Voltage-Gated and Ca2+-Activated Conductances Mediating and Controlling Graded Electrical Activity in Crayfish Muscle

Alfonso Araque1, Alain Marchand2, and Washington Buño1

1 Instituto Cajal, Consejo Superior de Investigaciones Científicas, E-28002 Madrid, Spain; and 2 Laboratoire de Neurobiologie et Mouvements, Centre National de la Recherche Scientifique, F-13402 Marseille, France

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Araque, Alfonso, Alain Marchand, and Washington Buño. Voltage-gated and Ca2+-activated conductances mediating and controlling graded electrical activity in crayfish muscle. J. Neurophysiol. 79: 2338-2344, 1998. Crayfish opener muscle fibers provide a unique preparation to quantitatively evaluate the relationships between the voltage-gated Ca2+ (ICa) and Ca2+-activated K+ (IK(Ca)) currents underlying the graded action potentials (GAPs) that typify these fibers. ICa, IK(Ca), and the voltage-gated K+ current (IK) were studied using two-electrode voltage-clamp applying voltage commands that simulated the GAPs evoked in current-clamp conditions by 60-ms current pulses. This methodology, unlike traditional voltage-clamp step commands, provides a description of the dynamic aspects of the interaction between different conductances participating in the generation of the natural GAP. The initial depolarizing phase of the GAP was due to activation of the ICa on depolarization above approximately -40 mV. The resulting Ca2+ inflow induced the activation of the fast IK(Ca) (<3 ms), which rapidly repolarized the fiber (<6 ms). Because of its relatively slow activation, the contribution of IK to the GAP repolarization was delayed. During the final steady GAP depolarization ICa and IK(Ca) were simultaneously activated with similar magnitudes, whereas IK aided in the control of the delayed sustained response. The larger GAPs evoked by higher intensity stimulations were due to the increase in ICa. The resulting larger Ca2+ inflow increased IK(Ca), which acted as a negative feedback that precisely controlled the fiber's depolarization. Hence IK(Ca) regulated the Ca2+-inflow needed for the contraction and controlled the depolarization that this Ca2+ inflow would otherwise elicit.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Ca2+-activated K+ conductances shape and control the electrical activity in neurons and muscle fibers (e.g., Barret and Barret 1976; Blatz and Magleby 1987; Gorman et al. 1981; Hille 1992; Madison and Nicoll 1984; Marty 1983; Yarom et al. 1985). Of the two types of Ca2+-activated K+ channels with small and large unitary conductances (SK and BK, respectively) (e.g., Latorre et al. 1989; Marty 1983), the latter controls action-potential (AP) repolarization and generates the early spike after hyperpolarization in invertebrate, sympathetic, and CA1 hippocampal pyramidal neurons (e.g., Adams et al. 1982; Crest and Gola 1993; Gola et al. 1990; Storm 1987). A Ca2+-activated K+ current (IK(Ca)) also controls the graded electrical activity of crayfish opener muscle fibers through BK channels that show large unitary conductances (Araque and Buño 1995; Araque and Buño, unpublished observations). The depolarizing phase of the graded AP (GAP) of opener fibers is exclusively mediated by a voltage-gated Ca2+ current (ICa) of the L type (e.g., Araque et al. 1994; Fatt and Ginsborg 1958; Mounier and Vassort 1975a). Therefore opener fibers provide a unique preparation to quantitatively evaluate the relationships between the waveforms and magnitudes of the ICa and IK(Ca) underlying the GAPs that typify these fibers.

We have taken advantage of this preparation to evaluate the contribution of ICa, of the delayed rectifier (IK), and especially of IK(Ca) to the GAP of opener fibers. Therefore we designed voltage-clamp experiments in which simulated voltage commands (i.e., "synthetic") imitated both the GAP evoked under current clamp in control conditions or the all-or-none AP (ANAP) elicited when IK(Ca) is blocked by intracellular bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) loading or extracellular application of charybdotoxin (CTX). This approach provides us with information regarding the contribution of the ionic currents to the electrical activity of opener fibers in physiological conditions of operation, permitting a quantitative description of the interactions between ionic conductances during the generation of the GAP, which is impossible using traditional voltage-clamp pulse commands.

