Calcium Channels in Xenopus Spinal Neurons Differ in Somas and Presynaptic Terminals

Weiyan Li, Christopher Thaler, and Paul Brehm

Department of Neurobiology and Behavior, State University of New York at Stony Brook, Stony Brook, New York 11794


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Li, Weiyan, Christopher Thaler, and Paul Brehm. Calcium Channels in Xenopus Spinal Neurons Differ in Somas and Presynaptic Terminals. J. Neurophysiol. 86: 269-279, 2001. Calcium channels play dual roles in cell signaling by promoting membrane depolarization and allowing entry of calcium ions. Patch-clamp recordings of calcium and calcium-dependent currents from the soma of Xenopus spinal neurons indicate key functional differences from those of presynaptic terminals. Both terminals and somas exhibit prominent high-voltage-activated (HVA) calcium current, but only the soma expresses additional low-voltage-activated (LVA) T-type current. Further differences are reflected in the HVA current; N- and R-type channels are predominant in the soma while the terminal calcium current is composed principally of N type with smaller contribution by L- and R-type channels. Potential physiological significance for these different distributions of channel types may lie in the differential channel kinetics. Activation of somatic HVA calcium current occurs more slowly than HVA currents in terminals. Additionally, somatic LVA calcium current activates and deactivates much more slowly than any HVA calcium current. Fast-activating and -deactivating calcium current may be critical to processing the rapid exocytotic response in terminals, whereas slow LVA and HVA calcium currents may play a central role in shaping the somatic firing pattern. In support of different kinetic behavior between these two compartments, we find that somatic calcium current activates a prominent slow chloride current not observed in terminal recordings. This current activates in response to calcium entering through either LVA or HVA channels and likely functions as a modulator of excitability or synaptic input. The restriction of this channel type to the soma lends further support to the idea that differential expression of fast and slow channel types in these neurons is dictated by differences in signaling requirements for somatic and terminal compartments.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Individual classes of neurons can often be distinguished on the basis of expression of distinct receptor and voltage-activated ion channel types. In each case, it is likely that the overall composition of channel types is dictated by the specific functional requirements of each cell type. Furthermore this principle appears to apply to the functionally differentiated compartments within the same neuron. In motorneurons for example, the functional requirements are thought to match the specific needs of each cellular compartment, differing in dendritic, somatic, axonal, or terminal regions (Mallart and Brigant 1982; Yuste and Tank 1996). The somatic compartment plays a central role in integrating synaptic inputs while the terminal compartment is principally responsible for release of transmitter in response to individual spikes. Of particular interest is the distribution of calcium channel isoforms within the motorneurons because these channels are the central players in both of these processes (Barish 1991; Katz and Miledi 1967). In the soma, calcium channels participate in shaping the depolarization as well as providing calcium signals for second-messenger signaling and gene activation. By contrast, calcium-triggered release of transmitter appears to be the principal responsibility of calcium channels at terminals (Catterall 1998). Functional segregation of calcium channel isoforms within cells was suggested by several studies indicating a differential distribution of channel subtypes between soma and dendrites (Christie et al. 1995; Mouginot et al. 1997; Westenbroek et al. 1990). However, electrophysiological comparisons of somatic and terminal calcium currents are difficult due to the small size of the terminals. In fact, direct electrophysiological recordings of presynaptic calcium currents have been made in only a handful of cell types (Borst et al. 1995; Llinas et al. 1981; Stanley and Goping 1991; Yawo and Momiyama 1993).

Recent studies have shown that it is possible to voltage clamp nerve terminals of Xenopus spinal neurons (Thaler et al. 2001; Yazejian et al. 1997), permitting direct comparison of somatic and terminal calcium channels. The results of such comparisons indicate that large differences in calcium channel isoforms exist between terminal and soma. Additionally, those isoforms that are shared by the two compartments exhibit differences in activation kinetics. We also identify a pronounced slow calcium-activated chloride current that is restricted to the soma. Our findings suggest that functional distinctions, principally involving differences in kinetics, dictate the types of channels expressed in somas and terminals.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture

Xenopus nerve and muscle co-cultures were prepared using the methods as previously described (Thaler et al. 2001). Briefly, myotomal muscle and spinal cord were removed from stage 20 to 22 Xenopus laevis embryos (Nieuwkoop and Faber 1956) and dissociated in Ca2+- and Mg2+-free solution. The cell suspension was placed on basement membrane matrix (Collaborative Biomedical Products)-coated glass coverslips in modified 60% Leibovitz's L-15 medium with the addition of 10 mM Na-HEPES, 0.5% horse serum (Life Technologies, Gaithersburg, MD), 100 U/ml penicillin/streptomycin, 20 nM Neurotrophic Factor-3 (Regeneron, Tarrytown, NJ), and 1 µM testosterone propionate (Sigma, St. Louis, MO). Cultures were kept in a dark environment at room temperature.

