omega -AgaIVA-Sensitive (P/Q-type) and -Resistant (R-type) High-Voltage-Activated Ba2+ Currents in Embryonic Cockroach Brain Neurons

Pascal Benquet,1 Janine Le Guen,1 Govindan Dayanithi,2 Yves Pichon,1 and François Tiaho1

 1Groupe de Neurobiologie, Equipe Canaux et Récepteurs Membranaires, UPRES-A Centre National de la Recherche Scientifique, Université de Rennes1, 35042 Rennes Cedex; and  2Centre National de la Recherche Scientifique-UPR, Biologie des Neurones Endocrines, Centre CNRS-INSERM de Pharmacologie-Endocrinologie, 34090 Montpellier Cedex 5, France


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INTRODUCTION
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Benquet, Pascal, Janine Le Guen, Govindan Dayanithi, Yves Pichon, and François Tiaho. omega -AgaIVA-Sensitive (P/Q-type) and -Resistant (R-type) High-Voltage-Activated Ba2+ Currents in Embryonic Cockroach Brain Neurons. J. Neurophysiol. 82: 2284-2293, 1999. By means of the whole cell patch-clamp technique, the biophysical and pharmacological properties of voltage-dependent Ba2+ currents (IBa) were characterized in embryonic cockroach brain neurons in primary culture. IBa was characterized by a threshold of approximately -30 mV, a maximum at ~0 mV, and a reversal potential near +40 mV. Varying the holding potential from -100 to -40 mV did not modify these properties. The steady-state, voltage-dependent activation and inactivation properties of the current were determined by fitting the corresponding curves with the Boltzmann equation and yielded V0.5 of -10 ± 2 (SE) mV and -30 ± 1 mV, respectively. IBa was insensitive to the dihydropyridine (DHP) agonist BayK8644 (1 µM) and antagonist isradipine (10 µM) but was efficiently and reversibly blocked by the phenylalkylamine verapamil in a dose-dependent manner (IC50 = 170 µM). The toxin omega -CgTxGVIA (1 µM) had no significant effect on IBa. Micromolar doses of omega -CmTxMVIIC were needed to reduce the current amplitude significantly, and the effect was slow. At 1 µM, 38% of the peak current was blocked after 1 h. In contrast, IBa was potently and irreversibly blocked by nanomolar concentrations of omega -AgaTxIVA in ~81% of the neurons. Approximately 20% of the current was unaffected after treatment of the neurons with high concentrations of the toxin (0.4-1 µM). The steady-state dose-response relationship was fitted with a Hill equation and yielded an IC50 of 17 nM and a Hill coefficient (n) of 0.6. A better fit was obtained with a combination of two Hill equations corresponding to specific (IC50 = 9 nM; n = 1) and nonspecific (IC50 = 900 nM; n = 1) omega -AgaTxIVA-sensitive components. In the remaining 19% of the neurons, concentrations >= 100 nM omega -AgaTxIVA had no visible effect on IBa. On the basis of these results, it is concluded that embryonic cockroach brain neurons in primary culture express at least two types of voltage-dependent, high-voltage-activated (HVA) calcium channels: a specific omega -AgaTxIVA-sensitive component and DHP-, omega -CgTxGVIA-, and omega -AgaTxIVA-resistant component related respectively to the P/Q- and R-type voltage-dependent calcium channels.


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INTRODUCTION
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In the CNS, voltage-dependent Ca2+ channels (VDCC) are involved in neurotransmission, regulation of cell excitability, and gene transcription. Multiple types of Ca2+ current (ICa) have been distinguished and named T-, L-, N-, P-, Q-, and R-type according to biophysical and pharmacological criteria (for review see Catterall et al. 1995; Dunlap et al. 1995; Olivera et al. 1994; Reuter 1996; Tsien et al. 1995). These currents have been subdivided in low-voltage-activated channels (LVA) for the T-type (Nowycky et al. 1985), mid-low-voltage-activated channel (M-LVA) for the R-type (Dunlap et al. 1995; Ellinor et al. 1993; Soong et al. 1993), and high-voltage-activated (HVA) for the L-, N-, P-, and Q-types (Llinas et al. 1989; Nowycky et al. 1985; Sather et al. 1993).

In the insect CNS, LVA or M-LVA (Baines and Bate 1998; Grolleau and Lapied 1996; Wicher and Penzlin 1997) and HVA calcium channels have been found similar to those in vertebrates. However, HVA calcium channels that were sensitive to phenylalkylamines (PAA) but insensitive to dihydropyridines (DHPs), unlike vertebrate L-type calcium channels, have been identified in several insect neurons, suggesting some difference between vertebrate and invertebrate calcium channels (Bickmeyer et al. 1994a; Pauron et al. 1987; Pearson et al. 1993; Pelzer et al. 1989; Wicher and Penzlin 1994). With the discovery of toxins that are specific blockers of vertebrate non-L-type HVA calcium channel blockers (Llinas et al. 1989; McCleskey et al. 1987; Mintz et al. 1992b), it has been shown that micromolar concentrations of omega -CgTxGVIA selectively block HVA Ca2+ currents in adult cockroach abdominal ganglion dorsal unpaired median (DUM) neurons (Wicher and Penzlin 1994), and that nanomolar concentrations of omega -AgaTxIVA partially block HVA Ca2+ currents in adult locust brain neurosecretory neurons (Bickmeyer et al. 1994b) and M-LVA Ca2+ currents in adult abdominal ganglion cockroach DUM neurons (Wicher and Penzlin 1997). Unfortunately, in all these experiments, the potencies and efficacies of these blockers were not determined by using dose-response curves. These parameters are necessary to assess the specificity of these blockers for VDCCs in insect neurons and to estimate the contribution of each type of channel to the macroscopic current.