We demonstrate that the initial ICa activation induced the depolarizing phase of the GAP and that the resulting Ca2+ inflow activated IK(Ca), which rapidly repolarized the fiber. IK was activated late, contributing to the final repolarization. In this muscle excitation-contraction, coupling is controlled by a Ca2+-induced Ca2+-release mechanism (Gyorke and Palade 1992), and the force of the contraction is governed exclusively by the degree of membrane depolarization (Bittner 1968; Orkand 1962). Consequently, IK(Ca) provides the negative feedback that controls the depolarization induced by ICa activation, thus precisely grading muscle contraction.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Preparation

Experimental procedures were as described previously (e.g., Araque and Buño 1995; Araque et al. 1994). Briefly, opener muscles from the first walking leg of crayfish (Procambarus clarkii) were isolated and transferred to a superfusion chamber (2 ml). Short muscle fibers (<400 mm) from the middle and proximal portions of opener muscles of small crayfish (<5 cm) were used.

Microelectrodes and recordings

Fibers were impaled with two 1 M KCl-filled micropipettes (1-5 MOmega ) and recorded under two-electrode voltage and current clamp with a Axoclamp-2A amplifier (Axon Instruments, Foster City, CA). The current electrode was substituted, after performing control recordings, by a new electrode filled with 0.16 M BAPTA neutralized with KOH (pH 7.2). BAPTA was ionophoretically injected with 500-ms, 100-nA current pulses, delivered at 0.2/s during 15 min. The final intracellular BAPTA concentration was estimated to be 2.5 mM (see Araque and Buño 1995). Pulse generation, data acquisition, and analysis were performed with a PC/AT personal computer (IBM) and pClamp-5.0 software (Axon Instruments) through a LabMaster TM-40 interface board (Scientific Solutions, Solon, OH). Data were obtained from six different fibers and expressed as means ± SD, unless stated otherwise.

Synthetic voltage-clamp command waveforms

The GAPs recorded under current clamp in response to 60-ms depolarizing current pulses in control conditions and the ANAP evoked after BAPTA-loading were simulated with steps and ramps, generated with the Clampex program of the pClamp-5.0 software, following smoothing with a 4-pole Bessel low-pass filter (Ithaco, model 4212). Step and ramp amplitudes, durations, slopes, and filter settings were adjusted to closely match the waveform and voltage of GAPs and the ANAP obtained under current clamp (e.g., Fig. 1A). Synthetic GAPs and the ANAP were used as voltage-command waveforms under voltage clamp; they mimicked responses obtained from the same fiber under current clamp. The use of computer-generated synthetic GAP and ANAP enabled the subtraction of linear capacitive and leak currents with the standard pCLAMP procedure. The holding potential was set at the value of the resting potential measured under current clamp in the fiber, usually -60 to -70 mV. It should be emphasized here that the traditional voltage-clamp methodology, as used by us with pulse commands and estimation of the voltage and time dependencies of the different isolated currents (cf., Araque and Buño 1994, 1995), does not give the time course and magnitude of the different ionic currents as they occur during the generation of GAPs. However, that information was obtained with the synthetic AP commands under voltage clamp used here.


View larger version (16K):
[in this window]
[in a new window]
 
FIG. 1. Voltage- and Ca2+-gated currents evoked by synthetic action-potential (AP) voltage commands. A: current-clamp responses (···) evoked by depolarizing current pulses (protocol shown above) in control [graded AP (GAP)] and bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA)-loaded conditions [all-or-none AP (ANAP)], superimposed with corresponding synthetic voltage commands (). B: currents evoked in Control and BAPTA conditions (left and right, respectively) by the corresponding GAP and ANAP, respectively. C: same as B, but currents evoked by GAP in BAPTA (left) and ANAP in Control conditions (right), respectively. D: IK evoked by GAP (left) and ANAPs (right) after Cd2+ superfusion, as indicated. To simplify the analysis, the 1st ANAP in the current-clamp response in A was simulated as if the pulse terminated during the AP rising phase and the Vm returned to the resting value after the AP falling phase; this was the synthetic ANAP.