Cells were visualized with a Zeiss 63× power LD Phase Achroplan objective and identified primarily on the basis of morphology. Muscles were easily distinguished by their prominent striations. Spinal neurons were rounded cells with diameters of 10-15 µm. The neurons projected long processes that occasionally exhibited swellings at locations where they contacted muscle cells. For the aim of side-by-side comparisons, most of the somatic and terminal recordings were performed during the third and the fourth day after plating when the accessibility to both the soma and terminal were optimal.

Electrophysiology

All voltage-clamp recordings of calcium current utilized the perforated-patch method (Horn and Marty 1988). Electrode tip (~2 µm OD after polished) was dipped in recording solution for 10 s. The electrode was then backfilled with a recording solution containing 200 µg/ml Amphotericin B and used immediately. Typically within 10 min after the gigaseal formation, the series resistance dropped below 20 MOmega , which was deemed acceptable for voltage-clamp recordings. The series resistance was routinely compensated by 50% with a 10-µs lag time.

Patch electrodes were made from borosilicate glass (Garner glass type: 7052) with Sutter Instrument P-97 micropipette puller and fire-polished before use to a pipette resistance of ~5 MOmega . The internal solution used to fill the electrode contained (in mM) 52 CsCH3SO4, 38 CsCl, 1 EGTA, 5 HEPES, and 50 glucose taken to a pH of 7.2 with CsOH.

To reduce the space-clamp artifacts resulting from the presence of prominent neurites, we used the puff-suck system to restrict the region of activation of calcium current (Thaler et al. 2001). As a variation of the method first introduced by Katz and Miledi (1967), we used a low-Ca2+ bath solution containing (in mM) 80 TEACl, 10 NaCl, 2 KCl, 5 MgCl2, 5 HEPES, 0.4 CaCl2, 3 glucose, 1 MnCl2, and 1 µM tetrodotoxin (Alomone Labs) taken to a pH of 7.2 with N-methyl-D-glucamine. A puffer pipette was used to deliver a 5 mM calcium bath solution (without Mg2+ and Mn2+) to the cell body or terminal under recording. A second sucking pipette was used to actively remove the high-calcium solution to avoid accumulation in the bath (for recording configuration see Fig. 1 in Thaler et al. 2001). By careful manipulations of the position and pressure of both puffing and sucking pipettes, a steady laminar flow of "high"-calcium solution could be created covering most part of the soma or terminal while avoiding the neurites. Calcium channels outside of the laminar flow were blocked by the manganese in the bath while those channels in the path of the laminar flow were relieved from block. Moving the calcium puffer a few microns off either the soma or the terminal led to a complete loss of calcium current (Thaler et al. 2001). With the combination of aforementioned pipette solution and puffing solution, voltage errors due to liquid junction potentials corresponded to 10 mV on the bases of estimated values (Barry and Lynch 1991) and direct measurements (Neher 1992) and all data were corrected for this error.

Drug delivery was achieved via a quartz filament placed within 2 mm of the tip of the puffing pipette. Full exchange of puffing solution could be completed within 2 min via injection of solution containing drugs. All drugs including omega -Ctx GVIA (Alomone Labs), omega -Ctx MVIIC (Peptides International), omega -Aga IVA (Peptides International) nitrendipine (Biomol), and niflumic acid were diluted from a stock solution to the working concentration just prior to use to ensure viability. Dihydropyridines were kept as stock solutions in DMSO.

Calcium currents were recorded using an EPC-9/2 dual patch-clamp amplifier (List Electronics, Darmstadt, Germany) and sampled at 10-µs intervals. Data were acquired and analyzed using HEKA Pulse + PulseFit software. Activation and inactivation time constants were measured by Hodgkin and Huxley fitting of calcium current traces. Prior to analysis the currents were leak-corrected by use of a P/10 protocol and refiltered with a 4-pole Bessel filter at 2 kHz. Statistical analysis, plotting and curve fitting were performed through the use of IGOR pro software. All statistical values represent means ± SD unless specified otherwise.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In dissociated cell cultures of Xenopus spinal neuron and muscle, both the somas and presynaptic terminals of neurons were accessible for voltage-clamp analysis. Dissociated spinal neurons represented a mixture of neuronal types, but we restricted our recordings to cholinergic neurons capable of eliciting muscle contractions when stimulated. The somatic diameter averaged 15 µm, whereas the terminals were usually <1 µm in diameter. However, occasional swellings in the terminals were observed in regions where the neurites contacted muscle. These regions were as large as 4 µm in diameter permitting whole cell voltage-clamp recording. These swellings were proved to be functional presynaptic terminals in previous study where direct stimulation produced excitatory postsynaptic currents (Thaler et al. 2001). Both terminal and somatic calcium currents were difficult to properly voltage clamp due to the large size of the calcium current and the presence of associated neuritic processes. These technical problems were ameliorated by focal application of calcium to the recording site. This technique restricts the physical boundaries of channel activation to a certain region under voltage clamp. For this purpose, a puffing pipette containing 5 mM calcium was repositioned until optimal recording of inward calcium current was achieved. The applied calcium was restricted to a local region of the cell through active removal by means of a second sucking pipette. Once the puffing and sucking pipettes were optimally positioned, they were not moved during the course of the experiment.