Embryonic cockroach brain neurons in primary culture have been shown to express voltage-dependent calcium currents in their soma (Christensen et al. 1988). The pharmacology of the corresponding channels was not studied in detail, and consequently, no information concerning their possible diversity was available. As a first step in understanding their physiological role in developing neurons, we have analyzed the contribution of each type of VDCC to the macroscopic current, by using specific blockers and toxins of vertebrate VDCCs. The results show that embryonic cockroach brain neurons in primary culture expressed VDCC that were specifically and potently blocked by omega -AgaTxIVA, similar to P/Q-like VDCCs in vertebrates. They also expressed DHP-, omega -CgTxGVIA-, and omega -AgaTxIVA-resistant VDCC, reminiscent of the vertebrate R-type. The physiological significance of the currents flowing through these two types of channels is discussed.


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Cell culture

The culture technique was derived from that of Chen and Levi-Montalcini (1970), as described by Beadle and Hicks (1985) and recently modified (Amar et al. 1991; Van Eyseren et al. 1998). Briefly, cells were isolated from 21-23-day-old egg case embryos that were stored in an incubator at 28-29°C in a humid atmosphere. The brains were then removed from the head capsules and transferred into a glass tube, where they were dissociated by gentle mechanical trituration with a Pasteur pipette (no enzyme treatment was necessary) in a defined volume of culture medium. In general, 30 brains were dissociated in 1 ml culture medium and yielded a density of 3 × 104 neurons/cm2 adhering to the bottom of the dish. The cultures were initiated in a medium (5 + 4) containing 5 parts of Schneider's revised Drosophila medium and 4 parts of Eagle's basal medium containing 100 IU/ml penicillin and 100 µg/ml streptomycin complemented with 6 mg/ml L-glutamine and 2.5 µg/ml fungizone. After 5 days, the first (5 + 4) culture medium was replaced by a second (L + G) medium made up of equal parts of Leibovitz's L-15 medium and Yunker's modified Grace medium containing penicillin, streptomycin, glutamine, and fungizone, supplemented with 10% fetal calf serum. This second medium was renewed every week. All culture media were obtained from Gibco (Cergy Pontoise, France).

Electrophysiology

Ca2+ currents of the neurons were studied using the whole cell configuration of the patch-clamp technique (Hamill et al. 1981). Before the experiments, the culture medium was replaced by a solution containing (in mM) 100 TEACl, 70 Tris-HCl, 10 4-AP, 10 BaCl2, 4 MgCl2, and 10 HEPES buffer adjusted to pH 7.2 with TEAOH. This experimental extracellular solution was designed to eliminate any contaminating voltage-dependent sodium current (INa+) and to markedly reduce the voltage-dependent potassium current (IK+). In addition, Ba2+ ions were used instead of Ca2+ in this solution to avoid the run-down of the current (ICa) through voltage-activated calcium channels (see also Wicher et Penzlin 1997). The patch electrodes were made of borosilicate 1.5-mm glass (Clark Electromedical) with a Flaming-Brown micropipette puller (Sutter Instruments). They were filled with a solution containing (in mM) 120 CsF, 25 CsOH, 2 MgCl2, 10 EGTA, 3 ATP-Mg2+, 0.5 guanosine 5'-triphosphate-Tris, and 10 HEPES buffer adjusted to pH 7.3, using CsOH, and their resistance ranged from 2 to 5 MOmega . Voltage-clamp experiments were performed with the patch-clamp amplifier RK300 (Biologic Science Instruments, Claix, France) at a holding potential (HP) of -70 mV, and all experiments were performed at room temperature (20-27°C). In some experiments, a ramp protocol was used to quickly assess the I-V relationship of IBa. In this protocol, the membrane potential was transiently and linearly varied from -100 to +50 mV for 500 ms.

Because the neurons studied (3-15 days in culture) had processes, the quality of the whole cell voltage clamp depended on the location of the calcium channels. Study of the transient current elicited by a hyperpolarizing voltage step (from -60 to -70 mV) provided information relevant to the quality of the voltage clamp (see Byerly and Leung 1988). In general, these transients varied progressively from a single exponential time constant [iota 1 = 0.3 ± 0.1 (SE) ms] for neurons that had been <5 days in culture) to a complicated time course that could be approximated by two exponential components (iota 1 = 0.3 ± 0.1 ms; iota 2 = 2 ± 1 ms) for older neurons. In the present experiments, we selected the neurons in which the amplitude of the slow exponential component, when present, was <25% of that of the fast component. Under these conditions, the control of the membrane potential was fast enough to enable an adequate recording of the currents. These neurons had an average input resistance of 2.2 ± 0.8 GOmega , and the estimated voltage error due to uncompensated series resistance was <5 mV in all the neurons used for the analysis (see also Tiaho et al. 1991).

Data analysis

The pClamp 5.5 program (Axon Instruments) was used for stimulation, data acquisition, and analysis. Furthermore, data were analyzed off-line using different software packages: Excel (Microsoft), Freelance Graphics (Lotus), and Sigmaplot (Jandel Scientific). Student's t-test was used for statistical analysis. The peak current-to-voltage relationship (I-V curve) was fitted with the following Boltzmann equation:
<IT>I</IT><IT>=</IT><IT>G</IT><SUB><IT>max</IT></SUB><IT> ∗ </IT>(<IT>V</IT><IT>−</IT><IT>E</IT><SUB><IT>Ba</IT></SUB>)<IT>&cjs0823;  {1+exp</IT>[(<IT>V</IT><SUB><IT>0.5</IT></SUB><IT>−</IT><IT>V</IT>)<IT>&cjs0823;  </IT><IT>K</IT>]<IT>}</IT> (1)
where Gmax is the maximal conductance of the global calcium channels, EBa is the reversal potential of IBa estimated by the curve-fitting program, V0.5 is the potential for half-maximal steady-state activation of the barium current, and K is a voltage-dependent slope factor. Steady-state activation curves were fitted with the following Boltzmann equation:
<IT>I</IT><IT>&cjs0823;  </IT><IT>I</IT><SUB><IT>max</IT></SUB><IT>=100&cjs0823;  {1+exp</IT>[(<IT>V</IT><SUB><IT>0.5</IT></SUB><IT>−</IT><IT>V</IT>)<IT>&cjs0823;  </IT><IT>K</IT>]<IT>}</IT> (2)
where I is the peak amplitude of IBa tail current on repolarization to -100 mV, Imax is the maximum IBa tail current, and V0.5 and K have the same meaning as in Eq. 1.