Solutions

The control solution had the following composition (in mM): 210 NaCl, 5.4 KCl, 13.5 CaCl2, 2.6 MgCl2, and 10 Tris buffer, pH adjusted to 7.2 with HCl. In Cd2+ solution, 5 mM CdCl2 was added in equimolar exchange with CaCl2. CTX was added to the control solution from a 8-mM stock and superfused at 0.4 mM. Experiments were performed at 21-23°C. All chemicals were purchased from Sigma.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Synthetic AP voltage-clamp commands

We analyzed the GAPs in crayfish opener muscle preparations under control current-clamp conditions. The amplitude of the initial peak and the steady components of the GAP elicited by 60-ms current pulses increased, and the delay to the initial response decreased with current pulse intensity. Moreover, the GAP evoked by different fibers were different depending on cell characteristics (e.g., Bittner 1968; Orkand 1962). Therefore, to simplify our analysis, the transmembrane current pulse stimulus was adjusted (107 ± 31.5 nA, mean ± SD) to evoke similar GAPs in the six fibers selected. The GAPs evoked above a membrane potential (Vm) threshold of -43.2 ± 4.5 mV, showed an initial peak within22.3 ± 5.2 ms (19 ± 5.8 mV take-off to peak amplitude,14.1 ± 5.7 ms duration), and slowly declined reaching a steady state within 41.8 ± 14.8 ms (e.g., Fig. 1A, GAP, discontinuous record). The results described below were essentially identical in the six fibers studied, but only one was selected to illustrate the results.

When IK(Ca) was suppressed after BAPTA loading or extracellular CTX application, the same depolarizing pulses elicited repetitive ANAPs (62.1 ± 1.5 mV amplitude, 9.5 ± 5.3 ms duration) above a -42.1 ± 4.4 mV threshold (Fig. 1A, ANAP, discontinuous record). Neither GAPs norANAPs were observed in 5 mM Cd2+ or Ca2+-free solutions, indicating their Ca2+-dependent nature (not shown, but see Araque and Buño 1995).

Both synthetic GAPs and the ANAP (Fig. 1A, ) were used under voltage clamp in control and Cd2+ solutions and after BAPTA loading. The reasons for using Cd2+ solutions were that they block voltage-gated Ca2+ conductances and therefore also Ca2+-activated K+ conductances. BAPTA loading was used because it chelates intracellular Ca2+, thus inhibiting Ca2+-activated K+ conductances without affecting voltage-gated Ca2+ conductances (however, see Synthetic GAP in BAPTA-loaded condition) (see Araque and Buño 1995).

Synthetic GAP in control condition

The currents evoked by the synthetic GAP in control conditions are an estimate of those elicited by current pulses when the fiber generated the GAP under current clamp. They consisted of a large, brief, inward component (-425.6 ± 102.7 nA; 3.5 ± 0.7 ms) that activated at -43.0 ± 7.5 mV, followed by a smaller (98.3 ± 18.5 nA) outward current (Fig. 1B, Control). The outward component activated rapidly, emerging directly from the decay phase of the inward current (see Isolation of ICa, IK(Ca) and IK), and then decayed slowly toward a low-magnitude steady state (80.8 ± 10.5 nA), which lasted throughout the slowly decaying phase of the GAP before falling to zero. It should be emphasized that the control current included ICa, IK(Ca), and IK (see Isolation of ICa, IK(Ca) and IK). Indeed, it has been previously demonstrated that an early inward current ICa, an early outward component mainly due to IK(Ca), and a late outward component primarily originated by IK were mixed in the total current evoked by traditional voltage command pulses (Araque and Buño 1994, 1995; Araque et al. 1994; Hencek et al. 1978; Mounier and Vassort 1975a,b).

Synthetic ANAP in BAPTA-loaded condition

The currents evoked by the synthetic ANAP under voltage clamp represent those underlying the response evoked by current pulses in BAPTA-loaded fibers under current clamp. They displayed a larger (78 ± 24.5%) and broader (1.3 ± 0.6 ms) inward component than in the control condition, in agreement with the higher AP amplitude and the IK(Ca) block (Fig. 1B, BAPTA). The inward current activated at-39.1 ± 5.3 mV. The ensuing outward current was also larger (65.0 ± 16.5%), although its activation was slower peaking 2.5 ± 0.5 ms later, and it returned to zero more rapidly.

