Instituto Cajal, Consejo Superior de Investigaciones, E-28002 Madrid, Spain
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ABSTRACT |
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Araque, Alfonso and
Washington Buño.
Fast BK-Type Channel Mediates the Ca2+-Activated
K+ Current in Crayfish Muscle.
J. Neurophysiol. 82: 1655-1661, 1999.
The role of the
Ca2+-activated K+ current
(IK(Ca)) in crayfish opener muscle fibers is
functionally important because it regulates the graded electrical
activity that is characteristic of these fibers. Using the
cell-attached and inside-out configurations of the patch-clamp
technique, we found three different classes of channels with properties
that matched those expected of the three different ionic channels
mediating the depolarization-activated macroscopic currents previously
described (Ca2+, K+, and
Ca2+-dependent K+ currents). We investigated
the properties of the ionic channels mediating the extremely fast
activating and persistent IK(Ca). These
voltage- and Ca2+-activated channels had a mean
single-channel conductance of ~ 70 pS and showed a very fast
activation. Both the single-channel open probability and the speed of
activation increased with depolarization. Both parameters also
increased in inside-out patches, i.e., in high Ca2+
concentration. Intracellular loading with the Ca2+ chelator
bis(2-aminophenoxy) ethane-N, N,N',N'-tetraacetic acid gradually reduced and eventually prevented channel openings. The channels opened at very brief delays after the pulse depolarization onset (<5 ms), and the time-dependent open probability was constant during sustained depolarization (560 ms), matching both the extremely fast activation kinetics and the persistent nature of the macroscopic IK(Ca). However, the intrinsic properties of
these single channels do not account for the partial apparent
inactivation of the macroscopic IK(Ca),
which probably reflects temporal Ca2+ variations in the
whole muscle fiber. We conclude that the channels mediating
IK(Ca) in crayfish muscle are voltage- and
Ca2+-gated BK channels with relatively small conductance.
The intrinsic properties of these channels allow them to act as precise
Ca2+ sensors that supply the exact feedback current needed
to control the graded electrical activity and therefore the contraction
of opener muscle fibers.
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INTRODUCTION |
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Ca2+-activated
K+ currents
(IK(Ca)) are of key functional
importance because they regulate the excitability of neurons and muscle fibers, participating in action potential repolarization, in the regulation of the graded electrical activity, and in AP frequency adaptation (e.g., Araque and Buño 1995;
Araque et al. 1998
; Blatz and Magleby
1987
; Crest and Gola 1993
; Gola et al.
1990
; Hille 1992
; Madison and Nicoll
1984
; Marty 1981
; Yarom et al.
1985
). On the basis of their single-channel conductance,
calcium sensitivity, voltage dependence, and pharmacology, the channels
underlying these currents have been classified in two main types, BK or
SK channels with high (>75 pS) and small (<20 pS) conductance,
respectively (Blatz and Magleby 1986
, 1987
;
Latorre et al. 1989
; Marty 1981
). In
addition, BK channels with different properties have been reported, suggesting the existence of BK channel subtypes (e.g., Hicks and Marrion 1998
; Kang et al. 1996
; Lagrutta
et al. 1994
; Reinhart et al. 1989
;
Sugihara 1994
). For example, in molluscan neurons, Ca2+-dependent K+ channels
with relatively small conductance are considered BK channels according
to their voltage dependence, selectivity, and pharmacology
(Crest et al. 1992
; Gola et al. 1990
;
Hermann and Erxleben 1987
).