Low-voltage-activated calcium current

All 232 somatic recordings revealed the presence of voltage-activated inward current. Removal of the calcium puffing pipette just prior to the termination of the experiment completely abolished the inward current, indicating that it was carried by calcium. In 79% of those somas tested, two components of inward current could be resolved on the basis of the differences in threshold and kinetics (Fig. 1A). Activation of a low-voltage-activated (LVA) calcium current occurred at an average potential corresponding to -63 ± 6 mV and was fully activated at -33 ± 7 mV (n = 183). A second high-voltage-activated (HVA) calcium current activated at -23 ± 7 mV, and this current peaked near 0 ± 6 mV (n = 232). These two currents were evident in the raw traces and in the current-voltage relations for peak current (Fig. 1A). Frequently, the HVA calcium current exceeded 3 nA, rendering adequate control over the voltage impossible and these data were discarded. All of the 49 terminal recordings were of HVA type; no evidence of any LVA current was obtained in any terminals tested (Fig. 1B).



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 1. Recordings of somatic (A) and terminal (B) calcium currents. A: somatic calcium currents and the associated current-voltage relations for early inward current. Calcium currents were elicited by depolarization from the holding potential of -90 mV to the indicated potentials. - - -, 0 current level. Note the presence of 2 components of calcium current in both the traces and I-V relations. B: terminal calcium currents and the associated I-V relations for recorded early inward current. The protocol differed from the one used in A in that a shorter test pulse was used to elicit inward current.

Inspection of the raw current records indicates that the LVA current inactivated more rapidly than the HVA calcium current (Fig. 1A). In all somatic recordings, kinetic analysis of LVA calcium current was complicated by the activation of HVA current at potentials above -23 mV. Addition of 1 µM omega -conotoxin (Ctx) GVIA irreversibly blocked much of the HVA current, leaving the LVA component untouched (Fig. 2). In most cells an incomplete block of HVA calcium current was observed, but in two cells, 1 µM omega -Ctx GVIA blocked 100% of the HVA component, permitting analysis of the pure LVA calcium current (Fig. 2). The activation and inactivation kinetics of LVA currents were both voltage dependent. The time constants of activation ranged from 8 ± 4 ms near threshold (-60 mV) to 3 ± 1 ms at potentials corresponding to maximal activation (Fig. 3, inset A; n = 10). Inactivation time constants were strongly voltage dependent, averaging 23 ± 6 ms at -60 mV and 12 ± 2 ms at -30 mV (Fig. 3, inset B; n = 10). Deactivation time constants at -90 mV were measured by exponential fitting of the tail currents associated with repolarizations from brief steps to -30 mV, prior to the inactivation of the current. The tails were well fit with a single exponential curve, yielding time constants of 2.5 ± 0.5 ms (n = 10).



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2. Block of somatic high-voltage-activated (HVA) calcium current by omega -Ctx GVIA. Shown are the calcium currents prior to (top left) and 3 min after (top right) addition of 1 µM omega -Ctx GVIA. The calcium currents were elicited by depolarization from -90 mV to the potentials indicated. Bottom: the associated current-voltage relations before () and after (open circle ) block of the HVA by omega -Ctx GVIA.



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 3. Properties of somatic low-voltage-activated (LVA) calcium current. The LVA calcium currents shown were elicited by 200-ms test pulses from a holding potential of -90 mV. The test pulses were stepped in 5-mV increments beginning at -60 mV. Inset A: the relationship between the time constants of activation for LVA current and membrane potentials. Inset B: the relationship between the time constants of inactivation for LVA current and membrane potentials. In both A and B, each data point represents the mean ± SE for 10 cells. The time constants in both data sets were obtained by Hodgkin-Huxley fitting of the traces at each potential using HEKA Pulsefit software. The relationships in A and B were each fitted with an exponential curve.

Inactivation of LVA calcium current was determined by use of 500-ms conditioning depolarizations prior to application of the test pulse. The midpoint of steady state availability was -67 mV (Fig. 4A), a potential that does not inactivate HVA calcium current. Comparisons of steady-state availability and normalized I-V relations from the same cell indicate a small region of overlap near threshold of the LVA current (Fig. 4A). At potentials corresponding to this region of overlap, persistent inward LVA calcium currents can be observed (Fig. 3). In cells with both LVA and HVA calcium current, it was possible to isolate the HVA component by means of long depolarizations to -50 mV, which inactivated the LVA current (Fig. 4B) as predicted by the steady-state inactivation curve in Fig. 4A. The resultant HVA current showed a reduction in inactivation reflecting the loss of the inactivating LVA component. However, the HVA current still showed modest inactivation (Fig. 4B, inset).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 4. Steady-state inactivation of the somatic LVA calcium current. A: , the steady-state inactivation of peak current as a function of prepulse potential. The fraction of available current was determined by use of a 500-ms conditioning prepulse to the indicated potential, followed by a test pulse to -30 mV. open circle , the normalized I-V relations for peak LVA calcium current determined by depolarization to the indicated potential from a holding potential of -140 mV. B: the current-voltage relations for total peak calcium current measured using 2 different holding potentials. The 2 I-V relations reflect the peak calcium current recorded from holding potentials corresponding to -90 mV () and -50 mV (open circle ). The inset shows representative current traces comparing the responses to step depolarizations to 0 mV from holding potentials of -50 and -90 mV.