Synthetic GAP in BAPTA-loaded condition

BAPTA-loaded cells fired ANAPs when depolarized under current clamp, whereas GAPs were impossible responses. However, the responses evoked by GAPs under voltage clamp provided an estimate of the ionic currents that were either in shortage or in excess in terms of generating the control and BAPTA-loaded current-clamp responses, respectively (see Isolation of ICa and IK(Ca)). The synthetic GAP evoked a large, wide, inward component (-1,420.5 ± 200.2 nA; 4.5 ± 1.1 ms) followed by a slowly rising outward current that decayed gradually to a low steady state (Fig. 1C, BAPTA). Interestingly, the inward component was larger and lasted longer than in controls, whereas the peak outward current was 36 ± 7.3% smaller and displayed a lower steady-state value (Fig. 1B, Control). The differences can be attributed to the presence of ICa, IK(Ca), and IK in the control and the lack of IK(Ca) in the BAPTA-loaded case.

Similar effects were observed when IK(Ca) was inhibited with 0.4 mM CTX (data not shown), indicating that possible errors in the estimation of IK(Ca) due to an increased ICa steady state under BAPTA (i.e., less Ca2+-dependent inactivation) were negligible. A similar conclusion had been reached previously with traditional pulse commands (Araque and Buño 1995).

Synthetic ANAP in control condition

Under current clamp in control conditions, GAPs rather than ANAPs were evoked by depolarization. However, the responses elicited by the ANAP in control conditions under voltage clamp enable us to determine the ionic currents either absent or in excess in terms of generating the control and BAPTA-loaded current-clamp responses, respectively (see Isolation of ICa, IK(Ca) and IK and Fig. 3). The synthetic ANAP evoked a relatively small, brief, inward current (-502.3 ± 50.6 nA; 2.2 ± 0.3 ms) followed by a large, outward component in control conditions (Fig. 1C, Control). Interestingly, whereas the outward component was much larger (94.4 ± 12.2%) and peaked 2.7 ± 0.6 ms earlier than that evoked by the synthetic ANAP in BAPTA-injected fibers, the inward current was considerably smaller (34.6 ± 6.3%) and 2.6 ± 0.2 ms briefer (see Fig. 1B, BAPTA). Again, the differences agree with the presence of ICa, IK(Ca), and IK in control and the lack of IK(Ca) in BAPTA-loaded conditions, respectively.


View larger version (28K):
[in this window]
[in a new window]
 
FIG. 3. ICa, IK(Ca), and IK evoked by different GAP amplitudes. A: GAP voltage commands. B-D: ICa, IK(Ca), and IK, respectively.

IK remaining under Cd2+

When the synthetic GAP was used as voltage command after Cd2+ superfusion, ICa and IK(Ca) were eliminated while IK remained. IK increased slowly to 135.9 ± 20.3 nA, then decayed gradually to a steady state and eventually dropped to zero (Fig. 1D, left). However, when the voltage command was the synthetic ANAP, the IK was larger (57.2 ± 15.8%) and faster, peaking 2.1 ± 1.2 ms earlier (Fig. 1D, right).

The relative amplitude and time course of the current components described above are shown in detail in Fig. 2B. With respect to these currents, the following points should be underlined. 1) The ANAP evoked larger currents than the GAP in control conditions, particularly noticeable in the outward currents (Fig. 2B, Control). 2) Although the inward currents evoked by the GAP and the ANAP in BAPTA-loaded conditions had similar magnitudes and durations, only the ANAP evoked an outward current (Fig. 2B, BAPTA). 3) The currents activated by the GAP in BAPTA-loaded conditions showed a larger and wider inward component and no outward component when compared with the control (GAP, Fig. 2B, Control, BAPTA). 4) The responses evoked by the ANAP in the presence of intracellular BAPTA showed a larger and briefer outward, and a smaller and briefer inward component when compared with the control case (ANAP, Fig. 2B, Control, BAPTA). It should be emphasized that these differences result from the loss of IK(Ca) on BAPTA loading, whereas in control conditions the currents evoked were the sum of ICa, IK(Ca), and IK. The IK activated late and was larger and briefer with the synthetic ANAP (Fig. 2B, Cd2+).


View larger version (20K):
[in this window]
[in a new window]
 
FIG. 2. Voltage- and Ca2+-gated currents and conductances during the synthetic AP peaks. A: synthetic voltage commands; dashed lines indicate analysis epoch. B: currents evoked by GAP (left) and ANAP (right) in Control, BAPTA, and Cd2+ conditions (thick solid, dotted, and thin solid lines, respectively). C: ICa, IK(Ca), and IK components (dotted, thick solid, and thin solid lines, respectively) elicited by GAP and ANAP (left and right, respectively). D: conductance modifications (gCa, gK(Ca), and gK) during the GAP. Individual conductances were calculated from the equation gx = Ix/(Vm - Ex), where × denoted the pervading ion and Ex its equilibrium potential. The estimated ECa and EK were +40 and -70 mV, respectively; see RESULTS for details. Top trace corresponds to the initial part of the GAP.