Steady-state inactivation curves were fitted with the following Boltzmann equation:
<IT>I</IT><IT>&cjs0823;  </IT><IT>I</IT><SUB><IT>max</IT></SUB><IT>=100&cjs0823;  {1+exp</IT>[(<IT>V</IT><IT>−</IT><IT>V</IT><SUB><IT>0.5</IT></SUB>)<IT>&cjs0823;  </IT><IT>K</IT>]<IT>}</IT> (3)
where I is the peak amplitude of IBa for a 0- or +10-mV test pulse after a conditioning prepulse of varying amplitude, Imax is the maximum IBa peak amplitude at a 0- or +10-mV test pulse after a conditioning prepulse, and V0.5 and K have the same meaning as in Eqs. 1 and 2.

To quantify the effect of calcium channel blockers, dose-response curve were fitted with a Hill equation:
<IT>I</IT><IT>&cjs0823;  </IT><IT>I</IT><SUB><IT>max</IT></SUB><IT>=100 ∗ </IT>[<IT>X</IT>]<SUP><IT>n</IT></SUP><IT>&cjs0823;  </IT>(<IT>IC</IT><SUP><IT>n</IT></SUP><SUB><IT>50</IT></SUB><IT>+</IT>[<IT>X</IT>]<SUP><IT>n</IT></SUP>) (4)
where I is the peak amplitude of IBa at 0- or +10-mV test pulse from an HP of -70 mV in the absence (Imax) and presence of varying concentrations ([X]) of blockers, and n is the Hill coefficient.

Drugs

Verapamil, amiloride, isradipine, nifedipine, and Bay K 8644 were purchased from Sigma (L'Isle d'Abeau Chesnes, France). Verapamil solutions were prepared immediately before use. Amiloride, isradipine, nifedipine, and Bay K 8644, were dissolved in DMSO to obtain a stock solution that was further diluted. The final test concentrations contained <= 0.1% DMSO. At this concentration the solvent had no significant effect on IBa. omega -CgTxGVIA, omega -CmTxMVIIC, and omega -AgaTxIVA (Neurex) were dissolved in distilled water at 10-4-10-3 M and stored at -70°C.


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Voltage-dependent activation of Ba2+ currents

We used extracellular and intracellular media defined to minimize the contribution of contaminating K+ outward and Na+ inward currents (see METHODS). Voltage-clamp pulses were applied from an HP of -70 mV to various depolarized levels in the whole cell configuration of the patch-clamp technique. As a result, net inward IBa at potentials between -30 mV and +40 mV was observed. After 2 days in culture, all neurons produced a detectable inward current. Typical recordings of IBa are shown in Fig. 1A. In this neuron, IBa was activated at -30 mV. Larger depolarizing pulses of <= 0 mV progressively increased IBa peak amplitude, and then further depolarizations beyond this potential decreased IBa peak amplitude. The peak current reversed at membrane potential more positive than +50 mV. These inward currents were completely blocked by millimolar concentrations of cadmium, nickel, and cobalt (data not shown) and had characteristics similar to those previously described by Christensen et al. (1988) in the same neurons. After a complete block of IBa by cadmium, nickel, and cobalt, a tiny time-independent, outwardly rectifying current reversing at -10 mV was generally observed (data not shown). Because this reversal potential does not correspond to that of any permeating ion, it was considered nonspecific. This current component can be seen in Fig. 1A for the depolarization to +50 mV, in which the current is inward at the peak and outward at the end of the pulse. A contamination of IBa with this nonspecific current could be detected at potentials more positive than +10 mV, but in general its average amplitude was small (<30% of IBa peak amplitude at +50 mV). The experiments presented here were performed at potential values at which this nonspecific current was negligible, the current traces were therefore not corrected for this contaminating component.



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Fig. 1. Time course and peak current-voltage (I-V) relationship of barium currents (IBa) recorded with 10 mM Ba2+ in the bathing medium from the soma of an embryonic cockroach brain neuron in primary culture. A: illustration of a typical family of IBa obtained by voltage pulses ranging from -30 to +50 mV from a holding potential (HP) of -70 mV. Horizontal dotted line indicates the 0-current level. No leak and capacitance subtraction was performed. Number near each trace represents the potential at which the current was obtained. Only voltage pulses showing a net inward current are shown. Note the voltage-clamp protocol used above the current traces. B: average I-V relationship of peak IBa currents recorded as indicated in A from 66 neurons. Peak IBa were measured relative to the zero-current level. Filled circles indicate measured peak IBa; Bars: SE. The smooth curve represents the fit of the data with Boltzmann Eq. 1, where G is 3 ± 1 nS, Erev is +47 ± 1 mV, V0.5 is -10 ± 1 mV, and K is 6 ± 1.

The average peak amplitude of IBa for 66 neurons is plotted against the membrane potential in Fig. 1B. The I-V curve was fitted with the Boltzmann Eq. 1 (see METHODS), assuming that the time-dependent inactivation of the current was negligible at the peak and that the current was flowing through a homogeneous population of calcium channels with the same steady-state voltage-dependent activation properties.

HVA and LVA calcium currents have been distinguished according to their voltage-dependent activation properties both in vertebrate and invertebrate neurons (Nowycky et al. 1985, and for review see Bean 1989; Hess 1990). They also differ by their inactivation properties: depolarized HP preferentially inactivate LVA currents, and their time-dependent inactivation kinetics are faster than those of HVA currents.

The activation of the current was therefore studied for three different HPs: -100 mV, -70 mV, and -40 mV. As illustrated in Fig. 2A, the currents were the same for -100- and -70-mV HPs. Furthermore, as illustrated in the left panel, no detectable current was seen for a depolarization from -100 mV or -70 mV to -40 mV, ruling out the existence of a low-voltage-activated current component that may have been hidden at -70 mV. For an HP of -40 mV, the current was significantly inactivated, and the amount varied from neuron to neuron by about one third to two thirds of the maximum peak current. The average peak amplitude of IBa for the three HPs versus the command potential for at least eight neurons is illustrated in Fig. 2B. Apart from the scaling factor, the I-V curves could also be fitted with the described Boltzmann activation equation with the same parameters (see legend of Fig. 2).