HVA calcium current

An incomplete block of somatic HVA current by omega -Ctx GVIA in the majority of cells tested suggested the existence of additional calcium channel isoforms (Fig. 5). Overall, 1 µM omega -Ctx GVIA blocked an average 62 ± 16% of HVA current (n = 25). Two lines of evidence indicate that the residual HVA current represents the presence of a omega -Ctx GVIA-insensitive calcium current rather than a sub-saturating block by the toxin. First, the time course of inhibition of the late steady-state inward current by 1 µM omega -Ctx GVIA indicated that maximal block was achieved within 2 min (Fig. 5A), and this block was irreversible (data not shown). Because this toxin blocks irreversibly over the time course of our experiment, the presence of a plateau block in Fig. 5A reflects a toxin-resistant component. As a second line of evidence, 6 µM omega -Ctx GVIA was tested on 13 cells, resulting in an average block of 66 ± 15% of the HVA current, a value not significantly different from the effects of 1 µM omega -Ctx GVIA (Student's t-test, P > 0.05).



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 5. Somatic HVA calcium current is sensitive to omega -Ctx GVIA. A: the time course of inhibition of calcium current during application of 1 µM omega -Ctx GVIA. The contribution by LVA current was minimized by measuring the late steady-state current near the end of the 100-ms depolarization. Maximal inhibition by omega -Ctx GVIA was observed after 2 min from the start of drug delivery. In this cell, the HVA was reduced by 65%. B: the current-voltage relations for early inward calcium current from the same soma shown in A before () and 2 min after (open circle ) the application of 1 µM omega -Ctx GVIA. Note that omega -Ctx GVIA reduced HVA current without affecting the LVA component. Inset: representative calcium currents before () and after (open circle ) application of 1 µM omega -Ctx GVIA.

Similar measurements from terminal calcium currents indicate that omega -Ctx GVIA blocks dihydropyridine (DHP)-sensitive channels in addition to N-type channels (Thaler et al. 2001). To further determine that the omega -Ctx GVIA sensitivity reflects N- and not L-type current in the soma, we tested the effects of another known N-type calcium channel antagonist, omega -Ctx MVIIC. This toxin also blocks P/Q type channels so it was necessary to first test for the presence of these latter channel types in the soma. For this purpose omega -agatoxin (Aga) IVA, a specific blocker of P/Q-type channel, was applied at a concentration of 500 nM. In five cells tested, omega -Aga IVA exerted no effects on calcium current (Fig. 6), consistent with the negative findings on terminal calcium current. Therefore any effects of omega -Ctx MVIIC were considered to be mediated through block of N-type calcium current. In 12 cells tested, 10 µM omega -Ctx MVIIC inhibited 51 ± 25% of the HVA calcium current (Fig. 7). Previously published dose dependence of inhibition of calcium current by omega -Ctx MVIIC in Xenopus spinal neurons showed that this concentration produces a steady-state block of 80% of the omega -Ctx MVIIC-sensitive current (Thaler et al. 2001). Therefore following correction, the estimated block by omega -Ctx MVIIC corresponded to 64%, which was not significantly different from the average block by 1 µM omega -Ctx GVIA. Taken together, these data indicate that ~60-65% of the somatic current is N-type and the balance of current is resistant to both toxins.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 6. Somatic currents are insensitive to omega -Aga IVA. Current-voltage relations for peak calcium current recorded before () and 5 min after (open circle ) application of 500 nM omega -Aga IVA. Inset: representative calcium current traces taken before () and after (open circle ) application of omega -Aga IVA.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 7. Somatic calcium currents are sensitive to omega -Ctx MVIIC. A: the time course of inhibition of HVA calcium current by omega -Ctx MVIIC. The steady-state current amplitude was measured just prior to termination of the test pulse to minimize the contribution by LVA calcium current. The decrease in HVA current corresponded to 41% in this cell. B: current-voltage relationship for the peak inward current recorded from the cell used in A. The current was measured before () and 6 min after (open circle ) the initial application of 10 µM omega -Ctx MVIIC. Inset: representative current traces taken before () and after (open circle ) application of omega -Ctx MVIIC.

