Large-Conductance Ca2+-Activated Potassium Channels in Secretory Neurons

Jesús Lara, Juan José Acevedo, and Carlos G. Onetti

Centro de Investigaciones Biomédicas, Universidad de Colima, Colima 28000, Mexico


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Lara, Jesús, Juan José Acevedo, and Carlos G. Onetti. Large-Conductance Ca2+-Activated Potassium Channels in Secretory Neurons. J. Neurophysiol. 82: 1317-1325, 1999. Large-conductance Ca2+-activated K+ channels (BK) are believed to underlie interburst intervals and contribute to the control of hormone release in several secretory cells. In crustacean neurosecretory cells, Ca2+ entry associated with electrical activity could act as a modulator of membrane K+ conductance. Therefore we studied the contribution of BK channels to the macroscopic outward current in the X-organ of crayfish, and their participation in electrophysiological activity, as well as their sensitivity toward intracellular Ca2+, ATP, and voltage, by using the patch-clamp technique. The BK channels had a conductance of 223 pS and rectified inwardly in symmetrical K+. These channels were highly selective to K+ ions; potassium permeability (PK) value was 2.3 × 10-13 cm3 s-1. The BK channels were sensitive to internal Ca2+ concentration, voltage dependent, and activated by intracellular MgATP. Voltage sensitivity (k) was ~13 mV, and the half-activation membrane potentials depended on the internal Ca2+ concentration. Calcium ions (0.3-3 µM) applied to the internal membrane surface caused an enhancement of the channel activity. This activation of BK channels by internal calcium had a KD(0) of 0.22 µM and was probably due to the binding of only one or two Ca2+ ions to the channel. Addition of MgATP (0.01-3 mM) to the internal solution increased steady state-open probability. The dissociation constant for MgATP (KD) was 119 µM, and the Hill coefficient (h) was 0.6, according to the Hill analysis. Ca2+-activated K+ currents recorded from whole cells were suppressed by either adding Cd2+ (0.4 mM) or removing Ca2+ ions from the external solution. TEA (1 mM) or charybdotoxin (100 nM) blocked these currents. Our results showed that both BK and KATP channels are present in the same cell. Even when BK and KATP channels were voltage dependent and modulated by internal Ca2+ and ATP, the profile of sensitivity was quite different for each kind of channel. It is tempting to suggest that BK and KATP channels contribute independently to the regulation of spontaneous discharge patterns in crayfish neurosecretory cells.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Calcium-activated potassium currents (KCa currents) have been observed in a number of neurons in invertebrates (Crest and Gola 1993; Deitmer and Eckert 1985) as well as in vertebrates (Kawai and Watanabe 1986; Lancaster et al. 1991; Pennefather et al. 1985; Smart 1987; Wang et al. 1998). Several types of potassium ion channels can mediate this KCa current: in addition to "small" conductance (SK) Ca2+-activated K+ channels, "big" conductance (200-300 pS) Ca2+-activated K+ (BK) channels have been described (Latorre et al. 1989). These channels couple the membrane potential to the intracellular Ca2+ concentration ([Ca2+]i) in such a way that an increase in internal Ca2+ leads to an efflux of K+ ions and a subsequent hyperpolarization of the membrane. Because of this property, Ca2+-activated K+ channels are believed to play a role in a number of different cellular processes that depend on the influx of Ca2+ through voltage-dependent pathways, including regulated secretion (McManus 1991; Petersen and Maruyama 1984). These channels also link internal Ca2+ and membrane excitability and thus are believed to play a role in regulating action potential frequency and duration in neurons. For instance, it has been proposed that BK channels underlie interburst intervals; therefore they could contribute to the control of hormone secretion in rat neurohypophysial terminals (Dopico et al. 1996).