In slowly contracting crustacean muscle fibers, such as those of the
opener muscle of crayfish that do not fire all-or-none action
potentials, the characteristic graded electrical activity is controlled
by an extremely fast-activating, voltage-sensitive, and
tetraethylammonium (TEA)- and charybdotoxin (CTX)-sensitive IK(Ca) (Araque and Buño
1995), suggesting that the macroscopic current is mediated by
BK type channels (see following text). Repeated activation of the
excitatory axon that innervates opener muscle fibers generates a graded
depolarization that activates an L-type voltage-gated
Ca2+ current
(ICa) (Araque et al. 1994
,
1998
). Besides adding to the membrane depolarization, the
Ca2+ inflow through the
ICa channels has two key functions, it
triggers contraction through a Ca2+-induced
Ca2+-release mechanism (Gyorke and Palade
1992
) and activates IK(Ca) (Araque et al. 1998
). The rate of membrane
depolarization due to the activation of
ICa is precisely regulated by the
negative feedback provided by the voltage- and
Ca2+-sensitive
IK(Ca) (Araque et al.
1998
). Therefore because the force of the contraction is
proportional to the degree of depolarization (Bittner
1968
; Orkand 1962
),
IK(Ca) is extremely important to the
function of this muscle because it prevents Ca2+
spiking and controls the graded depolarization, thus regulating the
force of contraction.
Many characteristics of the channels mediating
IK(Ca) can be deduced from the
analysis of the macroscopic current. Thus we have shown that to perform
its feedback regulatory function,
IK(Ca) must activate fast during the
rising phase of the graded depolarization and that the activation of
IK(Ca) lags that of
ICa by less than ~2 ms
(Araque et al. 1998). We also have reported that to
achieve this extremely fast activation (faster than previously
described BK type conductances), the channels mediating
ICa and
IK(Ca) must be very close together
(<200 nm) (Araque and Buño 1995
), and the gating
kinetics of the channels underlying
IK(Ca) must be very rapid. Finally, we
have demonstrated that to control the graded depolarization and the
sustained contractions that characterize this muscle, the
IK(Ca) must be noninactivating,
hypothesizing that the underlying channels do not inactivate
(Araque and Buño 1995
; Araque et al.
1998
).
To experimentally test the above conclusions, we characterized the intrinsic properties of the single channels mediating the IK(Ca) of crayfish opener muscle fibers. Special attention was paid to the ON kinetics and to the voltage and Ca2+ dependence of the ionic channels that may explain the extremely fast kinetics of the macroscopic IK(Ca). Finally, we discuss how the intrinsic properties of these BK channels contribute to the characteristics of the macroscopic IK(Ca).
We have found that BK channels mediating
IK(Ca) in crayfish muscle show voltage
and Ca2+ dependence, extremely fast activation
kinetics, and a persistent, noninactivating steady state. They have a
single-channel conductance similar to those found in molluscan cells
(~ 70 pS) (cf. Crest et al. 1992; Gola et al.
1990
). We also report that the BK channel activation depends on
membrane potential (Vm) and
Ca2+ and that at similar
Vm, the activation rate increases at
higher Ca2+ concentration, suggesting that the
Ca2+ influx is the rate-limiting step for the
IK(Ca) activation (cf. Araque
and Buño 1995
).
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METHODS |
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Preparation
Opener muscles from the propodite of the first walking leg of crayfish (Procambarus clarkii) were isolated and transferred to a 1-ml superfusion chamber placed on the stage of an inverted microscope. The propodite was glued to the glass bottom of the chamber with cyanoacrylate glue. The preparation was treated during 30-60 min. with control solution (see composition in the following text) containing 1 mg/ml of collagenase D.
Microelectrodes and recordings
Fire-polished patch electrodes (2-4 M) pulled from
thick-walled 1.5-mm diam borosilicate glass (A-M System, 3060) were
coated with silicone elastomer (Sylgard). The pipette solution and the extracellular control solution were the same and had the following composition (in mM): 210.0 NaCl, 5.4 KCl, 13.5 CaCl2, 2.6 MgCl2, and 10.0 Tris buffer; pH was adjusted to 7.2 with NaOH. Higher pipette and
extracellular K+ concentrations would tend to
symmetrical K+ concentrations on either side of
the membrane and would allow accurate estimations of
Vm and of the single-channel
conductance (e.g., Hamill et al. 1981
). However, they
could not be used because the fibers depolarized and contracted,
dislodging the patch electrode.
