ABSTRACT |
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Lara, Jesús,
Juan José Acevedo, and
Carlos G. Onetti.
Large-Conductance Ca2+-Activated Potassium Channels
in Secretory Neurons.
J. Neurophysiol. 82: 1317-1325, 1999.
Large-conductance Ca2+-activated
K+ channels (BK) are believed to underlie interburst
intervals and contribute to the control of hormone release in several
secretory cells. In crustacean neurosecretory cells, Ca2+
entry associated with electrical activity could act as a modulator of
membrane K+ conductance. Therefore we studied the
contribution of BK channels to the macroscopic outward current in the
X-organ of crayfish, and their participation in electrophysiological
activity, as well as their sensitivity toward intracellular
Ca2+, ATP, and voltage, by using the patch-clamp technique.
The BK channels had a conductance of 223 pS and rectified inwardly in symmetrical K+. These channels were highly selective to
K+ ions; potassium permeability
(PK) value was 2.3 × 1013 cm3 s
1. The BK channels
were sensitive to internal Ca2+ concentration, voltage
dependent, and activated by intracellular MgATP. Voltage sensitivity
(k) was ~13 mV, and the half-activation membrane
potentials depended on the internal Ca2+ concentration.
Calcium ions (0.3-3 µM) applied to the internal membrane surface
caused an enhancement of the channel activity. This activation of BK
channels by internal calcium had a KD(0) of
0.22 µM and was probably due to the binding of only one or two
Ca2+ ions to the channel. Addition of MgATP (0.01-3 mM) to
the internal solution increased steady state-open probability. The
dissociation constant for MgATP (KD) was 119 µM, and the Hill coefficient (h) was 0.6, according to
the Hill analysis. Ca2+-activated K+ currents
recorded from whole cells were suppressed by either adding
Cd2+ (0.4 mM) or removing Ca2+ ions from the
external solution. TEA (1 mM) or charybdotoxin (100 nM) blocked these
currents. Our results showed that both BK and KATP channels
are present in the same cell. Even when BK and KATP
channels were voltage dependent and modulated by internal Ca2+ and ATP, the profile of sensitivity was quite
different for each kind of channel. It is tempting to suggest that BK
and KATP channels contribute independently to the
regulation of spontaneous discharge patterns in crayfish neurosecretory cells.
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INTRODUCTION |
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Calcium-activated potassium currents
(KCa currents) have been observed in a number of
neurons in invertebrates (Crest and Gola 1993;
Deitmer and Eckert 1985
) as well as in vertebrates (Kawai and Watanabe 1986
; Lancaster et al.
1991
; Pennefather et al. 1985
; Smart
1987
; Wang et al. 1998
). Several types of
potassium ion channels can mediate this KCa
current: in addition to "small" conductance (SK)
Ca2+-activated K+ channels,
"big" conductance (200-300 pS)
Ca2+-activated K+ (BK)
channels have been described (Latorre et al. 1989
).
These channels couple the membrane potential to the intracellular
Ca2+ concentration
([Ca2+]i) in such a way
that an increase in internal Ca2+ leads to an
efflux of K+ ions and a subsequent
hyperpolarization of the membrane. Because of this property,
Ca2+-activated K+ channels
are believed to play a role in a number of different cellular processes
that depend on the influx of Ca2+ through
voltage-dependent pathways, including regulated secretion (McManus 1991
; Petersen and Maruyama
1984
). These channels also link internal
Ca2+ and membrane excitability and thus are
believed to play a role in regulating action potential frequency and
duration in neurons. For instance, it has been proposed that BK
channels underlie interburst intervals; therefore they could contribute
to the control of hormone secretion in rat neurohypophysial terminals
(Dopico et al. 1996
).
In the X-organ sinus gland (XO-SG) system, the most important
neurosecretory system of the crayfish, a periodic increase in [Ca2+]i in bursting
neurons modifies membrane conductances and regulates electrophysiological activity (Martínez et al.
