J Physiol Volume 510, Number 2, 361-370, July 15, 1998
The Journal of Physiology (1998), 510.2, pp. 361-370
© Copyright 1998 The Physiological Society
Ca2+ influx through voltage-gated Ca2+ channels regulates 5-HT3 receptor channel desensitization in rat glioma × mouse neuroblastoma hybrid NG108-15 cells
Susan Jones and Jerrel L. Yakel
Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, PO Box 12233, Research Triangle Park, NC 27709, USA
Received 6 January 1998; accepted after revision 25 March 1998.
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ABSTRACT |
- The kinetics of desensitization of the 5-HT3 receptor (5-HT3R)-gated ion channel were investigated using whole-cell and perforated-patch recording techniques in NG108-15 cells.
- Rapid application of 5-HT (50 µM) elicited a 5-HT3R-mediated inward current response that desensitized completely in the continued presence of agonist. In the whole-cell recording configuration (holding potential of -70 mV) while buffering internal calcium (Ca2+i) with 5 mM EGTA (0·5 mM added Ca2+; with an estimated free [Ca2+] of 30 nM), the rate of desensitization was initially rapid (with a half-time of ~230 ms), but dramatically slowed with time by 1120 ± 160 %.
- This slowing in the rate of desensitization was reduced by stronger Ca2+ buffering (20 mM BAPTA, without added Ca2+), or by the bath application of cadmium (100 µM) to block voltage-gated Ca2+ channels. The rate of desensitization was also dependent on membrane potential.
- In perforated-patch recordings, the rate of desensitization remained constant. However, a slowing in the desensitization rate could be induced by depolarizing cells immediately prior to the application of 5-HT.
- The depolarization-induced slowing was blocked by incubating cells with BAPTA-AM (a membrane-permeant analogue of BAPTA) or by the bath application of cadmium.
- These data suggest that Ca2+ influx through a cadmium-sensitive voltage-gated Ca2+ channel increases the cytoplasmic Ca2+ concentration ([Ca2+]i) and induces a dramatic slowing in the kinetics of desensitization of the 5-HT3R channel. These data provide evidence for cross-talk between voltage-gated Ca2+ channels and 5-HT3Rs in NG108-15 cells.
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INTRODUCTION |
The 5-HT3 receptor (5-HT3R) is a ligand-gated ion channel found in the central and peripheral nervous systems where it can mediate fast synaptic transmission and neurotransmitter release (see review by Jackson & Yakel, 1995). Like other ligand-gated ion channels, the 5-HT3R channel undergoes desensitization (i.e. a closed and/or inactivated state) in the continued presence of agonist (Katz & Thesleff, 1957). For excitatory and inhibitory synaptic responses in the CNS, desensitization of glutamatergic and GABAergic receptor channels is thought to significantly affect the shape of the synaptic responses (see review by Jones & Westbrook, 1996).
The molecular mechanism of desensitization is unknown and controversial (e.g. see Lin & Stevens, 1994; Colquhoun & Hawkes, 1995), but several factors are known to regulate the kinetics of desensitization. For many ligand-gated ion channels including the 5-HT3R channel, changes in the cytoplasmic Ca2+ concentration ([Ca2+]i) and/or Ca2+-dependent signal transduction cascades are known to play a role. Conditions that alter [Ca2+]i modulate desensitization of the NMDA glutamate receptors (Legendre, Rosenmund & Westbrook, 1993; Vyklick´y, 1993; Medina et al. 1995; Kyrozis, Goldstein, Heath & MacDermott, 1995; Kyrozis, Albuquerque, Gu & MacDermott, 1996), P2X ATP receptors (Khiroug, Giniatullin, Talantova & Nistri, 1997b), nicotinic ACh receptors (Khiroug, Giniatullin, Sokolova, Talantova & Nistri, 1997a) and vanilloid or capsaicin receptors (Docherty, Yeats, Bevan & Boddeke, 1996; Koplas, Rosenberg & Oxford, 1997). In addition, a dependence of desensitization kinetics on the Ca2+-calmodulin-regulated protein phosphatase, calcineurin, has been reported for the 5-HT3, NMDA, GABAA, nicotinic ACh and capsaicin receptors (see review by Yakel, 1997). The present study has investigated the role of [Ca2+]i in regulating desensitization of the 5-HT3R channel.