Pipettes were connected to a Cornerstone Series PC-ONE amplifier
(Dagan) and positioned with a mechanical micromanipulator under direct
visualization with a dissecting microscope. When the pipette's access
resistance increased after touching the fiber, a gentle suction was
applied through the electrode until a high-resistance (>1 G) seal
was obtained. Single-channel recordings (n = 28) were
obtained in this cell-attached configuration (Hamill et al. 1981
). In four cases, the inside-out configuration was obtained by gently pulling the electrode away from the fiber after recording in
the cell-attached mode. In many cases (n = 19), the
resting membrane potential (Vr) of the
patched fiber also was recorded with a sharp
K+-acetate (3 M)-filled micropipette (5-10 M
)
using an Axoclamp 2A amplifier (Axon Instruments) in the bridge mode.
These recordings provided an estimation of the mean
Vr, which was
70.5 ± 9.5 (SE) mV (cf. Araque and Buño 1994
, 1995
).
Because the holding potential of the pipette was set to 0 mV, for
simplicity, Vm was estimated to be
70 mV,
also in those cell-attached recordings from cells in which the
Vr was unknown. Even in these conditions the
small dispersion of measured Vr values
indicates that errors in the estimation of single-channel properties
introduced by Vr to
Vm differences would be small (see
RESULTS). Furthermore no significant differences were found
between the intrinsic properties of channels recorded from fibers in
which the Vr was known and set to
70 mV
and those in which the Vr was unknown and
estimated to be the mean Vr (i.e.,
70 mV).
Membrane potential is expressed conventionally as the difference
between the intracellular and extracellular side of the membrane.
In two experiments, the patched fiber was loaded with the fast
Ca2+ chelator 1,2-bis(2-aminophenoxy)
ethane-N,N,N',N'-tetraacetic acid (BAPTA) by
ionophoresis after impaling the fiber with a sharp micropipette filled
with 0.16 M BAPTA (see Araque and Buño 1995).
Stimulus pulse and ramp generation, data acquisition, and analysis were done with a PC 486-based computer and the pClamp software (Axon Instruments) through a LabMaster TM-100 (Scientific Solution) interface board. Currents were filtered >1 kHz and digitally sampled >2 kHz.
Uncompensated capacitive currents and ohmic leak currents were subtracted from the data using averaged currents obtained from voltage pulses that failed to evoke channel openings. A patch was considered to contain a single channel when openings to only a single conductance level were observed for several minutes at strong depolarization. The mean channel open probability was calculated by dividing the time spent in the open state by the total duration of the pulse. Experiments were performed at room temperature (21-23°C). Chemicals were purchased from Sigma-Aldrich (Spain). All values were expressed as means ± SE. Statistical differences were established using the Student's t-test.
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RESULTS |
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The depolarization-activated macroscopic current of the opener
muscle fibers shows three different components: an L-type voltage-gated Ca2+ current
(ICa), a voltage- and
Ca2+-dependent K+ current
(IK(Ca)), and a voltage-gated
K+ current (IK)
(Araque and Buño 1994, 1995
; Araque et al.
1994
; Erxleben and Rathmayer 1997
). Patch-clamp
recordings in the cell-attached configuration mode of the opener muscle
fibers, showed single channels with properties that matched those
expected of the three different ionic channels mediating the
macroscopic currents that we have described previously.
Three different voltage-gated ionic channels could be observed by
membrane depolarization from Vm = 70
to more than
30 mV. Two channels carried outward current, one
displayed a relative low conductance <40 pS (15.1 ± 3.6 pS; six
patches), and the other had a relative high conductance >40 pS
(67.6 ± 15.9 pS; 7 patches) and was Ca2+
dependent (see following text), suggesting that they corresponded to
channels mediating the macroscopic IK
and IK(Ca), respectively. The latter
channels will be termed BK channels because the electrophysiological and pharmacological properties of
IK(Ca) suggest that BK-type channels
mediate it (see following text) (see also Araque and Buño
1995
).