1991; Onetti et al. 1990
). Calcium sensitivity
is a very important phenomenon because the firing patterns have been
associated with the ability for secretion in neurons (Stuenkel
1985
). Several potassium currents contributing to modulate
action potential firing patterns have been previously described in
X-organ neurons: the delayed rectifier, the transient inactivating
outward current (Martínez et al. 1991
), and the
ATP-sensitive K+ current (García
et al. 1993
; Onetti et al. 1996
). Experimental evidence supports a key role of intracellular
Ca2+ in the modulation of these currents. It has
been suggested that KATP channels are involved in
the regulation of spontaneous spike firing, due to their sensitivity to
intracellular ATP and Ca2+ ions as well as to the
membrane potential. In whole cell patch-clamp studies performed earlier
in crayfish XO neurons, Ca2+-activated potassium
current was negligible because a cytoplasmic solution with EGTA was
used (Onetti et al. 1990
). However, recent experiments
with inside-out cell-free patches suggest the presence of
large-conductance Ca2+-activated potassium
channels (BK) in somata membranes from XO neurons. To study the
relative contribution of BK and KATP channels to
the macroscopic outward current and their participation in electrophysiological activity, we shall compare the properties of both
types of channels in terms of their sensitivity toward intracellular
Ca2+ and ATP as well as to the membrane potential.
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METHODS |
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The experiments were carried out in neurons of the XO-SG system
obtained from adult Procambarus clarkii crayfish of either sex. The dissection procedures and experimental conditions were described earlier (García et al. 1993). All
records were made in neuron bodies of the X-organ (XO) at room
temperature (20-22°C). In the experiments for macroscopic current
recording, the XO-SG nerve tract was severed at 200-300 µm after its
emergence from XO.
Electrophysiological recordings
The perforated patch-clamp technique (Horn and Marty
1988) was used to record membrane potential and macroscopic
currents in the current- and voltage-clamp mode. Single-channel current recordings were performed in cell-attached, outside-out, and inside-out patch configurations of the patch-clamp technique (Hamill et al. 1981
). All the records of membrane potential and membrane
currents obtained in perforated patch-clamp experiments were made using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). To
obtain single-channel currents we used a GeneClamp 500 amplifier (Axon
Instruments, Foster City, CA). Pipettes with resistance ranging from 4 to 6 M
were used for perforated patch-clamp experiments, and from 8 to 10 M
for single-channel recordings. Patch pipettes were pulled
from 1.5-mm borosilicate capillaries, coated with silicone elastomer
(Sylgard; Dow Corning, Midland, MI) to reduce background noise and
capacitive current and were fire polished.
Membrane potential and macroscopic current records were acquired with a
Digidata 1200 A/D converter (Axon Instruments, Foster City, CA) using
pClamp 6.0.2 software (Axon Instruments, Foster City, CA). Membrane
potential records were filtered at 5 kHz (3 dB). Macroscopic current
signals were filtered at 2 kHz and sampled at 5 kHz. Single-channel
currents were stored with a PCM Data Recorder 200 (A. R. Vetter,
Rebersburg, PA) for subsequent off-line acquisition and analysis. These
signals were filtered (1 kHz), amplified, and sampled at 10 kHz using a
Digidata 1200 A/D converter (Axon Instruments, Foster City, CA).
Analysis was made using a 486 microcomputer with pClamp 6.0.2 software
(Axon Instruments, Foster City, CA). The method described by
Davies et al. (1996)
was used to obtain current-voltage
(I-V) relations for single-channel currents. Briefly,
currents were elicited by 80-ms voltage ramps from
80 to 80 mV; a
leak trace, obtained by averaging sections that had no channel
openings, was subtracted from each individual record, and an ensemble
ramp was constructed by averaging sections containing a single opening.
The open-state probability (PO) values were obtained by using the method described by Quayle et al.
(1988)
. We used current records of 30 s to measure the
open-state probability and to obtain single-channel conductance values.
Single-channel data were filtered at 1 kHz (Bessel 4-pole) and sampled
at 10 kHz. Experimental values are presented as means ± SE. A
P value <0.05 was considered to be significant.
Solutions
For inside-out patch experiments, the intracellular membrane surface was bathed with solutions, the compositions of which are described in Table 1. Both cell-attached and inside-out patch pipettes were filled with 200 K solution (Table 1). Inside-out patch pipettes used in the experiments performed to test single-channel activity at quasi-physiological potassium gradient, were filled using 5 K solution. For outside-out patch current recording, the pipettes were filled with control solution, and the extracellular surface was bathed with 200 K solution (Table 1).