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Fig. 2. Effects of different HPs on the activating properties of IBa. A: typical current traces obtained with -40-, -10-, and +10-mV pulse potentials lasting 50 ms from three different HPs. Pulse potentials and HPs are indicated on the protocol above the current traces. IBa is superimposed at HPs of -100 and -70 mV. This 50-ms pulse did not reveal a significant fast-inactivation current component. B: average peak amplitude I-V curve obtained from different HPs (HP = -100, n = 8 neurons; HP = -70, n = 11 neurons; HP = -40, n = 10 neurons). Open circles, filled circle, and filled triangle are the measured data points obtained from HP of -100 mV, -70 mV and -40 mV, respectively. Bars: ±SE. The threshold of IBa is more positive than -40 mV and is not modified by the HP. Smooth curve represents the fit of the data obtained at each HP with the Boltzman equation (see legend to Fig. 1). The parameters for the equation were as follows: for HP = -100 mV, G is 8 ± 2 nS, Erev is 44 ± 4 mV, V0.5 is -10 ± 2 mV, and K is 7 ± 1; for HP = -70 mV, G is 7 ± 1 nS, Erev is 44 ± 2 mV, V0.5 is -10 ± 1 mV, and K is 7 ± 1; and for HP = -40 mV, G is 3 ± 1 nS, Erev is 44 ± 6 mV, V0.5 is -10 ± 3 mV, and K is 7 ± 2.

For suprathreshold potentials, IBa activation was fast (that is, in the ms range) and the time to peak decreased with increasing depolarizations. For the cell illustrated in Fig. 3A the times to peak were 6.8 ms at -20 mV, 5.7 ms at 0 mV, 4.4 ms at +20 mV, and 3.2 ms at +30 mV, indicating that activation kinetics were voltage dependent.



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Fig. 3. Steady-state, voltage-dependent activation and inactivation properties of Ba2+ currents. A: activation. Typical current traces of IBa evoked by a 9-ms test pulse from an HP of -70 mV to varying depolarization levels (from -40 mV to +20 mV in 20-mV increments) followed by a repolarization to -100 mV. Number near each trace represents the potential at which the current was obtained. Voltage-clamp protocol is indicated above the current traces. Tail currents settled within 3 ms. B: inactivation. Typical current traces of IBa evoked by an 800-ms test pulse to +10 mV. The test pulse was preceded by a 7-s prepulse ranging from -100 to -10 mV in 10-mV increments. Only the last 1 s of the prepulse current is shown. Numbers near each trace represent the conditioning prepulse potential. The voltage-clamp protocol is indicated above the current traces. Note that for conditioning prepulses between -100 and -50 mV, current traces were superimposed. C: steady-state activation and inactivation curves. Activation (open circle ): normalized tail currents from 10 neurons were plotted against the pulse potentials, and the data points (mean ± SE) fitted using Boltzmann Eq. 2 yielded V0.5 = -10 ± 2 mV and slope K = 9 ± 1. Inactivation (): normalized peak currents at +10 mV test potential from 20 neurons were plotted against the conditioning prepulse potentials, and the data points fitted with Eq. 3 yielded V0.5 = -30 ± 1 mV and slope K = 15 ± 1.

The steady-state activation properties of IBa were studied using short-duration pulses that were adequate to fully activate IBa and minimize time-dependent inactivation (Fig. 3A). Tail currents observed on hyperpolarizing the membrane to -100 mV (Fig. 3A) were used to estimate the steady-state conductance of the calcium channels (Fig. 3C). The mean potential of half-maximum activation of the conductance (-10 ± 2mV) was similar to that found for the peak conductance (-10 ± 1 mV, see legend to Fig. 1B). The small discrepancy found in the slope factor (K = 9 ± 1 for the steady-state activation curve against K = 7 ± 1 for the I-V curve) is within the limit of the values reported by different investigators (for review see Pelzer et al. 1990).

Voltage-dependent inactivation of IBa

We studied the steady-state, voltage-dependent inactivation properties of IBa using 7-s prepulses as illustrated in Fig. 3B. This duration was chosen because in most neurons complete time-dependent inactivation of IBa needed >1 second (see Fig. 1A). A typical illustration of the steady-state inactivation of IBa is illustrated in Fig. 3B. After conditioning prepulses of increasing positivity, the peak amplitude of the current evoked by a pulse to +10 mV progressively decreased and was almost completely suppressed for a -10 mV prepulse potential. The average peak amplitude was plotted against the membrane prepulse potential (Fig. 3C). The fit of the data with the Boltzmann Eq. 3 yielded a V0.5 of -30 ± 1 mV and a slope factor of K = 15 ± 1.

Pharmacological properties of IBa

LVA ICa (or IBa) that was sensitive to amiloride have been recorded in cockroach abdominal ganglionic neurosecretory DUM neurons (Grolleau and Lapied 1996), Drosophila CNS embryonic neurons (Baines and Bate 1998), and Drosophila larval muscle (Gielow et al. 1995). To determine whether an equivalent LVA IBa was absent from our preparation, we tested the effect of bath superfusion of the neurons with 1 mM amiloride for >= 2 min. The current was not significantly reduced (90 ± 17%, n = 6 neurons, of the control peak current; data not shown). In some preparations there appeared to be a gradual decrease in the peak amplitude of IBa. This, however, turned out to be statistically insignificant. The absence of effect of amiloride could not be attributed to the slow perfusion rate, because other pharmacological agents acted rapidly (for example, see effects of verapamil described later). We therefore concluded from the voltage-dependent activation and pharmacological properties that IBa was an HVA calcium channel current.

To identify the type of channel carrying this HVA IBa, we used agents that selectively block individual classes of both vertebrate and invertebrate VDCC.

