Fast BK-Type Channel Mediates the Ca2+-Activated K+ Current in Crayfish Muscle

Alfonso Araque and Washington Buño

Instituto Cajal, Consejo Superior de Investigaciones, E-28002 Madrid, Spain


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    METHODS
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega ) 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 GOmega ) 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 MOmega ) 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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1. Channel openings evoked by ramp membrane depolarization. A: ramp voltage command from -70 to 130 mV. B, 1-2: records showing single-channel openings. C: averaged (n = 25) single-channel current evoked by ramp depolarization. D: I-V relationship of a single-channel recording fitted to a linear regression (solid line). Single-channel conductance (g = 86.1 pS) was obtained from the slope of the linear regression, and the reversal potential was estimated from the 0 current value (-62.5 mV, *). E: I-V relationship of the averaged single-channel current shown in C. Horizontal line indicates the zero current and the slanted straight line the current that corresponds to the expected I-V relationship of single-channel openings (indicated by arrows). All records are from the same channel in cell-attached recording.

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
<IT>I</IT><IT>=</IT><IT>g</IT>(<IT>V</IT><SUB><IT>m</IT></SUB><IT>−</IT><IT>E</IT><SUB><IT>K</IT></SUB>)<IT>/</IT><IT>N</IT>
where N is the number of averaged responses, EK is the reversal potential for K+ and g is the single-channel conductance (both estimated from the linear I-V relationship of the single-channel current). Figure 1E shows that above about -30 mV, the I-V relationship of the averaged channel current increased nonlinearly over the value of single openings, indicating that the channel open probability increased with depolarization and, therefore that the channel was voltage sensitive.

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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Fig. 2. Single-channel activity evoked by pulse depolarization. A: cell-attached recordings of a single-channel evoked by depolarizing pulses from -70 to -20, 0, and 50 mV; protocol shown above as in Figs. 3 and 4. B: I-V relationship of the single-channel current amplitude. Data were fitted to a linear regression (---). Estimated conductance was 92.4 pS, and the reversal potential was -65.6 mV (*). C: mean single-channel open probability as a function of the membrane potential. Each data point represents the mean ± SE from 6 patches.

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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Fig. 3. Large-conductance channel is Ca2+ dependent. A: single-channel openings evoked by depolarizing pulses in cell-attached configuration (low Ca2+). B: same as A, but in high-Ca2+ conditions, i.e., after excising the patch and recording the channel in inside-out configuration (13.5 mM Ca2+). Single-channel current decreases in the inside-out configuration after the patch was excised from the membrane, probably because of the greatly diminished K+ concentration in the extracellular solution compared with that of the cytoplasm. Channel open probability increased and the channel tended to open at briefer delays from pulse onset. C and D, histograms of the latency between the onset of the depolarizing pulse and the 1st channel opening in low- and high-Ca2+ conditions, respectively. E: cumulative probability plot of the channel opening latencies in low- and high Ca2+-conditions (thin and thick lines, respectively). Histograms and cumulative probability plots were constructed from the responses to 100 depolarizing pulses from -70 to 50 mV. Bin width: 4 ms.

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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Fig. 4. Fast activation and deactivation characterize channel openings in cell-attached recordings. A: single-channel recordings (middle traces) showing brief opening delays (*), and averaged (n = 500) single-channel current also showing fast openings (down-arrow ; bottom) evoked by depolarizing pulses (top). B: single-channel recordings from a different patch showing responses to long-duration voltage pulses (middle traces); averaged single-channel current was calculated from 300 responses (bottom).

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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society