The ionic channel carrying inward current was encountered less frequently and probably corresponded to that mediating the macroscopic ICa. This channel showed a much lower conductance and was extremely difficult to resolve from the background noise (not shown).
The present study was focused on the characterization of the high-conductance BK-type channel, and the properties of other channels were not further analyzed.
Large-conductance channel is voltage sensitive
Depolarizing ramps (from 70 to 130 mV) applied in the
cell-attached configuration evoked BK channel openings above a
threshold Vm (e.g., Fig.
1B, 1 and
2). When a single channel was recorded in isolation
(n = 12) as shown in Fig. 1, the channel conductance and the voltage sensitivity of the open probability were estimated directly from responses evoked by ramp depolarizations. The intensity of the current flowing through the open channel increased linearly with
Vm depolarization (Fig. 1,
A and B). Current-voltage (I-V) relationships between the ramp Vm and
the open channel currents were constructed. Figure 1D shows
an example where the single-channel conductance, estimated from the
slope of the linear fit of the I-V curve, was 86.1 pS. The
linear I-V relationship showed a reversal potential at
62.5 mV, which fits with the K+ equilibrium
potential of opener muscle fibers (Araque and Buño 1994
).
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To investigate the voltage sensitivity of these channels, successive
responses evoked by Vm ramps were
averaged (Fig. 1C; n = 25 ramps). This
averaged channel current is, for single-channel recordings,
proportional to the single-channel current and the open probability as
a function of time (see Ganfornina and López-Barneo 1992). The I-V relationship between the averaged
single-channel currents and the corresponding
Vm were clearly nonlinear (Fig. 1E), suggesting that the channel open probability is voltage
dependent. Indeed, because the opening kinetics of these channels are
extremely fast (see following text), the time dependency of the open
probability can be neglected due to the relatively slow
Vm variation during the ramp.
Therefore the observed nonlinearity of the open probability reflects a
voltage dependence. Indeed, the slanted straight line represents the
expected I-V relationship of the single-channel openings
(arrows) and corresponds to the linear averaged channel current
expected for a single opening of the channel (Fig. 1E). It
was calculated from the equation
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In addition to voltage ramps, pulse depolarizations from
Vm = 70 mV also were used to
estimate the channel conductance and the voltage dependence of the open
probability (Fig. 2). Both the channel
current and open probability increased with increasing depolarization
to
20, 0, and 50 mV (Fig. 2). Channel current amplitudes were
measured by amplitude histogram analysis, where the values were fitted
by Gaussian distributions and the resulting mean amplitude was used.
The I-V curve of open channel currents evoked by pulse
depolarizations shows a linear relationship, where the slope gave a
single-channel conductance of 92.4 pS, and the reversal potential was
65.6 mV (Fig. 2B). On average, the mean channel
conductance obtained from seven different patches was 67.6 ± 15.9 pS, and the reversal potential of the single-channel current was
60.2 ± 3.6 mV. The single-channel open probability increased as
function of Vm, again demonstrating
that these BK channels are voltage dependent (Fig. 2C).
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Large-conductance channel is Ca2+ sensitive
In some experiments (n = 4), the large-conductance channel was recorded in the cell-attached configuration, then the patch was excised and the same channel was recorded in the inside-out mode (Fig. 3). In our cell-attached conditions, the [Ca2+]i is expected to be low because the cell is at rest and Vm is at the Vr, whereas in the inside-out mode a high Ca2+ concentration (13.5 mM) is in contact with the intracellular phase of the membrane. Figure 3 shows a representative example where the channel open probability at 50 mV increased from 0.05 in low [Ca2+]i to 0.84 in high Ca2+ conditions. On average, the mean single-channel open probability at 50 mV increased from 0.17 ± 0.06 to 0.58 ± 0.09 (n = 4; P < 0.01) in low and high Ca2+ conditions, respectively, indicating that in addition to their voltage dependence, these channels are also Ca2+ dependent. Further analysis is needed to elucidate the partial contribution of both variables to the behavior of the channel.