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For perforated patch-clamp experiments, the XO-SG system was continuously perfused with a saline solution of the following composition (mM): 200 NaCl, 5.4 KCl, 13.5 CaCl2, 2 MgCl2, and 10 HEPES for membrane potential measurements. For macroscopic current experiments, the cell bodies were bathed with a solution composed of (mM) 5.4 KCl, 13.5 CaCl2, 10 HEPES, and 200 N-methyl-D-glucamine. Charybdotoxin (100 nM), TEA (1 mM), or CdCl2 (0.4 mM) were added to external solutions. The tips of the pipettes used were filled with two different nystatin-free solutions (mM): 180 K-aspartate, 40 NaCl, and 10 HEPES for membrane potential recording, or 220 KCl and 10 HEPES for macroscopic currents. Nystatin (300 µg/ml; stock solution, 25 mg/ml in dimethylsulfoxide) was added to the corresponding filling solution. The pH of the external solution was adjusted to 7.4 with NaOH or HCl, and the pH of the pipette solutions was adjusted to 7.2 with KOH.
ATP magnesium salt (MgATP),
N-[2-hydroxyethyl]piperazine-N'-2-ethanesulfonic
acid (HEPES),
ethyleneglycol-bis[-aminoethylether]-N,N,N',N'-tetraacetic acid] (EGTA), nystatin, dimethylsulfoxide,
N-methyl-D-glucamine, and tetraethylammonium
(TEA) were all purchased from Sigma Chemical (St. Louis, MO).
Charybdotoxin was kindly provided by Dr. Ubaldo García.
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RESULTS |
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K+ selectivity, permeation, and blockade of BK channels
Unitary currents through Ca2+-activated K+ channels (BK) were obtained in both cell-attached and excised membrane patches from X-organ neurons. The BK channel activity observed in cell-attached was similar to that obtained on excising membrane patches. The open-state probability (PO) values were ~0.8 (VP = 0 mV) in the cell-attached configuration and ~0.9 (40 mV) in the inside-out patches at 1 µM Ca2+ on the internal side of the membrane.
The K+ selectivity of the channels was studied in
inside-out patches at different intracellular and extracellular
K+ concentrations. Throughout these experiments
the internal (bath) solution contained 1 µM free
Ca2+ without MgATP (control solution; Fig.
1A). In conditions of
symmetrical distribution of K+ ions
([K]o = [K]i = 200 mM),
a slight inward rectification was evident in the I-V
relationship. The unitary conductance, for membrane potential values
between 80 to 0 mV, was 223 ± 11 pS (n = 4),
and the reversal potential value was ~0 mV (Fig. 1B). In
this condition, the I-V relation in the linear region can be described by the Goldman-Hodgkin-Katz equation
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(1) |
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To explore the sensitivity of BK channels toward blocking agents specifically directed against particular types of K+ channels, we recorded the potassium currents in outside-out and whole cell patch configurations in the presence of tetraethylammonium chloride (TEA), charybdotoxin (CTX), or Cd2+. Also, we examined the effect of removing Ca2+ from the extracellular medium. The single currents from an outside-out patch were reversibly blocked by adding TEA (1 mM) or CTX (100 nM) to the extracellular side of the membrane patch (Fig. 3). Moreover, the transient component of macroscopic potassium currents through BK channels was blocked by the addition of Cd2+, TEA, or CTX. By replacing Ca2+ with an isosmotic amount of Mg2+, the current observed in the whole cell mode also diminished (Fig. 6A).
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Ca2+, MgATP, and voltage sensitivity of BK channels
To study Ca2+ and voltage dependence of BK
channels, we obtained the steady-state open probability in symmetrical
distribution of K+ ions, by changing the membrane
potential at different intracellular Ca2+
concentrations ([Ca2+]i)
in inside-out patches, in the absence of MgATP. When the membrane potential was held at 40 mV, increasing
[Ca2+]i from 0.3 to 3 µM surface caused an enhancement of the channel activity (Fig.