Isolation of ICa, IK(Ca), and IK

The suppression of ICa in the presence of Cd2+ and the abolition of IK(Ca) after BAPTA loading opens the possibility of isolating each current by subtraction (cf. Araque and Buño 1995). ICa (or BAPTA-Cd2+ current) evoked by both synthetic APs had similar peak values and overall profiles, except notably for the small, late inward component evoked by the sustained depolarizing segment in the GAP that was absent with the ANAP (Fig. 2C). ICa activated fast, peaked at similar values when evoked by the GAP and ANAP (-1,185.6 ± 105.8 nA), immediately before the GAP and the ANAP peaks (3.2 ± 0.7 ms and 2.5 ± 0.3 ms, respectively), and decayed rapidly (<6 ms). IK(Ca) (or control-BAPTA current; Fig. 2C) was larger in response to the synthetic ANAP than the GAP (603.0 ± 25.2 nA and 832.5 ± 46.7 nA, respectively), as expected from its voltage dependence (Araque and Buño 1995) and the driving force increase. Moreover, mixed with the late steady-state ICa evoked by the synthetic GAP, there was a late sustained IK(Ca) component that was absent with the synthetic ANAP. IK activated late and was important late during the sustained depolarization of the GAP (Fig. 2C; see Fig. 1D).

The findings described indicate that the main difference between the currents underlying the GAPs and the ANAP in current-clamp conditions was the IK(Ca), present in the former but absent in the latter (Fig. 2C). The differences indicate that under control conditions, the graded activity resulted from ICa activation. The resulting Ca2+ inflow evoked the early and fast IK(Ca) activation, which acted as a negative feedback during the GAP and controlled the evolving depolarization induced by the ICa activation. After BAPTA loading, when the IK(Ca) was absent, the uncontrolled depolarization induced by the ICa activation led to an ANAP, with IK activating slowly and controlling the late repolarizing phase of the ANAP.

Conductances underlying the GAP

Although the current magnitudes and waveforms are key variables in AP genesis (see above and DISCUSSION), it is also of interest to estimate the conductance changes associated with the GAP (Hodgkin and Huxley 1952). Indeed, the participation of driving force variations associated with the GAP voltage command were eliminated when the conductance (g) was calculated, thus the conductance waveforms provide direct information regarding the contribution of channel activation during the GAP in physiological conditions of operation. The corresponding gs were estimated by applying the formula gx Ix/(Vm - Ex), where x is the permeant ion and Ex its equilibrium potential. The corresponding Exs were estimated from the ICa reversal potential and the IK tail current amplitudes for IK(Ca) and IK (see Araque and Buño 1994, 1995; Araque et al. 1994).

Important g features for the different voltage and Ca2+-gated channels during the GAP were (Fig. 2D) 1) for gCa, its extremely rapid activation peaking in 3.4 ± 1.1 ms (peak value 16.2 ± 3.8 mS) and somewhat slower deactivation to a sustained steady value; 2) for gK(Ca), (peak value 8.3 ± 2.4 mS) the similarity of its overall profile with that of gCa and the small time shift of its activation to longer delay times (2.2 ± 0.1 ms), indicating its dependence on Ca2+ inflow and its extremely fast activation; 3) for gK its delayed, slow, activation, late during the GAP falling phase and its sustained activation thereafter.

Control of GAP amplitude

The amplitude of the synthetic GAP was changed (e.g., Figs. 3 and 4), and the relative contribution of ICa, IK(Ca), and IK to the changes in GAP amplitude that occurred in the natural condition were estimated (e.g., Araque and Buño 1995; Bittner 1968; Orkand 1962). The ICa (or BAPTA-Cd2+ current; Fig. 3B) increased and peaked at gradually decreasing delays with GAP voltage. The inward peak was followed by a lower amplitude sustained inward component that increased with GAP voltage. A brief decrease of the inward current was sometimes observed between the peak and the steady-state ICa at high AP voltages. This decrease was probably due to an incomplete block of IK(Ca), or to small modifications in the ICa inactivation rate in BAPTA-loaded conditions (see Araque and Buño 1995). The IK(Ca) (or Control-BAPTA current; Fig. 3C) gradually increased and peaked at briefer delays as the GAP voltage augmented. The outward peak was followed by a steady-state current that lasted throughout the depolarization and increased with GAP voltage. The IK increased gradually with GAP voltage (Fig. 3D), and it lasted throughout the GAP depolarization and activated slowly at long delays that did not change with GAP voltage.