Curiously, the proportion of toxin-resistant current appears to vary widely among somatic recordings. In 8 of 50 cells tested with either omega -Ctx GVIA or omega -Ctx MVIIC, more than 90% (95 ± 4%) of the HVA current was inhibited indicating almost complete contribution by N-type channels. However, in the other 42 cells tested with either toxin, an average 57 ± 14% of HVA current was inhibited, signaling a considerable contribution by calcium channels other than N-type (Fig. 8A). We considered that the balance of channel types in these cells might be DHP-sensitive due to the existence of this channel type in terminals. To test for the possible contribution by L-type channels, we applied nitrendipine to the soma. In the 15 cells tested, including the one shown in Fig. 8B, application of 1 µM nitrendipine for >= 3 min had no significant effect on peak calcium current. To ensure that nitrendipine was without effect on these neuronal somatic calcium currents we tested 2 µM (n = 6) and 5 µM (n = 2) nitrendipine and neither showed evidence of inhibition. As a positive control for nitrendipine inhibition, we applied 1 µM nitrendipine to GH3 pituitary cells expressing L-type calcium channels and observed a large inhibition of calcium current (data not shown). Furthermore, side-by-side recordings showed that this concentration of nitrendipine blocked ~40% of the calcium current in the terminals of these neurons (Thaler et al. 2001).



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 8. L-type channels do not contribute to somatic HVA calcium current. A: histogram of the percentage inhibition of somatic HVA calcium current by either omega -Ctx GVIA or omega -Ctx MVIIC (values after correction) measured for 50 cells. B: current-voltage relations of peak calcium current measured prior to () and 5 min after (open circle ) application of 1 µM nitrendipine. Inset: representative calcium currents showing the lack of effect by nitrendipine.

Concern over the observed lack of inhibition by nitrendipine was raised by previous studies attesting to the presence of verapamil-sensitive currents in the somas of these neurons (Barish 1991). Additionally, in our recordings of calcium current performed nearly 4 yr before completion of the study, we had observed partial inhibition of somatic calcium current by 1 µM nitrendipine. However, for reasons that are completely obscure to us, nitrendipine sensitivity was never observed in any cells tested over the past 2-yr period, despite our efforts to reproduce the exact conditions of those recordings.

The pharmacological criteria applied to HVA calcium current thus far point to contributions by both N-type and DHP/toxin-resistant channel isoforms. Each current component was further analyzed for possible differences in channel function. The omega -Ctx-GVIA-sensitive (N-type) calcium current activated at -21 ± 5 mV and peaked at -2 ± 7 mV (n = 10). The time constant of activation of N-type current averaged 4.6 ms at -10 mV and decreased at more depolarized potentials to a stable value of 0.8 ms at 30 mV (Fig. 9C; n = 10). The time constant of deactivation of N-type current at -90 mV averaged 0.47 ± 0.22 ms (n = 10). DHP/toxin-resistant (R-type) calcium current activated at -15 ± 6 mV and peaked at 5 ± 7 mV (n = 7). These values obtained for R-type current showed a slight right shift when compared with N-type currents, but the differences were not statistically significant (Student's t-test, P > 0.05). Activation time constants for R-type current were similar to N-type current, ranging from 5.0 ms at -10 mV to 0.8 ms at 30 mV (Fig. 9D; n = 6). Deactivation time constant for R-type calcium current at -90 mV measured 0.43 ± 0.36 ms (n = 6), again not statistically different from that of N-type current.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 9. Differences in activation kinetics for somatic and terminal HVA calcium current. A and B: normalized averaged traces for somatic ( · · · ) and terminal (---) are shown for depolarizations to -10 mV in A and +30 mV in B. The averaged traces were based on 6 individual records in A and 8 records in B. In A and B, only the omega -Ctx-GVIA-sensitive currents are shown to exclude any potential contamination by LVA calcium current. Note the differences in time scale between A and B. C-E: the time constants of calcium current activation were determined individually for somatic omega -Ctx GVIA sensitive in C, somatic omega -Ctx GVIA/DHP insensitive in D, and composite terminal calcium currents in E. The average time constants ± SE for each potential were determined on the basis of recordings from 6 to 10 cells. Each relationship was fitted with an exponential curve. F: direct comparisons of the curves obtained for C-E without the accompanying data points.

The small size of terminal calcium current impeded the reasonable fitting of separated omega -Ctx-GVIA- and DHP-sensitive currents, especially at lower membrane potentials. Therefore the activation kinetics were determined for overall terminal current to compare with somatic calcium current. Comparisons of normalized averaged traces revealed that terminal calcium current activated faster than somatic N-type current at potentials near threshold (Fig. 9, A and B). Activation time constants for terminal HVA currents averaged 2.6 ms at -10 mV and decreased at more depolarized potentials to a stable value of 0.5 ms at 30 mV (Fig. 9E; n = 10). Averaged activation time constants for terminal calcium current were smaller than those for somatic HVA currents at all membrane potentials tested (Fig. 9F). However, due to large variations, the difference was significant only at lower voltages (i.e., -10 and -5 mV; Student's t-test, P < 0.05).