The NG108-15 rat glioma × mouse neuroblastoma hybrid cell line was used as a model system to study 5-HT3R channel desensitization (Yakel, Shao & Jackson, 1991). NG108-15 cells have a number of mechanisms for increasing [Ca2+]i, including voltage-gated Ca2+ channels, release from internal Ca2+ stores, Ca2+-permeable ligand-gated ion channels, and a Na+-Ca2+ exchange pump (Freedman, Dawson, Villereal & Miller, 1984; Caulfield, Robbins & Brown, 1992; Kasai & Neher, 1992; Chueh & Kao, 1993; Lo & Thayer, 1993, 1995; Hsu, Chou & Chueh, 1995). In this report, utilizing whole-cell and perforated-patch recording techniques, we have demonstrated that an increase in [Ca2+]i, via the influx of Ca2+ through cadmium-sensitive voltage-gated Ca2+ channels, can dramatically decrease the rate of 5-HT3R channel desensitization. These data provide evidence for a novel mechanism of regulation of the 5-HT3R, and represent a new example of regulation of ligand-gated ion channels by voltage-gated Ca2+ channels.
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METHODS |
Cell culture
Undifferentiated NG108-15 cells (Nelson, Christian & Nirenberg, 1976) were maintained in culture in Dulbecco's modified Eagle's medium with 10 % fetal calf serum (Gibco), 100 µM hypoxanthine, 0·4 µM aminopterin, 16 µM thymidine, and a penicillin- streptomycin-neomycin (PSN) antibiotic mixture (Gibco; 100 × mixture contains (mg ml-1): 5 penicillin, 5 streptomycin, 10 neomycin) incubated in an atmosphere of 10 % CO2-90 % air at 37°C. Prior to experiments (1-3 days), cells were plated on glass coverslips coated with poly-D-lysine.
Electrophysiology
The whole-cell and nystatin perforated-patch (Horn & Marty, 1988) recording techniques were used to record membrane currents from NG108-15 cells that were maintained at room temperature (21-24°C) in a continuously flowing (
2 ml min-1) bathing solution of the following composition (mM): 150 NaCl, 3 KCl, 2·5 CaCl2, 1·2 MgCl2, 5 Hepes, 10 glucose, pH 7·4. The pipette solution for whole-cell recordings contained (mM): 140 potassium gluconate, 5 EGTA, 0·5 CaCl2, 2 MgCl2, 2 Mg-ATP, 10 Hepes, pH 7·2. The estimated free [Ca2+] of this solution is 30 nM. For some experiments, EGTA and CaCl2 was replaced by BAPTA (20 mM). For perforated-patch recordings, the pipette solution contained (mM): 140 potassium gluconate, 2 MgCl2, 10 Hepes, pH 7·2. A stock solution of nystatin was made up by sonicating nystatin in DMSO (50 mg ml-1), then nystatin was diluted into the pipette solution for a final concentration of 0·25 mg ml-1 nystatin (0·5 % DMSO). Patch pipettes were frontfilled with nystatin-free solution, then backfilled with the nystatin-containing solution. After obtaining a tight seal, the nystatin usually permeabilized the patch to give stable series resistances between 10 and 20 M
within 10 min, as monitored by the appearance of capacitance transients. Patch pipettes were made from Corning 7052 glass; the series resistance (usually < 10 M for whole-cell recordings) and membrane capacitance were compensated (70 %) with an Axopatch-1D amplifier (Axon Instruments, Foster City, CA, USA). Cells were held at a potential of -70 mV unless otherwise specified; all potentials were corrected for a junction potential of -10 mV. Voltage protocols and data analysis (including curve-fitting procedure) were done using the pCLAMP 6 software (Axon Instruments). Traces were digitized and filtered at 1 kHz. Data are expressed as means ± S.E.M. where appropriate and Student's t test was used to determine the significance in differences. P < 0·05 was considered significant.
5-HT (50 µM) was rapidly applied for 10 s through synthetic quartz tubing (Polymicro Technologies Incorp., Phoenix, AZ, USA; 320 µm i.d., 430 µm o.d.) placed 100 µm away from the cell; the application of the drug was controlled by computer-driven valves (General Valve Co., Fairfield, NJ, USA) and pCLAMP 6 software. Rise times (i.e. time required for activation of the response amplitude from 10-90 %) for 5-HT-activated responses averaged 28 ± 1 ms (n = 6). To allow complete recovery of 5-HT3 receptor desensitization, 5-HT was applied to the cell at 4 min intervals. As there was some variability in the kinetics of desensitization, data for experimental tests and control cells were always collected on the same day.
Chemicals
5-HT was obtained from Research Biochemicals International, BAPTA-AM was obtained from Calbiochem, and all other reagents were from Sigma. BAPTA-AM was stored as a 10 mM stock solution in DMSO.