In the X-organ sinus gland (XO-SG) system, the most important neurosecretory system of the crayfish, a periodic increase in [Ca2+]i in bursting neurons modifies membrane conductances and regulates electrophysiological activity (Martínez et al. 1991; Onetti et al. 1990). Calcium sensitivity is a very important phenomenon because the firing patterns have been associated with the ability for secretion in neurons (Stuenkel 1985). Several potassium currents contributing to modulate action potential firing patterns have been previously described in X-organ neurons: the delayed rectifier, the transient inactivating outward current (Martínez et al. 1991), and the ATP-sensitive K+ current (García et al. 1993; Onetti et al. 1996). Experimental evidence supports a key role of intracellular Ca2+ in the modulation of these currents. It has been suggested that KATP channels are involved in the regulation of spontaneous spike firing, due to their sensitivity to intracellular ATP and Ca2+ ions as well as to the membrane potential. In whole cell patch-clamp studies performed earlier in crayfish XO neurons, Ca2+-activated potassium current was negligible because a cytoplasmic solution with EGTA was used (Onetti et al. 1990). However, recent experiments with inside-out cell-free patches suggest the presence of large-conductance Ca2+-activated potassium channels (BK) in somata membranes from XO neurons. To study the relative contribution of BK and KATP channels to the macroscopic outward current and their participation in electrophysiological activity, we shall compare the properties of both types of channels in terms of their sensitivity toward intracellular Ca2+ and ATP as well as to the membrane potential.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The experiments were carried out in neurons of the XO-SG system obtained from adult Procambarus clarkii crayfish of either sex. The dissection procedures and experimental conditions were described earlier (García et al. 1993). All records were made in neuron bodies of the X-organ (XO) at room temperature (20-22°C). In the experiments for macroscopic current recording, the XO-SG nerve tract was severed at 200-300 µm after its emergence from XO.

Electrophysiological recordings

The perforated patch-clamp technique (Horn and Marty 1988) was used to record membrane potential and macroscopic currents in the current- and voltage-clamp mode. Single-channel current recordings were performed in cell-attached, outside-out, and inside-out patch configurations of the patch-clamp technique (Hamill et al. 1981). All the records of membrane potential and membrane currents obtained in perforated patch-clamp experiments were made using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). To obtain single-channel currents we used a GeneClamp 500 amplifier (Axon Instruments, Foster City, CA). Pipettes with resistance ranging from 4 to 6 MOmega were used for perforated patch-clamp experiments, and from 8 to 10 MOmega for single-channel recordings. Patch pipettes were pulled from 1.5-mm borosilicate capillaries, coated with silicone elastomer (Sylgard; Dow Corning, Midland, MI) to reduce background noise and capacitive current and were fire polished.

Membrane potential and macroscopic current records were acquired with a Digidata 1200 A/D converter (Axon Instruments, Foster City, CA) using pClamp 6.0.2 software (Axon Instruments, Foster City, CA). Membrane potential records were filtered at 5 kHz (-3 dB). Macroscopic current signals were filtered at 2 kHz and sampled at 5 kHz. Single-channel currents were stored with a PCM Data Recorder 200 (A. R. Vetter, Rebersburg, PA) for subsequent off-line acquisition and analysis. These signals were filtered (1 kHz), amplified, and sampled at 10 kHz using a Digidata 1200 A/D converter (Axon Instruments, Foster City, CA). Analysis was made using a 486 microcomputer with pClamp 6.0.2 software (Axon Instruments, Foster City, CA). The method described by Davies et al. (1996) was used to obtain current-voltage (I-V) relations for single-channel currents. Briefly, currents were elicited by 80-ms voltage ramps from -80 to 80 mV; a leak trace, obtained by averaging sections that had no channel openings, was subtracted from each individual record, and an ensemble ramp was constructed by averaging sections containing a single opening. The open-state probability (PO) values were obtained by using the method described by Quayle et al. (1988). We used current records of 30 s to measure the open-state probability and to obtain single-channel conductance values. Single-channel data were filtered at 1 kHz (Bessel 4-pole) and sampled at 10 kHz. Experimental values are presented as means ± SE. A P value <0.05 was considered to be significant.

Solutions

For inside-out patch experiments, the intracellular membrane surface was bathed with solutions, the compositions of which are described in Table 1. Both cell-attached and inside-out patch pipettes were filled with 200 K solution (Table 1). Inside-out patch pipettes used in the experiments performed to test single-channel activity at quasi-physiological potassium gradient, were filled using 5 K solution. For outside-out patch current recording, the pipettes were filled with control solution, and the extracellular surface was bathed with 200 K solution (Table 1).