4A). To describe the voltage
dependence of channel activity, we fitted the experimental values of
Po, obtained at different membrane
potentials, to the Boltzmann equation
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(2) |
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The number of Ca2+ binding sites on the BK
channel can be estimated using the following equation (Wong et
al. 1982)
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(3) |
Figure 5A is a typical example
of the activating action on the BK channels by the addition of 0.1 and
1 mM MgATP to the internal solution at 1 µM
Ca2+ (Vm = 40 mV). To study the sensitivity of the BK channel to MgATP, we used
NPO rather than
PO due to the possibility that MgATP
can affect both of these parameters (Albarwani et al.
1994). A plot of the number of functional channels multiplied
by their open probability (NPo),
against MgATP concentration ([MgATP]) is shown in Fig. 5B.
The relationship NPo/[MgATP] can be
described by the Hill equation
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(4) |
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These results show that the BK channels are sensitive to internal Ca2+ concentration, voltage dependent, and activated by intracellular MgATP.
Contribution of BK and KATP channels to the whole current
To study the contribution of BK and KATP channels to the whole current, we obtained macroscopic current records by using the perforated patch-clamp technique in the voltage-clamp mode. We carried out these experiments under control conditions, blocking the BK currents with TEA or CTX or suppressing the calcium current by adding CdCl2 to the external solution or by replacing Ca2+ with Mg2+. The macroscopic current, obtained in the control solution, exhibited three components: the first one was inward, and the other two were outward. One of the outward current components was transient and the other was sustained. Suppressing the inward current by adding Cd2+ or by removing external Ca2+, the transient outward current was blocked, whereas the sustained component was not modified (Fig. 6, Aa and Ab). Moreover, the addition of 1 mM TEA or 100 nM CTX to the external solution decreased the transient outward current without a significant change in either the inward current or the sustained outward current (Fig. 6, Ac and Ad). The I-V relationship of the outward current obtained in the control solution, of the current in the presence of 0.4 mM of CdCl2, and of the subtraction of the currents obtained with CdCl2 from those obtained in the control solution are shown in Fig. 6B. It is evident that the addition of TEA or CTX suppresses, at least in part, the transient outward currents in the whole cells. This suggests that the outward current blocked by TEA or CTX can be carried through the BK channels. These results, together with those obtained in excised membrane patches (Fig. 3), show that part of the transient potassium currents could depend on Ca2+ influx and membrane potential, whereas the sustained outward current was only voltage dependent. In excised patch membranes the BK channel activity was persistent because [Ca2+]i was constant throughout the entire recording. In contrast, the BK macroscopic currents, obtained in whole cell condition, were transient because calcium currents are transient in themselves.
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ATP-sensitive potassium channels (KATP) from
X-organ neurons are also modulated by intracellular
Ca2+ and membrane potential (Onetti et al.
1996). To determine whether both kinds of channels (BK and
KATP) coexist in the same cell, we carried out
experiments recording whole currents using blockers of BK and
KATP channels. Figure
7A shows recordings obtained
from a representative X-organ neuron in the presence of glybenclamide (50 µM), both with and without CdCl2 (0.4 mM)
in the perfusion solution. To isolate the current component blocked by
glybenclamide or by Cd2+, the currents obtained
with glybenclamide were subtracted from those obtained in the control
solution; the same procedure was applied to the currents recorded with
glybenclamide and Cd2+ and to those obtained in
the presence of glybenclamide, alone. Figure 7B shows the
I-V relationship of K+ currents
obtained from a neuron, in the control solution, in the presence of
glybenclamide, glybenclamide plus Cd2+, and the
subtractions mentioned above. These results show that BK and
KATP channels are distinct entities that could
coexist in the same cell of the X-organ. Furthermore, our observations suggest that K+ currents, carried through
KATP channels, may contribute to the delayed
rectifier in these neurons.