Calcium-activated chloride current

Recordings from somas in the presence of external calcium revealed a current that was not observed when barium was substituted for calcium. This pronounced current initially complicated our measurements of calcium tail currents because of its large size and slow kinetics (Fig. 10). The current was observed in 40% of cells tested, and it appeared as a slowly decaying tail current following repolarization from potentials positive to -50 mV. At -90 mV, this tail current decayed with two time constants corresponding to 22 ± 8 and 0.4 ± 0.2 ms (n = 10). The fast-decaying component of the tail currents exhibited time constants similar to N- and R-type calcium currents. In the spinal neurons exhibiting the slow-decaying tail currents, larger somas generally tended to have larger amplitude tail current. Additionally, the tail current also showed a clear dependence on time in culture. The slow component of tail current was absent in recordings performed within 2 days after plating. However, 60% of cells tested on day 5 showed slow components of tail current.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 10. The long-lasting tail current is carried by Cl-. A: the dependence of the tail current on potential was determined during repolarizations from 0 mV to membrane potentials between -90 and -35 mV. At potentials positive to -35 mV, the HVA calcium current was activated precluding measurements of the tail current. The tail current decreased in amplitude with more positive membrane potentials. B: the long-lasting component of the tail current is shown before () and 5 min after (open circle ) the initial application of 200 µM niflumic acid.

This tail current was carried by chloride as the estimated reversal potential for this current (-30 ± 8 mV; Fig. 10A; n = 10) was close to the theoretical equilibrium potential (-26 mV) for chloride in our recording solutions. Also, niflumic acid, an inhibitor of chloride channels (White and Aylwin 1990), fully blocked the slow tail current at a concentration of 200 µM while sparing both the calcium current and the fast decaying component of the tail (Fig. 10B; n = 4).

Furthermore several lines of evidence identified this chloride tail current as calcium-activated. First, removal of Ca2+ by lifting the puffer eliminated both the inward calcium current and the tail current (Fig. 11A); second, when depolarizing pulses neared ECa (e.g., +60 mV), this tail current also disappeared (Fig. 11B); third, substitution of barium for calcium eliminated the long-lasting tail while sparing the fast component of tail current (Fig. 11C). Finally, the sensitivity to niflumic acid (Fig. 10B) is consistent with previously reported effects on calcium-dependent chloride channels.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 11. The chloride tail current requires calcium entry for activation. A: current traces are shown in response to depolarization from -90 to 0 mV in the presence of 5 mM () and 200 µM (open circle ) calcium. In 5 mM calcium, a pronounced HVA inward current was observed during the depolarization and a biphasically decaying tail current followed repolarization to -90 mV. When 200 µM calcium was substituted for 5 mM calcium, most of the inward current as well as the associated long-lasting tail current were reversibly abolished. B: comparisons of long-lasting tail current amplitude in response to depolarizations to 0 mV () and +60 mV (open circle ). Depolarizations to +60 mV resulted in smaller inward HVA currents and reduced tail current responses. C: substituting 5 mM barium (open circle ) for 5 mM calcium () resulted in less decay of HVA current during a depolarization from -90 to 0 mV as well as a reduction in the amplitude of the tail current associated with repolarization.

In cells exhibiting slowly decaying tail currents, the inward current during the test pulse further indicates contribution by calcium-activated chloride current. In Fig. 10B, treatment with 200 µM niflumic acid reduced the apparent inactivation of inward calcium current following depolarization to +10 mV. Similar reduction in inward current decay was observed following substitution of 5 mM barium for extracellular calcium. These actions reflect an inhibition of an outward chloride current during the test pulse that resulted from maintained entry of calcium.