                              
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Table 1. Composition of solutions for single-channel experiments (in mM)

For perforated patch-clamp experiments, the XO-SG system was continuously perfused with a saline solution of the following composition (mM): 200 NaCl, 5.4 KCl, 13.5 CaCl2, 2 MgCl2, and 10 HEPES for membrane potential measurements. For macroscopic current experiments, the cell bodies were bathed with a solution composed of (mM) 5.4 KCl, 13.5 CaCl2, 10 HEPES, and 200 N-methyl-D-glucamine. Charybdotoxin (100 nM), TEA (1 mM), or CdCl2 (0.4 mM) were added to external solutions. The tips of the pipettes used were filled with two different nystatin-free solutions (mM): 180 K-aspartate, 40 NaCl, and 10 HEPES for membrane potential recording, or 220 KCl and 10 HEPES for macroscopic currents. Nystatin (300 µg/ml; stock solution, 25 mg/ml in dimethylsulfoxide) was added to the corresponding filling solution. The pH of the external solution was adjusted to 7.4 with NaOH or HCl, and the pH of the pipette solutions was adjusted to 7.2 with KOH.

ATP magnesium salt (MgATP), N-[2-hydroxyethyl]piperazine-N'-2-ethanesulfonic acid (HEPES), ethyleneglycol-bis[beta -aminoethylether]-N,N,N',N'-tetraacetic acid] (EGTA), nystatin, dimethylsulfoxide, N-methyl-D-glucamine, and tetraethylammonium (TEA) were all purchased from Sigma Chemical (St. Louis, MO). Charybdotoxin was kindly provided by Dr. Ubaldo García.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

K+ selectivity, permeation, and blockade of BK channels

Unitary currents through Ca2+-activated K+ channels (BK) were obtained in both cell-attached and excised membrane patches from X-organ neurons. The BK channel activity observed in cell-attached was similar to that obtained on excising membrane patches. The open-state probability (PO) values were ~0.8 (VP = 0 mV) in the cell-attached configuration and ~0.9 (40 mV) in the inside-out patches at 1 µM Ca2+ on the internal side of the membrane.

The K+ selectivity of the channels was studied in inside-out patches at different intracellular and extracellular K+ concentrations. Throughout these experiments the internal (bath) solution contained 1 µM free Ca2+ without MgATP (control solution; Fig. 1A). In conditions of symmetrical distribution of K+ ions ([K]o = [K]i = 200 mM), a slight inward rectification was evident in the I-V relationship. The unitary conductance, for membrane potential values between -80 to 0 mV, was 223 ± 11 pS (n = 4), and the reversal potential value was ~0 mV (Fig. 1B). In this condition, the I-V relation in the linear region can be described by the Goldman-Hodgkin-Katz equation
<IT>I</IT><SUB><IT>K</IT></SUB><IT>=</IT><IT>P</IT><SUB><IT>K</IT></SUB> <FR><NU><IT>F</IT><SUP><IT>2</IT></SUP><IT>V</IT><SUB><IT>m</IT></SUB></NU><DE><IT>RT</IT></DE></FR> [<IT>K</IT>] (1)
where PK is the channel permeability to K+ ions and Vm is the membrane potential. R, T, and F have their usual meaning. The PK value obtained by fitting Eq. 1 was 2.3 × 10-13 cm3 s-1. When the KCl concentration on the intracellular surface of the membrane patches was reduced to 40 mM (160 mM NaCl, low K solution) the reversal potential of the unitary current was shifted to 41 ± 4 mV (Fig. 2) in all five patches studied. This value is close to the equilibrium potential for K+ ions predicted by the Nernst equation (40 mV), and an inward rectification was evident. When the potassium gradient was closer to physiological conditions ([K]o = 5 and [K]i = 200 mM), a nonlinear I-V relation was found, exhibiting outward rectification. In this condition, the unitary current was outwardly directed at all membrane potentials explored (-80 to 80 mV). Single-channel ensemble ramp currents at different internal and external K+ concentrations demonstrate that the BK channels are highly permeable to K+ ions (Fig. 2). These results suggest these channels are selective to K+ ions.



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Fig. 1. Current-voltage relationship for the BK channels. A: single-channel currents recorded from an inside-out patch at 2 different membrane potentials (indicated at the right of recordings) in symmetrical K+ ([K]o = [K]i = 200 mM). Closed (c) and open (o) current levels are indicated to the left of the traces. B: experimental current-voltage plots for the BK channel obtained in symmetrical K+. Dotted line corresponds to experimental data fittings using the Goldman-Hodgkin-Katz equation. The intracellular membrane surface was bathed with control solution. Data are presented as means ± SE (n = 5).