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How the BK and KATP channels participate in electrophysiological activity
To determine how the BK and KATP channels participate in the activity of XO neurons that discharge action potential bursts, we obtained membrane potential records with the perforated patch-clamp technique in the current-clamp mode. By adding TEA (1 mM) to the perfusion solution, an increase in the duration of action potentials (25 ± 1%, n = 7) and bursts (38 ± 9%, n = 7) was produced. Only a slight increase in the burst frequency (6 ± 2%, n = 7) was observed. The effects on the duration of action potentials and bursts, as well as on the burst frequency produced during the addition of CTX (100 nM), were similar to those produced by TEA addition; they increased 31 ± 12, 9 ± 2, and 10 ± 2% (n = 5), respectively. Figure 8A shows typical membrane potential recordings, at two different time scales, in the control solution and after the addition of CTX. The first action potentials of a burst in the control solution and in the presence of CTX are shown in Fig. 8B. On the other hand, besides a membrane depolarization, the addition of glybenclamide (50 µM) produced an increase of duration (55 ± 10%, n = 5) and frequency (110 ± 15%, n = 5) of the bursts; also, a slight increase in action potential duration (6 ± 1, n = 5) was observed. These effects can be seen in the records shown in Fig. 8 (C and D), obtained from a neuron with burst activity. The increase in the action potential and burst duration and burst frequency produced by the addition of TEA, CTX, or glybenclamide to the bath solution, was significant (P < 0.01).
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In summary, the most noticeable effect on the electrophysiological activity, produced by the blockage of the BK channels (with TEA or CTX), was an increase in the duration of bursts and action potentials. Nevertheless, the most noticeable effect found to block the KATP channels (with glybenclamide), besides that of membrane depolarization, was an increase in the burst duration as well as in the burst frequency.
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DISCUSSION |
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We have characterized the biophysical properties of the BK channels and their contribution to the macroscopic outward current as well as their participation in the electrophysiological activity from secretory neurons of the crayfish by using the patch-clamp technique.
The electrophysiological properties and the calcium
sensitivity of the BK channels in XO neurons are similar to
those from other BK channels (McManus 1991),
particularly the BK channels from rat neurons
(Franciolini 1988
; Safronov and Vogel
1998
). Single-channel kinetics in cell-attached
configuration were similar to those observed when the membrane
patch was excised from the cell, at 1 or 3 µM
Ca2+ concentration; even the I-V
relationships of the BK channels were identical in both configurations.
This leads one to think that the internal Ca2+
concentration of intact X-organ neurons could fall within this interval
(1-3 µM).
Permeation and blocking of the BK channels
The unitary conductance of the BK channels in symmetrical
K+ (~220 pS) is within the range reported
(180-290 pS) for BK channels in other tissues (McManus
1991). Comparison of theoretical equilibrium potentials with
reversal potentials obtained experimentally by changing the
K+ gradient (Fig. 2), reveals that BK channels in
excised inside-out patches are highly selective for
K+ ions. Like BK channels in other tissues
(Latorre et al. 1989
) these channels display a high
K+ permeability (2.3 × 10
13 cm3
s
1); however, under symmetrical
K+ gradient, the BK channels in XO neurons do not
have a linear conductance, as the slope falls below that of a straight
line at high positive voltage (Fig. 1B). For the time being
we cannot explain this effect. We speculate that this nonlinearity
could be attributed to a rapid voltage-dependent block, perhaps by
H+ ions. It could also be produced by the
intrinsic permeability properties of the BK channel, or by
diffusion-limited ion flow (Yellen 1984
). An inward
rectification is seen when 40 mM K+ (160 mM
Na+) is present in the internal solution; an
outward rectification is observed when using quasi-physiological
K+ concentrations (Fig. 2); this is consistent
with the I-V relationship predicted by the
Goldman-Hodgkin-Katz field equation.
As previously reported for other tissues (Anderson et al.
1988; Kehl and Wong 1996
; Safronov and
Vogel 1998
), the current carried by BK channels can be blocked
by tetraethylammonium or charybdotoxin applied to the extracellular
medium (Figs. 3 and 8A).
Voltage dependence, and Ca2+ and MgATP sensitivity of BK channels
Steady-state activation, as a function of membrane potential for
the BK channels described here, was analyzed by fitting the Po/Vm
relationship to the Boltzmann equation at different
[Ca]i (Fig. 4B). The voltage
sensitivity of the BK channels from XO neurons was similar to that
reported for neurons (Safronov and Vogel 1998) and other
tissues (Albarwani et al. 1994
; Kehl and Wong
1996
; Latorre et al. 1989
; Singer
and Walsh 1987
). It became evident that these channels are
acutely sensitive to voltage (k = 13 mV); also, the
half-activation point depends on the internal Ca2+ level
(
5,
26, and
44 mV, at 0.3, 1, and 3 µM [Ca]i,
respectively). Increasing the Ca2+ concentration produced a
significant shift of the activation curve to more negative membrane
potentials, even when the sensitivity factor (k)
remained unaffected. It is possible that an increase in internal
Ca2+ shifts the voltage activation threshold for BK
channels without affecting their voltage sensitivity. However, another
possibility is that the affinity of BK channels for Ca2+,
itself, is modulated by voltage; other authors (Albarwani et al.