The relationship between the amplitude of calcium current and chloride tail current was determined by comparing the current-voltage relations of these two currents. For this purpose, the peak calcium current and the amplitude of the slow component of tail current were plotted as a function of test potential. Both LVA and HVA calcium currents were capable of activating chloride tail current (Fig. 12A). However, the activation of calcium-activated chloride current also depends on the accumulated calcium entry (Fig. 12B). Test pulses of increasing durations led to cumulative increases in the amplitude of chloride tail current. Comparison of tail current amplitude with the integrated calcium charge entry indicated a nonlinear region for pulses briefer than 5 ms. However, longer pulses resulted in a linear relationship between chloride current and calcium charge entry. (Fig. 12B). Because individual short pulses were seemingly less effective in triggering calcium-dependent chloride currents, we explored the possibility that under physiological conditions most of these channels are recruited only during high-frequency stimulation. Stimulation of cells using a series of high-frequency short-duration pulses resulted in an apparent reduction in inward calcium current, while the size of the slow chloride tail current exhibited cumulative increases in amplitude during the train (Fig. 12C).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 12. Relationship between calcium current and chloride tail current. A: the current-voltage relations measured for peak calcium current () and the peak chloride tail current (open circle ) are compared. The chloride current was measured in response to repolarization from the indicated potential to -90 mV. B: the dependence of the amplitude of the chloride tail current on calcium entry was established by varying the duration of the excursion to 0 mV (inset). The relationship between calcium charge entry and amplitude of chloride tail current is shown. The calcium charge entry was measured as the integral of inward current during the depolarization, and any contribution by the calcium component of tail current was ignored. The amplitude of the chloride current was measured by fit of the slow component of the tail current associated with repolarization to -90 mV. C: successive repetitive depolarizations result in a decline of inward calcium current and the cumulative enhancement of chloride tail current. A single pulse proceeds the stimulus train to show clearly the slow component of the tail current. This pulse is followed, after 100 ms, by a 20-Hz train of 10 depolarizations to 0 mV.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Comparisons of somatic and terminal voltage-clamp recordings have revealed differences in the distribution of functionally distinct calcium channels. The most obvious difference is reflected in the contribution by LVA calcium current (Barish 1991; Yazejian et al. 1997). Approximately 80% of the somas tested exhibited prominent LVA current, whereas no terminals tested showed any sign of LVA calcium current. Analysis of the voltage dependence of activation and the kinetics both indicate potential reasons for the exclusion of functional LVA calcium channels from terminals. First, the low-threshold opening by these calcium channels results in the potential for activation near resting membrane levels (Gu and Spitzer 1993). Such openings, even by single channels, would likely result in unwanted transmitter release (Stanley 1993). Additionally, the LVA channels activate three times slower and deactivate six times slower than HVA channels, consistent with previous findings on Xenopus spinal neuron somas (Barish 1991; Gu and Spitzer 1993). Because the majority of calcium enters during the repolarizing phase of action potential (Thaler et al. 2001; Yazejian et al. 1997), fast activation and deactivation of current in these terminals is advantageous. Moreover, fast kinetics provide for synchronized entry of calcium by ensuring that conductance is maximal at the time of calcium entry and that entry is terminated quickly on repolarization (Sabatini and Regehr 1999). At half-amplitude, the duration of the presynaptic spike measures 1.4 ± 0.2 ms (Thaler et al. 2001), which is far too short in duration for full activation of LVA current. Also the slow deactivation or LVA calcium current would be expected to prolong postrepolarization entry of calcium.

One potential solution to the problems posed by LVA calcium current in terminals would be to uncouple the calcium entry associated with LVA current from the exocytotic process. While slow deactivation of LVA current would still slow repolarization, the calcium entering would not result in persistent transmitter release. In fact, some calcium channel types are thought to couple more effectively to transmitter release machinery (Butz et al. 1998; Leveque et al. 1994; Sheng et al. 1994). However, it appears that all three types of calcium channels (N, L, and R type) present in spinal motor nerve terminals couple to exocytosis (Thaler et al. 2001). On the basis of these findings, it seems unlikely that terminal LVA current could be present and fail to participate in transmitter release. Therefore the solution adopted by these neurons was to differentially target LVA channels to the somatic compartment. In agreement with our findings, LVA calcium channel is not present in most of the characterized presynaptic terminals studied to date, such as squid giant synapse (Charlton and Augustine 1990), the calyx of Held (Doughty et al. 1998), the calyx of the chick ciliary ganglion (Stanley and Goping 1991), and axon terminal of goldfish bipolar cells (Tachibana et al. 1993). One exception is in leech heart interneurons, where LVA currents were shown to support specifically graded synaptic transmission in which a prolonged subthreshold neurotransmitter release is evident (Ivanov and Calabrese 2000; Lu et al. 1997). This further agrees with our hypothesis that LVA calcium current can couple to release, yet they are not suited for a synchronized fast synaptic transmission.

The likely importance of fast channel kinetics to transmitter release is underscored by our finding that HVA current activation is significantly slower in the soma than in the terminal at membrane potentials near the threshold level. Direct comparisons of calcium current in response to rectangular command pulses indicate consistently faster rise of inward HVA calcium current in terminals at these potentials. The average activation time constants of terminal HVA calcium current also appear smaller than somatic HVA current at more positive potentials, yet the differences were not significant due to a large variability. Although direct comparisons of calcium channel kinetics between somas and terminals in other preparations are lacking, similar differences have been suggested by studies on calyx of Held (Borst and Sakmann 1998). As in our preparation, the mechanisms underlying the faster activation of calcium channels in terminals have not been identified. However, the established effects of auxiliary subunits (Wakamori et al. 1999) and G-protein-mediated modulation (Delmas et al. 2000; Dolphin 1998) on calcium current kinetics point to possible regulatory mechanisms.

In contrast to the terminals, LVA calcium channels have been shown to exist in the somas of various neurons (Tsien et al. 1991). Based on their low threshold and slow kinetics, the proposed functional roles of somatic LVA calcium currents in different neurons have included regulation of the threshold for spike generation (Huguenard 1996), contribution to depolarizing envelope underlying burst firing (Llinas and Yarom 1981), and promotion of intrinsic oscillatory behavior (McCormick and Huguenard 1992). In Xenopus spinal neurons, the function of LVA calcium currents likely lies in their ability to regulate spike initiation and synaptic integration. For example, in these neurons, LVA channels were shown to function as the trigger responsible for the spontaneous elevations of intracellular calcium, which seems essential for their development (Gu and Spitzer 1993). In mature neurons, these channels may function to enhance postsynaptic integration via activation by subthreshold EPSPs and therefore lower the threshold for action potential initiation.