HVA L-type currents are characterized by their selective sensitivity to DHPs and PAAs (for review see Bean 1989; Hess 1990). After a >= 2-min superfusion of the neurons with the DHP agonist Bay K 8644 (1 µM, n = 7 neurons) and the antagonist isradipine (10 µM, n = 7 neurons), the peak amplitude of IBa was not significantly changed. In the presence of the DHPs the peak amplitudes of IBa were 88 ± 6% (n = 7 neurons) and 91 ± 15% (n = 7 neurons) of the control values with Bay K 8644 and isradipine, respectively (data not shown). However, larger DHP concentrations (100 µM nifedipine) were found to partially block IBa (data not shown).

HVA calcium currents of several insect neurons exhibit a component that is blocked by PAA but is insensitive to DHP (Bickmeyer et al. 1994a; Pearson et al. 1993; Pelzer et al. 1989; Wicher and Penzlin 1997). Superfusion of neurons with the PAA verapamil in the bath solution at concentrations <10 µM had no significant effect on the peak amplitude of IBa. A block was seen at concentrations starting at 10 µM (Fig. 4B). For concentrations that were just suprathreshold (50 µM), the reduction of the current at the end of an 800-ms test pulse was often larger than that observed at the peak amplitude of the current (Fig. 4, A and C; see also Wicher and Penzlin 1997: Fig. 5A). This effect is reminiscent of the open-channel-blocking properties of the PAAs (for review see Hondeghem and Katzung 1984). The effect of verapamil on IBa peak amplitude was dose-dependent (Fig. 4B) and could be fitted using the Hill equation (see METHODS). This fit yielded an IC50 of 170 µM and a Hill coefficient of 0.96. A near complete block was achieved at millimolar concentrations. At all tested concentrations, a steady-state block was achieved within 40 s, and the effect of verapamil was at least partially reversed by washing. At our usual HP (-70 mV) the blocking effect of verapamil was independent of the test membrane potential (Fig. 4D). That IBa was insensitive to DHPs and weakly sensitive to PAA suggests that it could be different from an L-type IBa.



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Fig. 4. Effects of verapamil on IBa. A: typical current traces showing the concentration-dependent reduction of IBa during bath perfusion of increasing verapamil concentrations, as indicated. Currents were elicited by 800-ms pulses from -70 mV (HP) to +5 mV. For 50 µM verapamil, the current at the end of the 800-ms test pulse is more reduced than the peak current. B: dose-response curve representing the percentage of inhibition of the peak current at different verapamil concentrations. Filled circles represent the mean reduction obtained for 10, 50, 100, 500, and 1000 µM verapamil and from 12, 6, 4, 7, and 4 neurons, respectively. Bars: ±SE. Smooth line represents the fit of the data points with the Hill equation; IC50 = 170 µM, n = 0.96. C: kinetics of reduction of IBa at the peak () and at the end of the 800-ms pulse (open circle ) during superfusion with verapamil. Horizontal bars indicate the perfusion period of the drug. Number above each bar represents the applied concentration. The effect was rapid and was quickly but only partially reversible. The initial run-up (first 3-4 min) followed by a slow run-down of the current which was observed in most experiments can also be seen. D: average percentage of block obtained at different membrane potentials with 100 µM (, n = 5 neurons) and 500 µM (black-square, n = 5 neurons) verapamil. Bars: ±SE. The current was generated using a ramp protocol from -100 to +50 mV and lasting 500 ms. HP = -70 mV.



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Fig. 5. Effect of different toxins on IBa. A: current traces illustrating the effect of 1 µM omega -CgTxGVIA (GVIA) plus 1 µM omega -CmTxMVIIC (MVIIC) and omega -AgaTxIVA (AgaIVA) on IBa after superfusion of one neuron. The neuron was first superfused with the combination of 1 µM omega -CgTxGVIA plus 1 µM omega -CmTxMVIIC for 2 min and then with 200 nM omega -AgaTxIVA for an additional 2 min. The voltage-clamp protocol used to elicit IBa is shown above the current traces. Because the effect of omega -AgaTxIVA was slow, it was important in these experiments to check the quality of the seal, accomplished by use of a short prepulse to -100 mV, as shown in the top trace (protocol). The slight reduction of IBa seen in the presence of both toxins omega -CgTxGVIA and omega -CmTxMVIIC was not significant and may represent a run-down of IBa seen in this time scale. B: illustration of the time-dependent evolution of IBa peak amplitude before, during simultaneous superfusion of 1 µM omega -CgTxGVIA and 1 µM omega -CmTxMVIIC (GVIA + MVIIC), and during superfusion of 200 nM omega -AgaTxIVA (AgaIVA). The neuron used for this illustration is different from the one used in A. The voltage-clamp protocol was the same as in A. Arrow indicates the time at which the neuron was depolarized repetitively at 1 Hz (ten 30-ms pulses to +150 mV), to try to reverse the effect of omega -AgaTxIVA. Note the initial run-up of IBa. C: residual peak amplitude of IBa normalized to control values after pretreatment of the neurons for >= 10 min with the following toxins: 1 µM omega -CgTxGVIA (GVIA), 1 µM omega -CmTxMVIIC (MVIIC), and 400 nM omega -AgaTxIVA (AgaIVA). D: dose-response curve representing the percentage of inhibition of IBa peak current in the presence of increasing concentration of omega -AgaTxIVA. Neurons were incubated with the toxin for >= 10 min, and the extent of the block was determined compared with control neurons of the same culture. Filled circles represent the average peak current obtained from a set of 5-10 treated neurons. Bars: ±SE. Smooth dashed line represents the fit of the data points with a single Hill equation: IC50 = 17 nM and Hill coefficient, n = 0.6. Smooth straight line represents the fit of data points with the a combination of two Hill equations with n = 1: IC50 = 9 nM and 900 nM, representing, respectively, 80 and 20% of the total IBa.

In vertebrate neurons the toxins omega -CgTxGVIA, omega -CmTxMVIIC, and omega -AgaTxIVA are specific blockers of N-, Q-, and P- type currents, respectively. These toxins were tested using two protocols.