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Fig. 2. Selectivity of the BK channel. Single-channel ensemble ramp currents in the inside-out configuration obtained at different K+ gradients: [K]o = 5 mM and [K]i = 200 mM (a), [K]o = [K]i = 200 mM (b), and [K]o = 200 mM and [K]i = 40 mM (c). The intracellular membrane surface was bathed with control (a and b) or low K (c) solutions. The pipettes were filled with 200 K (b and c) or 5 K (a) solutions. Ramp currents in high and low intracellular K+ concentrations were obtained from the same patch (b and c).

To explore the sensitivity of BK channels toward blocking agents specifically directed against particular types of K+ channels, we recorded the potassium currents in outside-out and whole cell patch configurations in the presence of tetraethylammonium chloride (TEA), charybdotoxin (CTX), or Cd2+. Also, we examined the effect of removing Ca2+ from the extracellular medium. The single currents from an outside-out patch were reversibly blocked by adding TEA (1 mM) or CTX (100 nM) to the extracellular side of the membrane patch (Fig. 3). Moreover, the transient component of macroscopic potassium currents through BK channels was blocked by the addition of Cd2+, TEA, or CTX. By replacing Ca2+ with an isosmotic amount of Mg2+, the current observed in the whole cell mode also diminished (Fig. 6A).



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Fig. 3. Tetraethylammonium (TEA) and charybdotoxin (CTX) effects on the BK channel activity. Single-channel activity recorded from an outside-out patch with symmetrical K+ concentrations (Vm = 40 mV). Channel activity was abolished after exposure to 1 mM of TEA. When the patch was exposed to 100 nM of CTX, after wash out TEA, the channel activity was abolished. The channel activity was partially recovered after wash out (bottom trace). Closed (c) and open (o) current levels are indicated to the left of the traces. The extracellular membrane surface was bathed with 200 K solution, and the internal face of the membrane (pipette) was exposed to the solution that contained 1 µM Ca2+ (control solution).

Ca2+, MgATP, and voltage sensitivity of BK channels

To study Ca2+ and voltage dependence of BK channels, we obtained the steady-state open probability in symmetrical distribution of K+ ions, by changing the membrane potential at different intracellular Ca2+ concentrations ([Ca2+]i) in inside-out patches, in the absence of MgATP. When the membrane potential was held at 40 mV, increasing [Ca2+]i from 0.3 to 3 µM surface caused an enhancement of the channel activity (Fig. 4A). To describe the voltage dependence of channel activity, we fitted the experimental values of Po, obtained at different membrane potentials, to the Boltzmann equation
<IT>P</IT><SUB><IT>o</IT></SUB><IT>/</IT><IT>P</IT><SUB><IT>max</IT></SUB><IT>=</IT>[<IT>1+</IT><IT>e</IT><SUP>(<IT>V</IT><SUB><IT>0.5</IT></SUB><IT>−</IT><IT>V</IT><SUB><IT>m</IT></SUB>)<IT>/</IT><IT>k</IT></SUP>]<SUP><IT>−1</IT></SUP> (2)
where Po is determined experimentally at a specific membrane potential (Vm). V0.5 is the membrane potential at which Po is one-half of its maximum (Pmax) and k is the logarithmic potential sensitivity. The e-fold increase in Po (k) did not significantly change (12.6-12.8 mV) at different [Ca2+]i. However, an increase in [Ca2+]i produced a shift to more negative potentials of Po/Vm relationship. The half-activation membrane potentials (V0.5) were -5, -26, and -44 mV at 0.3, 1, and 3 µM of [Ca2+]i, respectively (Fig. 4B).