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We confirmed this Ca2+ dependence by loading the
patched fibers with the Ca2+ chelator BAPTA
(n = 2), which reduced the
[Ca2+]i and prevented
IK(Ca) activation (see Araque
and Buño 1995; Araque et al. 1998
). In
these conditions, channel openings evoked by pulse depolarization (from
70 to 30 mV) were reduced gradually and eventually abolished (not
shown), confirming the Ca2+ dependence of the BK channels.
We have hypothesized that the rate of activation of the macroscopic
IK(Ca) was limited by the
Ca2+ inflow rather than by membrane
depolarization (Araque and Buño 1995). Figure 3
shows that at similar depolarization channels tended to activate at
briefer delays in the inside-out configuration, i.e., in high
Ca2+ conditions. The latency histograms between
the onset of the depolarizing pulse and the first channel openings in
low and high Ca2+ conditions, respectively, show
the clearly different latency distributions (Fig. 3, C and
D). In low Ca2+ conditions, the
histogram was asymmetric, having most values grouped at brief latencies
and showing a tail of few long latency values (Fig. 3C). The
long latency values disappeared in high Ca2+
conditions, and values grouped at brief latencies (Fig. 3D). The different channel opening latencies in high and low
Ca2+ conditions are also obvious when comparing
their respective cumulative probability plots (P < 0.001, Kolmogorov-Smirnov test), again demonstrating that these
channels are Ca2+ dependent (Fig. 3E).
These results confirm previous data on the
IK(Ca) activation kinetics obtained by
analysis of the behavior of the macroscopic current and indicate that
the binding of Ca2+ is the rate limiting step for
the opening of BK channels (cf. Araque and Buño
1995).
Large-conductance channel activates fast and does not inactivate
In agreement with the extremely fast activation kinetics of the
macroscopic IK(Ca), the underlying
large-conductance BK channels displayed fast open kinetics (Fig.
4). Successive responses evoked by
Vm pulses (from 70 to 30 mV) show
that channels could occasionally open in <5 ms from the onset of the
pulse (Fig. 4A). Moreover, the average of successive
responses (n = 500) shows that the open probability
increased markedly in the first 10 ms after the
Vm pulse onset (Fig. 4A,
bottom). Likewise, longer pulse depolarizations revealed that the
open probability increased steeply during the initial 50 ms (i.e.,
reaching 90% of the maximum probability in ~10 ms) and tended to
stabilize thereafter. The averaged channel current evoked by long
Vm pulses reached a persistent steady
state as shown in Fig. 4B (bottom), where
n = 300 responses where averaged from a patch
containing a channel with a relatively high open probability. The
uniform late (>50 ms) averaged channel current indicates an invariable
open probability during the constant
Vm depolarization. It is noteworthy
that the open probability also remained stable during the depolarizing
pulse in inside-out patch recordings (not shown).
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Therefore these BK channels open with extremely fast activation kinetics and show a persistent noninactivating state that lasts as long as the depolarizing pulse, during which the channel open probability is invariable.
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DISCUSSION |
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Ca2+-dependent K+
channels have been described in most excitable cells (Blatz and
Magleby 1987; Hille 1992
; Latorre et al.
1989
) and have been classified in two main groups according to
their single-channel conductance, calcium sensitivity, voltage
dependence, and pharmacology. SK channels have small unitary
conductance (<20 pS) (Blatz and Magleby 1986
, 1987
;
Lang and Ritchie 1987
; Latorre et al.
1989
), are generally voltage independent (Barret et al. 1982
; Marty 1981
; Moczydlowski and
Latorre 1983
) and are sensitive to apamin (Blatz and
Magleby 1986
; Latorre et al. 1989
; Romey and Lazdunski 1984
). BK channels have high unitary conductances (ranging from 75 to 250 pS) (e.g., Blatz and Magleby
1987
; Lang and Ritchie 1987
; Reinhart et
al. 1989
; Wang et al. 1998
), are voltage
dependent (Barret et al. 1982
; Marty
1981
; Moczydlowski and Latorre 1983
), and are
sensitive to TEA and CTX (Blatz and Magleby 1984
;
Crest et al. 1992
; Hermann and Erxleben
1987
; Latorre et al. 1989
; Miller et al.