Bath perfusion (superfusion protocol) of 1 µM omega -CgTxGVIA or 1 µM omega -CmTxMVIIC for <= 5 min left the peak amplitude of IBa virtually unchanged (not shown). This was confirmed by coapplication to the same neuron of 1 µM omega -CgTxGVIA and 1 µM omega -CmTxMVIIC (n = 8 neurons; Fig. 5, A and B). However, subsequent superfusion of the same neurons with 200 nM omega -AgaTxIVA resulted in a significant reduction of IBa peak amplitude after 1 min (Fig. 5, A and B). For a population of 26 neurons, we found that concentration of omega -AgaTxIVA >= 100 nM, had no detectable effect on 2 neurons, a hardly detectable effect on 3, an effect of between 25 and 80% on 8, and an effect >80% on 13. This result illustrates some heterogeneity of the neurons regarding their sensitivity to the toxin with a clear continuum between neurons that are barely (19%, n = 5 neurons) and neurons that were highly (50%, n = 13 neurons) sensitive to the toxin. Considering the neurons with clear sensitivity to the toxin, the average steady-state reduction of IBa peak amplitude for concentrations of omega -AgaTxIVA >= 100 nM was 74 ± 4% (n = 21 neurons). The effect of omega -AgaTxIVA was not reversed after a >= 5-min washing (data not shown) and was not reversed by depolarizing prepulses (Fig. 5B) in contrast with the observation of Mintz et al. (1992a) in rat neurons. The persistence of a residual current in high concentrations of omega -AgaTxIVA (>= 100 nM) suggests the presence in these neurons of an omega -AgaTxIVA-resistant current component.

To reduce the amount of toxin used for the experiment and avoid the run-down of IBa, we used a second protocol. In this (incubation) protocol, the neurons were incubated with the toxins before any recording for >= 10 min. At that time we assumed that the effect of the toxins had reached a steady state, at least in large concentrations. The peak amplitude of IBa was measured for 5-10 neurons per concentration and per culture dish. This protocol had the advantage of reducing the number of culture dishes needed per experiment but had the minor drawback of increasing the values of the SE (Fig. 5, C and D). The effect of the toxins was estimated from a comparison of maximum peak amplitudes of IBa (obtained from the I-V curves) of treated and untreated (control) neurons of the same age originating from the same culture. For the experiments illustrated in Fig. 5C, the average maximum amplitude of the current in 1 µM omega -CgTxGVIA was -195 ± 22 pA (n = 6 neurons) compared with -214 ± 52 pA (n = 6) in control neurons, the average maximum amplitude of the current in 1 µM omega -CmTxMVIIC was -192 ± 30 pA (n = 19 neurons) compared with -158 ± 17 pA (n = 24 neurons) in control neurons, and the average maximum amplitude of the current in 400 nM omega -AgaTxIVA was -15 ± 8 pA (n = 7 neurons) compared with -149 ± 40 pA (n = 5 neurons) in control neurons. In the first two cases, the difference between the treated and untreated neurons was not statistically significant (P = 0.4 for 1 µM omega -CgTxGVIA and P = 0.08 for 1 µM omega -CmTxMVIIC). On the contrary, the blocking effect of 400 nM omega -Aga-IVA (81 ± 5%, n = 7 neurons) was highly significant (P = 0.002).

These results are in perfect agreement with those obtained by superfusion: IBa is insensitive to micromolar concentrations of omega -CgTxGVIA and omega -CmTxMVIIC, and part of the current is highly sensitive to omega -AgaTxIVA. Furthermore, these experiments showed that most neurons exhibited two current components of IBa: an omega -Aga-IVA-sensitive component that represented ~80% of the macroscopic current and a DHP-, omega -CgTxGVIA-, and omega -AgaTxIVA-resistant current component representing ~20% of the total current.

The incubation protocol was used to study the dose-response relationship for omega -AgaTxIVA. As illustrated in Fig. 5D, the effect of this toxin was dose-dependent. The parameters of the fit of this curve with the Hill equation were, respectively, 17 nM for the IC50 and 0.6 for the Hill coefficient n (Fig. 5D, dashed line). This low value of the Hill coefficient in addition to the fact that the block was incomplete at high toxin concentrations suggests that the overall response was made of the combination of at least two populations of calcium channels with clearly different affinities for the toxin. This hypothesis was tested, and it was found that the data points could be equally well fitted with the combination of two Hill equations with a Hill coefficient of 1 and an IC50 of, respectively, 9 nM and 900 nM, corresponding to high-affinity and low-affinity components (Fig. 5D, straight line). The low-affinity omega -AgaTxIVA component obtained from the fit represented 20% of IBa and could be suggestive of its nonspecific effect on the omega -AgaTxIVA-resistant component at higher concentrations.

In the next series of experiments, we used the incubation protocol to compare the time course and the voltage-dependent activation properties of the barium current before and after a 10-min exposure to 400 nM omega -Aga-IVA, which completely blocks P/Q-type calcium currents in vertebrate neurons (Mintz et al.1992a; Zhang et al. 1993). We found that the kinetics of the current for test-potential values ranging from -40 to +40 mV were apparently not different between treated and untreated neurons (data not shown) and that the current-voltage relationship of the residual peak IBa was not altered by the toxin (Fig. 6C). Similar results were obtained using the superfusion protocol in conditions under which the residual currents were larger and could therefore be analyzed with more precision. The kinetics were apparently not altered, as illustrated in Fig. 6A, for a depolarization to +5 mV. The omega -AgaTxIVA-sensitive component had the same inactivation kinetics as the current recorded before the superfusion. The I-V curve was not significantly modified by omega -AgaTxIVA superfusion, as illustrated in Fig. 6B, and the percentage of block was found to be insensitive to membrane potential between -30 and +20 mV (data not shown). We concluded from these experiments that the IBa of embryonic cockroach brain neurons possesses omega -AgaTxIVA-sensitive and -resistant components bearing similar voltage-dependent properties. The distribution of the two components varied from neuron to neuron.