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Fig. 4. Ca2+ and voltage dependence of the BK channels. A: single current records obtained from an inside-out patch (Vm = 40 mV) in symmetrical K+ gradient at 3 different internal Ca2+ concentrations (shown to the right of the traces). Closed (c) and open (1, 2, 3) current levels are indicated to the left of the traces. B: channel activity (Po/Pmax) as a function of membrane potential, obtained at 3 different intracellular Ca2+ concentrations (black-down-triangle : 0.3 µM, n = 4; open circle : 1 µM, n = 5; : 3 µM, n = 4) in symmetrical K+ gradient. Because the reversal potential value is close to 0 mV in symmetrical K+ gradient, the 0-mV points on the curves are missing. Curve lines correspond to Boltzmann fits for experimental values of Po/Pmax. The intracellular membrane surface was bathed with control, 0.3 Ca, or 3 Ca solutions. Data are presented as means ± SE. C: half activation potential (V0.5) divided by the slope factor (k) as a function of [Ca2+]i to estimate the number of Ca2+ binding sites on the BK channel. Values for V0.5 and k were taken from fittings shown in Fig. 4B. The line corresponds to the best fit of the Eq. 3 (see text) to values of V0.5/k.

The number of Ca2+ binding sites on the BK channel can be estimated using the following equation (Wong et al. 1982)
<IT>V</IT><SUB><IT>0.5</IT></SUB><IT>/</IT><IT>k</IT><IT>=</IT><IT>N</IT><SUB><IT>Ca</IT></SUB><IT> ln </IT><FR><NU><IT>K</IT><SUB><IT>D</IT></SUB>(<IT>0</IT>)</NU><DE>[<IT>Ca</IT><SUP><IT>2+</IT></SUP>]<SUB><IT>i</IT></SUB></DE></FR> (3)
where KD(0) is the concentration of Ca2+ that induces 50% of maximum channel activity at 0 mV, and NCa is the number of binding sites that is analogous to the Hill coefficient. Values for V0.5 and k were taken from the experiments of Fig. 4B. From the fitting of those data NCa was 1.32 and KD(0) was 0.22 µM (Fig. 4C).

Figure 5A is a typical example of the activating action on the BK channels by the addition of 0.1 and 1 mM MgATP to the internal solution at 1 µM Ca2+ (Vm = 40 mV). To study the sensitivity of the BK channel to MgATP, we used NPO rather than PO due to the possibility that MgATP can affect both of these parameters (Albarwani et al. 1994). A plot of the number of functional channels multiplied by their open probability (NPo), against MgATP concentration ([MgATP]) is shown in Fig. 5B. The relationship NPo/[MgATP] can be described by the Hill equation
<IT>NP</IT><SUB><IT>o</IT></SUB><IT>=</IT><IT>NP</IT><SUB><IT>omax</IT></SUB> <FR><NU>[<IT>MgATP</IT>]<SUP><IT>h</IT></SUP></NU><DE>[<IT>MgATP</IT>]<SUP><IT>h</IT></SUP><IT>+</IT><IT>K</IT><SUP><IT>h</IT></SUP><SUB><IT>D</IT></SUB></DE></FR> (4)
where NPomax, is the maximum number of functional channels multiplied by their open probability, KD is the half-activating concentration of MgATP, and h is the Hill coefficient. By fitting Eq. 4 to the NPo values obtained at various [MgATP], ranging from 0.01 to 3 mM, the dissociation constant for MgATP (KD) was 119 µM and the Hill coefficient (h) was 0.6 (Fig. 5B).



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Fig. 5. Activation of the BK channels by MgATP. A: single-channel currents recorded from an inside-out patch (Vm = 40 mV) at 3 different MgATP concentrations (shown to the right of the traces) in symmetrical K+ gradient. Closed (c) and open (1 to 5) current levels are indicated to the left of the traces. B: number of functional channels multiplied by their open probability (NPo) as a function of MgATP concentration ([MgATP]) from inside-out patches in symmetrical K+ concentrations (Vm = 40 mV). The curved line corresponds to the best fit of the Hill equation to NPo values. The intracellular membrane surface was bathed with control solution. Data are presented as means ± SE (n = 3).

These results show that the BK channels are sensitive to internal Ca2+ concentration, voltage dependent, and activated by intracellular MgATP.