1985
; Tauc et al. 1993
; Villarroel et al.
1988
).
The macroscopic IK(Ca) of crayfish
opener muscle fibers is fast activating, persistent, voltage and
Ca2+ dependent, and TEA and CTX sensitive
(Araque and Buño 1995), suggesting that the
current is mediated by BK type channels. Our present results
demonstrate that single channels with relatively large conductance are
Ca2+ and voltage sensitive, activate fast, and do
not inactivate, matching most of the properties of the macroscopic
IK(Ca).
We have found that BK channels in crayfish muscle show voltage and
Ca2+ dependence. Such voltage dependence is not
merely apparent (due to the voltage dependence of the associated
Ca2+ currents) because the single-channel open
probability increased monotonically as a function of
Vm (from 50 to 50 mV; Fig. 2). If
such voltage dependence was due to the voltage dependence of the
Ca2+ inflow and the subsequent rise in
[Ca2+]i, the channel open
probability first would increase and then decrease as
ICa initially rises and subsequently
drops at positive Vm when the reversal
potential of Ca2+ is approached. Because the
channel open probability increased monotonically with
Vm, we conclude that these BK channels
are also voltage dependent (see Araque and Buño 1995
).
In most cells studied, BK channels have a very high single-channel
conductance, ranging from 100 to 250 pS. However, channels with BK
properties but with smaller conductance (40-100 pS) have been reported
in molluscan neurons (Crest et al. 1992; Gola et al. 1990
; Hermann and Erxleben 1987
) and
vertebrate smooth muscle cells (Van Renterghem and Lazdunski
1992
). Our present results show that BK channels in crayfish
opener muscle have a single-channel conductance of ~70 pS. Therefore
in invertebrate cells, although BK channels in insect muscle have a
high single-channel conductance (>100 pS) (Gorczynska et al.
1996
), BK channels found in molluscs (cf. Crest et al.
1992
; Gola et al. 1990
) and crustacea (our
present work) show similar and relatively small single-channel
conductances (<100 pS).
The current results show that the intrinsic properties of the ionic
channels mediating the IK(Ca) of
crayfish opener muscle fibers explain most of the characteristics of
the macroscopic current (Araque and Buño 1995).
Indeed, the channels are voltage and Ca2+
sensitive and they activate fast, in harmony with the similar sensitivities and fast activation kinetics of the macroscopic IK(Ca) (Araque and Buño
1995
). In agreement with the behavior of the macroscopic
IK(Ca), which increased and activated
faster with increasing membrane depolarization as a result of the
augmented Ca2+ inflow caused by the increased
activation of ICa (Araque and Buño 1995
), the channels mediating
IK(Ca) opened faster and the open
probability increased in high-Ca2+ conditions.
Therefore these intrinsic channel properties would favor the extremely
fast activation that typifies the macroscopic IK(Ca).
The open probability of these channels tended to be invariant during a
prolonged depolarizing pulse, in accord with the persistent property of
the macroscopic IK(Ca). However,
although the properties of BK channels explain the persistent nature of
IK(Ca), they do not correspond with
the complex profile of the macroscopic current in response to
depolarizing pulses. Indeed, we have reported that this
IK(Ca) showed an incomplete
inactivation, declining from its maximum value to reach a persistent
steady state within 10 ms, but we could not elucidate if inactivation
of the macroscopic IK(Ca) was due to
intrinsic channel properties or simply reflected temporal
[Ca2+]i variations
(Araque and Buño 1995). Present results show that the single-channel open probability displayed a fast initial increase to a steady state without peaks in both low- and
high-Ca2+ conditions, indicating the BK-type
single channels mediating this IK(Ca)
exhibit different behaviors when activated by patch depolarization as
compared with depolarization of the whole fiber. Therefore the complex
profile of the macroscopic IK(Ca) may
be due to rapid changes of the
[Ca2+]i (Araque
and Buño 1995
).