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Fig. 6. Time course and voltage-dependent properties of omega -AgaTxIVA-sensitive and -resistant current components of IBa. A: time course of omega -AgaTxIVA-sensitive and -resistant current components of IBa. Typical current traces illustrating the effect of 100 nM omega -AgaTxIVA on IBa during an 800-ms test pulse from an HP of -70 mV to a membrane potential of +5mV before and after superfusion of omega AgaIVA (a). omega -AgaIVA-sensitive current is deduced by subtraction (b). Note that the inactivation kinetic of IBa was not significantly modified by the toxin. B: I-V relationship of IBa in control conditions and after superfusion of 100 nM omega -AgaTxIVA on the same neuron using a ramp from -100 to +50 mV and lasting 500 ms. HP = -70 mV. C: comparison of I-V relationship of IBa peak amplitude in control neurons without () and with 400 nM omega -AgaTxIVA (black-triangle). omega -AgaTxIVA was preincubated for >= 10 min in the bathing medium. The smooth lines represent the fit of the data points with the Boltzmann equation. The parameters of this equation were: in control conditions, (n = 7) G = 4.4 ± 1 nS; Erev = 52.5 ± 2 mV; V0.5 = -3.8 ± 2 mV; K = 6.3 ± 1. A good fit of the data in the presence of omega -AgaTxIVA (n = 7) was obtained with the same parameters by reducing the value of G from 4.4 to 0.8 nS.

It is well known that, in vertebrate CNS neurons, the omega -AgaTxIVA-sensitive current components are also sensitive to omega -CmTxMVIIC. The effect of this latter toxin could need an incubation time of >= 30 min to reach steady state (Sabatier et al. 1997), and therefore the absence of effect seen with 1 µM of omega -CmTxMVIIC could reflect an insufficient incubation time. To verify this possibility, we decided to probe the effects of the application of the usual 1-µM concentrations of omega -CmTxMVIIC but for a much longer period (1-h incubation). Under these conditions, the mean peak amplitude of the current was reduced from 204 ± 37 pA (n = 6 neurons) for untreated neurons to 126 ± 19 pA (n = 8 neurons), a 38% reduction (data not shown). The blocking effect of omega -CmTxMVIIC was not accompanied by a significant modification of the activation and inactivation kinetics or of the peak current-voltage relationship (data not shown).


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Our patch-clamp study of the barium currents provide new and interesting results concerning the biophysical and pharmacological properties of the voltage-dependent calcium channels of embryonic cockroach brain neurons. The main findings can be summarized as follows.

The voltage-dependent activation and inactivation properties of IBa in these neurons were typical of HVA calcium currents seen in other preparations. Their pharmacological properties, which were studied quantitatively using both fast-bath superfusion and incubation techniques, were original in several respects. The current was not significantly affected by DHP agonists or antagonists but was efficiently and reversibly reduced by verapamil, a PAA.

The toxin omega -CgTxGVIA had no significant effect, micromolar concentrations of omega -CmTxMVIIC partly reduced the peak amplitude of the current after a 1-h incubation, and nanomolar concentrations of omega -AgaTxIVA irreversibly blocked the major part of the current in the majority (81%) of the tested neurons (in which 400 nM omega -AgaTxIVA reduced the current by 80%). A small proportion of the neurons (19%) were significantly less sensitive to that toxin (their barium current was unaffected by 100 nM toxin).

Biophysical properties: HVA versus LVA currents

Our experiments clearly demonstrate that, in embryonic cockroach neurons, the macroscopic barium current activates at potential values more positive than -50 mV, in agreement with previous observations on the same preparation (Christensen et al. 1988) and on adult cockroach brain neurons (Amar and Pichon 1992). Changes in the HP between -100 and -60 mV failed to modify this threshold or the time course of the current and did not shift the I-V relationship. Furthermore, amiloride which has been shown to inhibit LVA currents in other insect preparations (Baines and Bate 1998; Gielow et al. 1995; Grolleau and Lapied 1996) did not modify the current. Conversely, agents that are known to modify HVA currents in other preparations, namely omega -AgaTxIVA and verapamil, blocked the current in embryonic cockroach brain neurons. Altogether, these results strongly suggest that embryonic cockroach brain neurons in primary culture express only HVA calcium channels. They differ in that respect from the neurosecretory DUM neurons of this same species which also exhibit one or two LVA/M-LVA-activated components (Grolleau and Lapied 1996; Wicher and Penzlin 1997) that are likely to play an important role in the generation of their pacemaker activity. They also differ from developing neurons from Drosophila embryos (Baines and Bate 1998). The origin of this discrepancy remains speculative. It is tempting, however, to suggest that it may be because our cultures were grown in high-potassium media (see METHODS) in which the cells were likely to have a low resting potential (between -40 and -20 mV, Lees et al. 1985) and that at these potential levels, which are known to inactivate LVA currents (Baines and Bate 1998; Grolleau and Lapied 1996; Wicher and Penzlin 1997), the expression of LVA currents is repressed.

Sensitivity to DHPs, PAAs, and omega -CgTxGVIA

Our observation that the barium current was not significantly affected by micromolar concentrations of the DHP isradipine (10 µM) or Bay K 8644 (1 µM) but was blocked by the PAA verapamil is consistent with earlier observations on insect neurons (Bickmeyer et al. 1994a; Pearson et al. 1993; Pelzer et al. 1989; Wicher and Penzlin 1997). In this respect, insect HVA calcium channels differ from their vertebrate L-type counterparts, which are sensitive to both DHP and PAA. As previously shown by Mills and Pitman (1997), larger DHP concentrations (100 µM) block IBa, but this effect is considered nonspecific. That a relatively large concentration of verapamil was needed (IC50 of 170 µM) to block the current is consistent with the observations of Byerly and Leung (1988) on Drosophila embryonic neurons and suggests that the very-high-affinity PAA-binding sites detected by Pauron et al. (1987) in Drosophila head membranes (Kd = 4.7 pM for [3H]-verapamil) may not correspond to the channel-blocking sites in native membranes (see also Pelzer et al. 1989). Low-affinity blocking sites have also been observed in some vertebrate preparations in which non-L-type HVA calcium currents have been blocked by supramicromolar concentrations of verapamil (Diochot et al. 1995; Ishibashi et al. 1995).