Contribution of BK and KATP channels to the whole current

To study the contribution of BK and KATP channels to the whole current, we obtained macroscopic current records by using the perforated patch-clamp technique in the voltage-clamp mode. We carried out these experiments under control conditions, blocking the BK currents with TEA or CTX or suppressing the calcium current by adding CdCl2 to the external solution or by replacing Ca2+ with Mg2+. The macroscopic current, obtained in the control solution, exhibited three components: the first one was inward, and the other two were outward. One of the outward current components was transient and the other was sustained. Suppressing the inward current by adding Cd2+ or by removing external Ca2+, the transient outward current was blocked, whereas the sustained component was not modified (Fig. 6, Aa and Ab). Moreover, the addition of 1 mM TEA or 100 nM CTX to the external solution decreased the transient outward current without a significant change in either the inward current or the sustained outward current (Fig. 6, Ac and Ad). The I-V relationship of the outward current obtained in the control solution, of the current in the presence of 0.4 mM of CdCl2, and of the subtraction of the currents obtained with CdCl2 from those obtained in the control solution are shown in Fig. 6B. It is evident that the addition of TEA or CTX suppresses, at least in part, the transient outward currents in the whole cells. This suggests that the outward current blocked by TEA or CTX can be carried through the BK channels. These results, together with those obtained in excised membrane patches (Fig. 3), show that part of the transient potassium currents could depend on Ca2+ influx and membrane potential, whereas the sustained outward current was only voltage dependent. In excised patch membranes the BK channel activity was persistent because [Ca2+]i was constant throughout the entire recording. In contrast, the BK macroscopic currents, obtained in whole cell condition, were transient because calcium currents are transient in themselves.



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Fig. 6. Contribution of the BK channels to the whole cell outward current. A: current records obtained from 4 different axotomized neurons, using the perforated patch-clamp technique (Vm = 0 mV). Holding potential -50 mV. The application of 0.4 mM CdCl2 (a), the substitution of Ca2+ by Mg2+ (b), or the application of 1 mM TEA (c) or 100 nM of charybdotoxin (d) decrease the transient outward current. B: current-voltage relationships of outward current obtained in control solution (), in the presence of 0.4 mM of CdCl2 (open circle ), and the Cd2+-sensitive component ( =  - open circle ).

ATP-sensitive potassium channels (KATP) from X-organ neurons are also modulated by intracellular Ca2+ and membrane potential (Onetti et al. 1996). To determine whether both kinds of channels (BK and KATP) coexist in the same cell, we carried out experiments recording whole currents using blockers of BK and KATP channels. Figure 7A shows recordings obtained from a representative X-organ neuron in the presence of glybenclamide (50 µM), both with and without CdCl2 (0.4 mM) in the perfusion solution. To isolate the current component blocked by glybenclamide or by Cd2+, the currents obtained with glybenclamide were subtracted from those obtained in the control solution; the same procedure was applied to the currents recorded with glybenclamide and Cd2+ and to those obtained in the presence of glybenclamide, alone. Figure 7B shows the I-V relationship of K+ currents obtained from a neuron, in the control solution, in the presence of glybenclamide, glybenclamide plus Cd2+, and the subtractions mentioned above. These results show that BK and KATP channels are distinct entities that could coexist in the same cell of the X-organ. Furthermore, our observations suggest that K+ currents, carried through KATP channels, may contribute to the delayed rectifier in these neurons.



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Fig. 7. BK and KATP channels coexist in the same cells. A: current records obtained from an axotomized neuron using the perforated patch-clamp technique in control solution (a), after the addition of 50 µM glybenclamide (b), and in the presence of glybenclamide (50 µM) plus 0.4 mM CdCl2 (c). Membrane potential values are signaled near of traces. Holding potential -50 mV. B: current-voltage relationships of outward current obtained in control solution (), in presence of 50 µM glybenclamide (), 50 µM glybenclamide plus 0.4 mM CdCl2 (black-down-triangle ), and the subtractions: open circle  =  - ,  =  - black-down-triangle . Outward currents obtained with subtractions correspond to the currents carried by BK () and KATP (open circle ) channels.