View larger version (18K):
[in this window]
[in a new window]
 
FIG. 4. ICa, IK(Ca), and IK during GAP steady-state depolarization; effects of GAP amplitude. A: GAP waveforms (dashed lines indicate the epoch analyzed). B: ICa and IK(Ca) (dotted and solid lines, respectively) evoked at different GAP amplitudes. C: current-voltage relationships of ICa, IK(Ca), and IK (bullet , black-square, and black-triangle, respectively) measured at the beginning of the analysis epoch (vertical bars indicate SE; n = 6).

The contribution of ICa, IK(Ca), and IK(Ca) during the final steady-state segment of the GAP are functionally important because they confer the sustained contraction that typifies opener fibers (Bittner 1968; Orkand 1962). All three currents were active during the steady-state depolarization and increased with GAP voltage (Fig. 4; see also Fig. 3). ICa and IK(Ca) increased with GAP voltage (Fig. 4, B and C, bullet  and black-square, respectively), and the similarity of both current magnitudes (i.e., ICa and IK(Ca)) at all GAP voltage values should be emphasized (see DISCUSSION). IK also increased with GAP voltage (Fig. 4C, black-triangle; see also Fig. 3D).

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

The above results show that, with the use of synthetic AP waveforms under voltage clamp and pharmacological agents that block specific conductances, it is possible to examine the contributions of different voltage and Ca2+-gated currents to the genesis of the GAP that typifies the electrical activity of crayfish opener muscle fibers. The procedure depends on the 1) similarities between real and synthetic APs; 2) blocking specificity of the agents used; and 3) absence of voltage-dependent block. The real and synthetic APs do show small differences; however, these were insignificant at depolarized values above -40 mV, which corresponded to the activation threshold of voltage-gated currents. Thus the synthetic APs appear to approximate well to the current-clamp situation. Although BAPTA may exert an effect on ICa magnitude, the predominant, if not exclusive, effect of BAPTA was the suppression of IK(Ca). The results obtained from CTX experiments are qualitatively similar to those obtained with BAPTA injection (e.g., generation of ANAPs) confirming the validity of BAPTA results. However, because the CTX-mediated IK(Ca) block is voltage dependent (Mackinnon and Miller 1988), the quantitative analysis presented here was based on the effects of BAPTA. For a more complete discussion of possible problems associated with ICa measurement after BAPTA injection, and the efficacy of CTX block, see Araque and Buño (1995).

Although the procedure we have adopted appears to fulfill the criteria, it should be emphasized that the analysis centered on the postsynaptic mechanisms that control the generation of the GAP. Thus it did not take into account the functional importance of the continuous feedback regulation of excitatory synaptic input by the coactivation of inhibitory synaptic outflow in controlling muscle contraction (e.g., Smith 1974; Wilson and Davies 1965).

IK(Ca) involvement in the regulation of the GAP and dependence on IK(Ca)

In agreement with our previous demonstration using traditional pulse commands under voltage clamp (Araque and Buño 1995), the BK channels mediating IK(Ca) had extremely brief opening latencies. IK(Ca) activated so fast that it appeared before the peak ICa activation was reached (2-ms lag) during the GAP (Fig. 2, C and D), thus enabling an extremely quick regulation of the depolarization and subsequent Ca2+ inflow. The IK(Ca) of crayfish muscle fibers is faster than other previously reported BK-type currents, and it inactivates partially, declining rapidly to a persistent steady state, probably reflecting temporal intracellular Ca2+ concentration ([Ca2+]i) variations (Araque and Buño 1995).

The fast IK(Ca) activation (Figs. 2 and 3) acted as a negative feedback that controlled the subsequent ICa activation by restricting the depolarization and obstructing the generation of the ANAP that would otherwise occur. This negative feedback permits the precise regulation of the Ca2+ influx and a gradual and smooth control of the ensuing muscle contraction during the GAP (see below). The added negative feedback provided by the late IK activation also contributes to this. Interestingly, the feedback regulation by IK and IK(Ca) depended on Vm and on both [Ca2+]i and Vm, respectively. Thus the two related variables were under continuous feedback control during the GAP.