Two confronting dynamic mechanisms control [Ca2+]i in opener fibers during depolarization, namely, the Ca2+ influx through ICa channels and the intracellular Ca2+-buffering mechanisms. The interactions between these two dynamic processes may result in rapid variations of the [Ca2+]i that explain the complex macroscopic IK(Ca) profile. Because these variations are absent in cell-attached and inside-out conditions, our present data indicate that the apparent incomplete inactivation of IK(Ca) corresponds to temporal [Ca2+]i variations and is not due to the intrinsic properties of the channels mediating this current.
BK channel inactivation has been reported in several cell types such as
vertebrate skeletal muscle (Pallotta 1985), hippocampal pyramidal neurons (Hicks and Marrion 1998
), and rat
adrenal chromaffin cells (Solaro et al. 1995
). Our data
indicate that BK channels in crayfish muscle are noninactivating,
matching the behavior of BK channels in most cells that exhibit a
sustained activation in the presence of a constant
[Ca2+]i (e.g.,
Barret et al. 1982
; Blatz and Magleby
1986
; Latorre et al. 1989
).
Two findings were interesting and unexpected and should be underscored
because they could be of key functional importance. First, in the
cell-attached mode, openings could be evoked by depolarization at very
negative potentials of about 50 mV, well below the activation
threshold of the L-type ICa
(Araque and Buño 1994
, 1995
; Araque et al.
1994
). Second, in these conditions of low
[Ca2+]i, openings were
fast and channels could open at latencies <5 ms and reached 90% of
the maximum open probability in ~10 ms. This results suggest that BK
channels in crayfish muscle may be activated by very low
[Ca2+]i. Very high
Ca2+ sensitivity of BK channels has been reported
in mammalian salivary gland cells (Maruyama et al. 1983
)
and recently in locust muscle (Gorczynska et al. 1996
),
although its physiological relevance in these cells is unclear.
However, such a property may be of key importance for the functional
role of IK(Ca) in the crayfish muscle.
We have proposed that IK(Ca) provides
a rapid and continuous feedback that controls the depolarization-evoked
Ca2+ inflow, thereby regulating the
depolarization and the ICa activation during the graded action potentials that typify these muscle fibers (Araque and Buño 1995
; Araque et al.
1998
). This feedback allows a graded and persistent
Ca2+ inflow needed for the graded and sustained
contraction and prevents the uncontrolled depolarization that this
Ca2+ inflow would otherwise evoke (Araque
et al. 1998
). Accordingly, the high Ca2+
sensitivity of these channels, in addition to their voltage dependence, contributes to the extremely fast activation kinetics of the
macroscopic IK(Ca) (even at low
resting [Ca2+]i).
Therefore these channels act as precise Ca2+
sensors, providing the exact feedback current needed to control the
graded electrical activity and the contraction of these muscle fibers.
In conclusion, we have demonstrated that Ca2+- and voltage-dependent BK-type channels mediate the IK(Ca) in opener crayfish muscle. We show that the intrinsic properties of these channels are responsible for most of the characteristics of the macroscopic current. However, we report that owing to these intrinsic properties, the behavior of these channels is different when studied isolated (i.e., in single-channel recordings) than when studied in the whole cell, when the interaction with other channel types is significantly relevant.
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ACKNOWLEDGMENTS |
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This work was supported by Dirección General de Investigación Científica y Tecnológica, Ministerio de Educación y Cultura, and Fundación Areces Grants to W. Buño. A. Araque was a Fundación Areces postdoctoral fellow.
Present address of A. Araque: Dept. of Zoology and Genetics, Iowa State University, Ames, IA 50011.
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FOOTNOTES |
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Address for reprint requests: W. Buño, Instituto Cajal, Consejo Superior de Investigaciones, Av. Dr. Arce 37, E-28002 Madrid, Spain.
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 25 May 1999.
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REFERENCES |
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