How the BK and KATP channels participate in electrophysiological activity

To determine how the BK and KATP channels participate in the activity of XO neurons that discharge action potential bursts, we obtained membrane potential records with the perforated patch-clamp technique in the current-clamp mode. By adding TEA (1 mM) to the perfusion solution, an increase in the duration of action potentials (25 ± 1%, n = 7) and bursts (38 ± 9%, n = 7) was produced. Only a slight increase in the burst frequency (6 ± 2%, n = 7) was observed. The effects on the duration of action potentials and bursts, as well as on the burst frequency produced during the addition of CTX (100 nM), were similar to those produced by TEA addition; they increased 31 ± 12, 9 ± 2, and 10 ± 2% (n = 5), respectively. Figure 8A shows typical membrane potential recordings, at two different time scales, in the control solution and after the addition of CTX. The first action potentials of a burst in the control solution and in the presence of CTX are shown in Fig. 8B. On the other hand, besides a membrane depolarization, the addition of glybenclamide (50 µM) produced an increase of duration (55 ± 10%, n = 5) and frequency (110 ± 15%, n = 5) of the bursts; also, a slight increase in action potential duration (6 ± 1, n = 5) was observed. These effects can be seen in the records shown in Fig. 8 (C and D), obtained from a neuron with burst activity. The increase in the action potential and burst duration and burst frequency produced by the addition of TEA, CTX, or glybenclamide to the bath solution, was significant (P < 0.01).



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Fig. 8. BK and KATP channel participation in the bursting activity of X-organ neurons. A and B: action potential records shown at different time scales obtained from a neuron in control solution and after the addition of 100 nM charybdotoxin (CTX). The first action potentials of 2 bursts, one of these obtained in the control solution and the other in the presence of charybdotoxin (CTX) are shown in B. C and D: action potential records obtained from another neuron in control solution and after the addition of 50 µM glybenclamide (Glyb). The 1st action potentials of 2 bursts, one of these obtained in the control solution and the other in the presence of glybenclamide (Glyb), are shown in D.

In summary, the most noticeable effect on the electrophysiological activity, produced by the blockage of the BK channels (with TEA or CTX), was an increase in the duration of bursts and action potentials. Nevertheless, the most noticeable effect found to block the KATP channels (with glybenclamide), besides that of membrane depolarization, was an increase in the burst duration as well as in the burst frequency.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have characterized the biophysical properties of the BK channels and their contribution to the macroscopic outward current as well as their participation in the electrophysiological activity from secretory neurons of the crayfish by using the patch-clamp technique.

The electrophysiological properties and the calcium sensitivity of the BK channels in XO neurons are similar to those from other BK channels (McManus 1991), particularly the BK channels from rat neurons (Franciolini 1988; Safronov and Vogel 1998). Single-channel kinetics in cell-attached configuration were similar to those observed when the membrane patch was excised from the cell, at 1 or 3 µM Ca2+ concentration; even the I-V relationships of the BK channels were identical in both configurations. This leads one to think that the internal Ca2+ concentration of intact X-organ neurons could fall within this interval (1-3 µM).

Permeation and blocking of the BK channels

The unitary conductance of the BK channels in symmetrical K+ (~220 pS) is within the range reported (180-290 pS) for BK channels in other tissues (McManus 1991). Comparison of theoretical equilibrium potentials with reversal potentials obtained experimentally by changing the K+ gradient (Fig. 2), reveals that BK channels in excised inside-out patches are highly selective for K+ ions. Like BK channels in other tissues (Latorre et al. 1989) these channels display a high K+ permeability (2.3 × 10-13 cm3 s-1); however, under symmetrical K+ gradient, the BK channels in XO neurons do not have a linear conductance, as the slope falls below that of a straight line at high positive voltage (Fig. 1B). For the time being we cannot explain this effect. We speculate that this nonlinearity could be attributed to a rapid voltage-dependent block, perhaps by H+ ions. It could also be produced by the intrinsic permeability properties of the BK channel, or by diffusion-limited ion flow (Yellen 1984). An inward rectification is seen when 40 mM K+ (160 mM Na+) is present in the internal solution; an outward rectification is observed when using quasi-physiological K+ concentrations (Fig. 2); this is consistent with the I-V relationship predicted by the Goldman-Hodgkin-Katz field equation.

As previously reported for other tissues (Anderson et al. 1988; Kehl and Wong 1996; Safronov and Vogel 1998), the current carried by BK channels can be blocked by tetraethylammonium or charybdotoxin applied to the extracellular medium (Figs. 3 and 8A).