The relative magnitudes of ICa and IK(Ca) were usually similar and increased in parallel as the GAP voltage was incremented (Fig. 4) (see also Araque and Buño 1995). This strict correspondence could account for the precise regulation of the graded electrical activity of opener muscle fibers. With relatively more or less IK(Ca), the Vm would either be clamped at relatively hyperpolarized Vms, at which ICa would be insufficiently activated or not activated at all, or the fiber would fire ANAPs, and a smooth regulation of [Ca2+]i would be impossible (H. Chagneux, A. Araque, W. Buño, and M. Gola, unpublished observations). The similar ICa and IK(Ca) magnitudes could enable the correct control of Vm within the activation range of ICa and thus of the degree of depolarization and of muscle contraction. Therefore the tuning of the fast kinetics and relative magnitudes of ICa and IK(Ca) demonstrated above would be decisive in determining the final [Ca2+]i that set the force of the contraction.

All-or-none activity following IK(Ca) block

BAPTA loading or extracellular CTX evoked the ANAP instead of the GAP variant (Araque and Buño 1995). Similar results were obtained with ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) loading in barnacle muscle fibers (Hagiwara and Naka 1964). However, EGTA was ineffective in crayfish muscle fibers (Araque and Buño 1995). BAPTA is a much faster Ca2+ chelator than EGTA; therefore the differences in IK(Ca) sensitivity in both muscles probably indicate that Ca2+ and Ca2+-activated K+ channels were more closely localized in the membrane of crayfish muscle fibers (Araque and Buño 1995).

ICa was essentially identical in control and BAPTA-loaded fibers, whereas IK(Ca) was absent in treated cells, indicating that IK(Ca) regulated the graded depolarization under control conditions, and that ICa was nonessential in determining the different behaviors (e.g., Fig. 2C). However, the inward current was smaller in the control than in BAPTA-loaded conditions because the added, fast, IK(Ca) was activated before ICa peak activation occurred (e.g., Fig. 2B), and it tended to repolarize the fiber. Therefore the large, early, IK(Ca) inhibited the ANAP in control conditions. When IK(Ca) was absent, the magnitude of the inward component was larger and was only controlled by the comparatively modest and slow IK. In those conditions the regenerative nature of the uncontrolled ICa activation elicited ANAPs.

Functional implications

When the cell is depolarized by excitatory postsynaptic potential (EPSP) barrages, the voltage-dependent ICa is activated and a Ca2+ inflow leads to a Ca2+-induced Ca2+ release, which triggers contraction (Gyorke and Palade 1992). The graded contraction depends linearly on the degree of depolarization (Bittner 1968; Orkand 1962). We propose that the continuous and rapid feedback provided by IK(Ca) controls the Ca2+ inflow, thereby regulating the depolarization and the ICa activation during GAPs. The precise feedback relied on similar magnitudes of IK(Ca) and ICa, which allows the persistent Ca2+ inflow needed for sustained contraction while preventing the uncontrolled depolarization that this Ca2+ inflow would otherwise evoke. The important contribution of the leak current that tends to stabilize the system due to its high amplitude (see Araque and Buño 1994) should be emphasized. The leak conductance must be constantly overcome by the EPSPs and other active responses to reach a stable depolarized Vm.

    ACKNOWLEDGEMENTS

  We thank Dr. Euan Brown for suggestions and the correction of an initial version of the manuscript and Dr. Mark Sefton for the correction of the final version.

  This work was supported by Direción General de Investigación Cientifica y Técnica/Ministerio de Educación y Cultura, Fundación Areces (Spain), European Commission CI1*-CT90-0861VY and ERBCHRXT930190, and National Atlantic Treaty Organization grants, and an Hispano-French Integrated Action to W. Buño. A. Araque was a Fundación Areces postdoctoral fellow.

  Present addresses: A. Marchand, Laboratoire de Psychophysiologie, CNRS-URA 1295, F-67000 Strasbourg, France; A. Araque, Dept. of Zoology and Genetics, Iowa State University, Ames, IA 50010.

    FOOTNOTES

  Address for reprint requests: W. Buño, Instituto Cajal, CSIC, Av. Dr. Arce 37, E-28002 Madrid, Spain.

  Received 8 July 1997; accepted in final form 21 January 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society