1994
; Barrett et al. 1982
; Moczydlowski
and Latorre 1983
) have suggested this possibility. The number
of Ca2+ binding sites (NCa = 1.3) for the BK channel from XO neurons, estimated using Eq. 3 (Fig. 4C), suggests that the activation is due
to binding of only one or two Ca2+ ions to the channel.
Similar values for NCa have been reported in
rat plasma membrane (Moczydlowski and Latorre 1983
),
smooth muscle (Benham et al. 1986
; Inoue et al.
1985
), and rat hippocampus neurons (Franciolini
1988
). The dissociation constant at 0 mV [KD(0) = 0.22 µM] for these
channels was similar to those obtained in other tissues, such as smooth
muscle (Benham et al. 1986
; Carl and Sanders
1989
; Inoue et al. 1985
; Kume et al.
1990
) and rat melanotrophs (Kehl and Wong 1996
).
In XO neurons, intracellular MgATP activates the BK channels. Using a
symmetrical distribution of K+, this activation is observed
when 0.01-3 mM of internal MgATP is present (Fig. 5). Similar behavior
has been found for the BK channels from the main pulmonary artery of
the rat (Albarwani et al. 1994). Some authors have
explained this activation as a result of phosphorylation, rendering BK
channels more sensitive to Ca2+ ions (Albarwani et
al. 1994
; Robertson et al. 1992
). It is possible that the MgATP activation mechanism plays a physiological role in XO
neurons enhancing BK channel activity and therefore producing an
increase in the hyperpolarization after a burst of action potentials. It is known that intracellular ATP concentration is strictly related to
the availability of energy sources; levels of extracellular glucose can
determine internal ATP concentration. In XO neurons, metabolic balance
could change in such a manner as to render BK channels more sensitive
to Ca2+ due to an increase in intracellular ATP
concentration (KD = 119 µM). However,
it is not known whether the intracellular ATP concentration falls
within the level at which BK channels are most sensitive to
Ca2+.
We conclude that the BK channels in XO neurons are voltage dependent, sensitive to internal Ca2+, and activated by intracellular MgATP; it is possible that these factors interact to play an important role in the functional profile of these neurons.
Physiological implication of BK and KATP channel coexistence on electrophysiological activity
It seems reasonable to assume that in spontaneously bursting XO
neurons, internal Ca2+ concentration will attain
the optimal level necessary to activate both BK and
KATP channels. Due to the influx of
Ca2+ ions during a spike burst, through
voltage-dependent Ca2+ channels, the BK channels
can regulate both spike burst and action potential duration (Fig. 8).
Similar phenomena have been described in neurons from other species
(Crest and Gola 1993; Wang et al. 1998
).
Depending on the cellular metabolic state, the
KATP channels can control not only the resting
potential, but both the duration and frequency of bursts. Because BK
and KATP channels are distinct entities and could
coexist in the same neuron of the X-organ, spike shaping and
spontaneous firing pattern regulation would depend on the balance
between the level of activity of both channels.
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ACKNOWLEDGMENTS |
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The authors express their gratitude to Dr. Esperanza García for critical review of the manuscript.
This work has been partially supported by Grants 1913P-N and 29471-N from Consejo Nacional de Ciencia y Tecnología, Secretaría de Educación Pública and Fondo para el Mejoramiento de la Educación Superior: 98-07-01 from Subsecretaría de Educación Superior e Investigación Cientifica, Secretaría de Educación Pública, Mexico.
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FOOTNOTES |
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Address for reprint requests: C. G. Onetti, Centro de Investigaciones Biomédicas, Universidad de Colima, Apdo. Postal 97, Colima, Col. 28000, Mexico.
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 5 March 1999; accepted in final form 19 May 1999.
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REFERENCES |
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