Voltage dependence, and Ca2+ and MgATP sensitivity of BK channels

Steady-state activation, as a function of membrane potential for the BK channels described here, was analyzed by fitting the Po/Vm relationship to the Boltzmann equation at different [Ca]i (Fig. 4B). The voltage sensitivity of the BK channels from XO neurons was similar to that reported for neurons (Safronov and Vogel 1998) and other tissues (Albarwani et al. 1994; Kehl and Wong 1996; Latorre et al. 1989; Singer and Walsh 1987). It became evident that these channels are acutely sensitive to voltage (k = 13 mV); also, the half-activation point depends on the internal Ca2+ level (-5, -26, and -44 mV, at 0.3, 1, and 3 µM [Ca]i, respectively). Increasing the Ca2+ concentration produced a significant shift of the activation curve to more negative membrane potentials, even when the sensitivity factor (k) remained unaffected. It is possible that an increase in internal Ca2+ shifts the voltage activation threshold for BK channels without affecting their voltage sensitivity. However, another possibility is that the affinity of BK channels for Ca2+, itself, is modulated by voltage; other authors (Albarwani et al. 1994; Barrett et al. 1982; Moczydlowski and Latorre 1983) have suggested this possibility. The number of Ca2+ binding sites (NCa = 1.3) for the BK channel from XO neurons, estimated using Eq. 3 (Fig. 4C), suggests that the activation is due to binding of only one or two Ca2+ ions to the channel. Similar values for NCa have been reported in rat plasma membrane (Moczydlowski and Latorre 1983), smooth muscle (Benham et al. 1986; Inoue et al. 1985), and rat hippocampus neurons (Franciolini 1988). The dissociation constant at 0 mV [KD(0) = 0.22 µM] for these channels was similar to those obtained in other tissues, such as smooth muscle (Benham et al. 1986; Carl and Sanders 1989; Inoue et al. 1985; Kume et al. 1990) and rat melanotrophs (Kehl and Wong 1996).

In XO neurons, intracellular MgATP activates the BK channels. Using a symmetrical distribution of K+, this activation is observed when 0.01-3 mM of internal MgATP is present (Fig. 5). Similar behavior has been found for the BK channels from the main pulmonary artery of the rat (Albarwani et al. 1994). Some authors have explained this activation as a result of phosphorylation, rendering BK channels more sensitive to Ca2+ ions (Albarwani et al. 1994; Robertson et al. 1992). It is possible that the MgATP activation mechanism plays a physiological role in XO neurons enhancing BK channel activity and therefore producing an increase in the hyperpolarization after a burst of action potentials. It is known that intracellular ATP concentration is strictly related to the availability of energy sources; levels of extracellular glucose can determine internal ATP concentration. In XO neurons, metabolic balance could change in such a manner as to render BK channels more sensitive to Ca2+ due to an increase in intracellular ATP concentration (KD = 119 µM). However, it is not known whether the intracellular ATP concentration falls within the level at which BK channels are most sensitive to Ca2+.

We conclude that the BK channels in XO neurons are voltage dependent, sensitive to internal Ca2+, and activated by intracellular MgATP; it is possible that these factors interact to play an important role in the functional profile of these neurons.

Physiological implication of BK and KATP channel coexistence on electrophysiological activity

It seems reasonable to assume that in spontaneously bursting XO neurons, internal Ca2+ concentration will attain the optimal level necessary to activate both BK and KATP channels. Due to the influx of Ca2+ ions during a spike burst, through voltage-dependent Ca2+ channels, the BK channels can regulate both spike burst and action potential duration (Fig. 8). Similar phenomena have been described in neurons from other species (Crest and Gola 1993; Wang et al. 1998). Depending on the cellular metabolic state, the KATP channels can control not only the resting potential, but both the duration and frequency of bursts. Because BK and KATP channels are distinct entities and could coexist in the same neuron of the X-organ, spike shaping and spontaneous firing pattern regulation would depend on the balance between the level of activity of both channels.


    ACKNOWLEDGMENTS

The authors express their gratitude to Dr. Esperanza García for critical review of the manuscript.

This work has been partially supported by Grants 1913P-N and 29471-N from Consejo Nacional de Ciencia y Tecnología, Secretaría de Educación Pública and Fondo para el Mejoramiento de la Educación Superior: 98-07-01 from Subsecretaría de Educación Superior e Investigación Cientifica, Secretaría de Educación Pública, Mexico.


    FOOTNOTES

Address for reprint requests: C. G. Onetti, Centro de Investigaciones Biomédicas, Universidad de Colima, Apdo. Postal 97, Colima, Col. 28000, Mexico.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5 March 1999; accepted in final form 19 May 1999.


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ABSTRACT
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