Our study points to contributions by two calcium channel types to HVA current in the soma. The predominant calcium channel type is N type based on the sensitivity to both omega -Ctx GVIA and omega -Ctx MVIIC. N-type current contributes an average 65% of overall current, and the balance of HVA current is contributed by channels that are insensitive to both conotoxins. P/Q- and L-type channels are ruled out on the basis of insensitivity to omega -Aga IVA and nitrendipine. Given the unusual pharmacological profile, we tentatively refer to these channels as resistant or R type. An appreciable, but much smaller proportion of R-type current was observed in terminals. However, there is no direct evidence to suggest that the R-type currents in the soma and terminal represent the same channel types. R-type was originally defined as "resistant" to all the known calcium channel antagonists. It has been generally established via expression studies that alpha 1E subunit forms the pore for R-type calcium channels (Hilaire et al. 1997; Piedras-Renteria and Tsien 1998; Zhang et al. 1993). However, recent pharmacological studies have demonstrated that R-type current is not homogenous across cell types (Newcomb et al. 1998) and multiple subtypes of R-type calcium channels may co-exist in the same neuron (Tottene et al. 2000; Wilson et al. 2000).

In the process of identifying differences in calcium current between somatic and terminal compartments, a prominent slowly deactivating inward tail current surfaced. This current was deemed to be calcium-activated based on its dependence on calcium entry for activation and the absence of activation when barium was substituted for calcium. Furthermore the current was carried by chloride on the basis of reversal potential measurements and its sensitivity to niflumic acid, an inhibitor of chloride channels (White and Aylwin 1990). Our results agree with previously published studies showing that calcium-activated currents in Xenopus spinal neurons appear over time in culture (Hussy 1991, 1992). We now show that these currents do not activate fully in response to a short-duration pulse corresponding to a single action potential. A steep nonlinear dependence between stimulus duration and chloride charge entry exists for pulse lengths between 1 and 4 ms, whereas a shallow linear dependence of charge entry on pulse duration exists beyond 5 ms. We interpret the nonlinear region of the relationship to represent the local concentration dependence of chloride channel activation. Pulse durations in excess of 5 ms may result in cumulative calcium concentrations sufficient to fully activate local chloride channels. The mechanisms causal to the linear region of this relationship are less clear and may represent diffusion of calcium and subsequent activation of distal chloride channels in the cell soma.

In cultured Xenopus embryonic spinal neurons, the chloride equilibrium potential is approximately -60 mV, close to the resting potential (Bixby and Spitzer 1984). Therefore the calcium-activated chloride current was speculated to either promote repolarization or to modulate excitability and/or synaptic input (Hussy 1991). However, our results show that amplitude of chloride current activated by single short pulses is minimal. It is unlikely that this current has significant physiological role during the repolarization phase of the action potential considering the large size of the normal repolarizing potassium current. Furthermore we showed that high-frequency stimulation recruited more chloride currents depending on an accumulative calcium entry. It is therefore reasonable to hypothesize that these channels may only respond to elevation of intracellular calcium induced by high-frequency firing, exerting an inhibitory effect on synaptic integration and spike initiation.

The complete absence of this current in the terminal may reflect the different functional requirements of this cellular compartment. As discussed for calcium current, fast activation and deactivation may be required for synchronous transmitter release. If the calcium-activated chloride current were present in terminals, it would result in persistent activation of an inhibitory current. Such a current could potentially alter the presynaptic spike waveform thereby further altering calcium entry. While this might be an attractive mechanism for presynaptic modulation, it may not be suitable for a follower synapse such as the neuromuscular junction. This was indirectly supported by the very fast kinetics of calcium-activated potassium channel identified in the presynaptic terminal of the same preparation (Yazejian et al. 2000). The lack of calcium-activated chloride current in terminals may reflect differential targeting to the somatic compartment. Alternatively, the chloride channels may be present in terminals but fail to activate due to the strong calcium buffering shown to occur in presynaptic terminals. In either case, it is clear that this current plays a specific role in firing of somatic spikes.

Overall, our data have indicated that the composition and possibly modulation of calcium channels are tailored to serve the specific functions for different subcellular compartments. Between the cell body and terminal of Xenopus spinal neurons, the issue of speed seems to be the dominating principle such that slow kinetics supports synaptic input integration in the soma while fast kinetics provides synchronized release of neurotransmitter at presynaptic terminals.


    ACKNOWLEDGMENTS

The authors thank Regeneron for the generous donation of NT-3.

This research was supported by National Institute of Neurological Disorders and Stroke Grant NS-18205.


    FOOTNOTES

Address for reprint requests: W. Li (E-mail: weili{at}ic.sunysb.edu).

Received 8 January 2001; accepted in final form 20 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society