The insensitivity of the embryonic cultured neurons to omega -CgTxGVIA, a selective blocker of the neuronal vertebrate N-type calcium channels (McCleskey et al. 1987) that also blocks the HVA component of the calcium current in insect adult DUM neurons (Grolleau and Lapied 1996; Wicher and Penzlin 1997), is an interesting feature of our preparation. These differences among various neuronal populations in the same species could result from cell-specific differential expression of the channels, which ought to be studied in more detail. It could also reflect the growth conditions in vitro or a difference between embryonic and adult neurons.

Sensitivity to omega -AgaTxIVA: evidence for two populations of HVA calcium channels

The experiments clearly indicate that most neurons (81%) were sensitive to omega -AgaTxIVA but that a fraction of the current (varying between 10 and 75%, mean = 25%) in these neurons was resistant to the toxin. Furthermore, a significant proportion of the neurons (19%) was much less sensitive to the toxin. These results strongly suggest that the total current was associated with the opening of two different populations of HVA calcium channels, the proportion of which varied from neuron to neuron. The first (omega -AgaTxIVA-sensitive) would be of the P/Q type (i.e., resistant to DHP and omega -CgTxGVIA), the second of the R type (i.e., resistant to all). Interestingly, the dose-response curve of the omega -AgaTxIVA effect could be fitted with a combination of two Hill equations assuming a low (IC50 = 9 nM) and high affinity (IC50 = 900 nM) for the toxin. The low-affinity binding site would correspond to the nonspecific effect of omega -AgaTxIVA on the R-type calcium channels when higher concentrations of the toxin were used (>= 100 nM).

Here again, our results differ in several respects from those reported for other insect preparations. Thus, whereas an omega -AgaTxVIA-sensitive component has also been observed in adult locust neurosecretory cells (Bickmeyer et al. 1994b), this component was present in only 30% of the neurons (compared with 81% in our experiments) and, importantly, the blocking effects were reversed by a brief train of strong depolarizations (as shown earlier by Mintz et al. 1992a in rat central and peripheral neurons). omega -AgaTxIVA also blocked part of the barium current in cockroach DUM neurons, but omega -AgaTxIVA was less potent than omega -CmTxMVIIC, its effects were reversed after washing out the toxins, and the fraction of the current affected by omega -AgaTxIVA had the activation and inactivation properties of an M-LVA current (Wicher and Penzlin 1997).

The pharmacological properties of the omega -AgaTxIVA-sensitive component of the embryonic cockroach brain neurons resemble that of the vertebrate P-type calcium currents (Llinas et al. 1989; Mintz et al. 1992a) rather than that of the Q-type calcium channel (Sather et al. 1993; Wheeler et al. 1994). However, this current component differs from the vertebrate P-type calcium current in the nonreversibility of the blocking effect of omega -AgaTxIVA after application of conditioning depolarizations. In contrast with data reported in vertebrates for the P/Q-type and R-type, the two components of the barium current exhibited indistinguishable voltage-dependent activation and inactivation properties. It would be interesting to see whether this is also true at the single-channel level.

Physiological significance of the P/Q and R-type currents

The precise role of the calcium current in the embryonic cockroach brain neurons in primary culture remains speculative. Under normal conditions (i.e., in cockroach saline), these neurons, similar to most insect neurons, have been shown to be electrically silent, partly because of the small size of the TTX-sensitive sodium current, and partly because of its very large delayed rectifier current (see Christensen et al. 1988).

In the culture medium, however, the external potassium concentration was much higher: 16 mM in the first medium and 30 mM in the second medium (see METHODS). Under these conditions, the neurons were depolarized (estimated resting potentials of ~ -40 and -20 mV; see Lees et al. 1985). The window potential of the barium current that can be calculated from the steady-state activation and inactivation curves (Fig. 2C) lay between -40 mV and +20 mV in 10 mM Ba2+. In the culture dish, there was no barium, and the calcium concentration was ~2 mM. Under these conditions, the activation and inactivation curves were displaced by ~10 mV toward more hyperpolarized potentials, and the window current was shifted (between -50 and +10 mV), a potential range that enabled calcium to enter the cell. One role for the observed calcium current could be to enable survival and differentiation of the neurons in culture. This hypothesis is strengthened by the report that, in vertebrate cultures, a better survival after growth factor deprivation is obtained in high-K+ solutions, which induces membrane depolarization and thereby activates voltage-dependent calcium channels (Collins et al. 1991; Koike et al. 1989; for review see Finkbeiner and Greenberg 1998). We observed, in agreement with Beadle and Hicks (1985), that our second culture medium (L + G), which contained more potassium, enabled better development of the neurons than the first medium (5 + 4). The recent findings that CNS neurons during the early embryonic development of Drosophila express voltage-dependent calcium channels (Baines and Bate 1998) and that the mutation of the genes encoding for these channels is lethal (Smith et al. 1996), also suggest that calcium channels play a prominent role during embryogenesis. Interestingly, preliminary experiments showing that verapamil induces the death of all cultured neurons in 72 h also suggest that calcium channels are important for survival and growth of embryonic cockroach neurons. Because the R-type calcium channels are present in all neurons, it is tempting to suggest that they could be involved in neuronal survival and differentiation shared by all neurons, whereas the P/Q type channels, which are expressed in only a subset of neurons, are implicated in another cellular function.


    ACKNOWLEDGMENTS

We thank Drs. F. Grolleau, B. Lapied, and S. Richard for comments on the manuscript.


    FOOTNOTES

Address for reprint requests: F. Tiaho, Equipe Canaux et Récepteurs Membranaires, UPRES-A 6026, Campus de Beaulieu, Bât. 13, 35042 Rennes Cedex, France.

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 7 July 1999.


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