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RESULTS |
5-HT responses desensitize with a rate that slows dramatically with time
The application of 5-HT (50 µM, for 10 s) elicited a rapid inward current response, via activation of the 5-HT3R (Yakel & Jackson, 1988), in undifferentiated NG108-15 cells held at a holding potential of -70 mV; the mean amplitude was 1·55 ± 0·2 nA (36 cells). The amplitude of the response generally varied with time such that both increases and decreases in amplitude were observed; typically by 20 min after break-in, changes in amplitude of 0-50 % were observed. Using the whole-cell recording configuration while buffering internal free Ca2+ with EGTA (5 mM) and Ca2+ (0·5 mM), the initial response to 5-HT desensitized rapidly (Fig. 1A); the time required for the response to decay by 50 % (t½) immediately after break-in (t = 0 min) averaged 227 ± 37 ms (22 cells). As previously reported, the onset of desensitization was a biphasic process (Yakel et al. 1991). At t = 0 min, the fast time constant of decay (
fast) averaged 185 ± 23 ms (20 cells), the slow time constant of decay (slow) averaged 3390 ± 400 ms, and the ratio of the zero time intercepts of the fast and slow components (Rf/s) averaged 5·0 ± 0·5.
5-HT was applied at 4 min intervals to allow complete recovery from 5-HT3R desensitization. Although initially rapid, the rate of desensitization of the 5-HT responses dramatically slowed with time in whole-cell recordings, a phenomenon previously observed under similar but not identical conditions (Yakel et al. 1991). At 8 min after break-in, t½ increased on average to 1640 ± 240 ms, with a maximum percentage increase per cell of 1120 ± 160 %. (Fig. 1). The extent and time course of the slowing in the rate of desensitization was variable from cell to cell. In general, the maximum slowing occurred between 8 and 20 min.
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Figure 1. Increased [Ca2+]i buffering prevented the slowing in the rate of 5-HT3R desensitization
A, responses elicited by a 10 s application of 50 µM 5-HT (indicated by the horizontal bar) at a holding potential of -70 mV at 0 and 8 min after break-in in the same cell when buffering internal free Ca2+ with EGTA (5 mM) and Ca2+ (0·5 mM). The values of t½, fast, slow and Rf/s at 0 min for this cell were 145 ms, 138 ms, 2850 ms and 4·1, respectively. The value of t½ at 8 min was 927 ms. B, responses in a different cell when buffering internal free Ca2+ with BAPTA (20 mM). The values of t½ at 0 and 8 min for this cell were 138 and 165 ms, respectively. C, plot of the mean values of t½ with time in EGTA- (22 cells) or BAPTA-dialysed (24 cells) cells.
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The fast time constant of decay also increased dramatically with time; on average fast was 430 ± 57 ms (20 cells) at 8 min, with a maximum percentage increase of 348 ± 59 %. In contrast, the value of slow did not significantly change with time. The value of Rf/s decreased significantly with time under these conditions; by 8 min the value of Rf/s averaged 1·1 ± 0·4. This decreased ratio indicates that the contribution of the fast decay phase to the overall decay of the response decreased with time. Therefore, the overall slowing in the rate of desensitization was accounted for by an increase in the value of fast and a decrease in the contribution of fast (vs. slow) with time.
High internal BAPTA blocks slowing of desensitization
To examine whether changes in cytoplasmic Ca2+ levels ([Ca2+]i) were responsible for the slowing in the desensitization rate, a stronger Ca2+-buffering internal solution was used by replacing EGTA (5 mM; and added Ca2+) with a high concentration (20 mM) of the Ca2+ chelator, BAPTA. Intracellular BAPTA significantly reduced the extent of slowing of the desensitization rate (Fig. 1B). On average (24 cells), t½ was 233 ± 50 ms at 0 min and 592 ± 124 ms at 8 min under these conditions, with a maximum percentage increase in t½ of 257 ± 80 % (24 cells); this value was significantly less than that of EGTA-dialysed cells (1120 ± 160 %). The value of t½ was significantly reduced by BAPTA versus EGTA dialysis from 4-20 min (Fig. 1C).
The increase in the fast time constant of decay with time was also reduced significantly in BAPTA-dialysed cells. On average (21 cells), fast was 195 ± 23 ms at 0 min and 308 ± 35 ms at 8 min, with a maximum percentage increase of 126 ± 4 %; this value was significantly less than that of EGTA-dialysed cells (348 ± 59 %). The value of slow in BAPTA-dialysed cells was similar (3290 ± 320 ms at 0 min) to that in EGTA-dialysed cells, and also did not significantly change with time. The decrease in the value of Rf/s with time was also decreased significantly in BAPTA-dialysed cells. On average Rf/s decreased from 6·2 ± 0·8 at 0 min to 2·5 ± 0·4 at 8 min; this 8 min value was significantly higher than that of EGTA-dialysed cells (1·1 ± 0·4).
Cadmium blocks the slowing in the rate of desensitization
To determine whether Ca2+ influx through voltage-gated Ca2+ channels might be a source of the Ca2+-dependent slowing in the rate of desensitization, the voltage-gated Ca2+ channel blocker cadmium (100 µM; Fig. 2A) was added to the bathing solution prior to break-in; cadmium significantly reduced the slowing in the rate of desensitization (Fig. 2). The value of t½ for the cadmium-treated cells averaged 114 ± 12 ms at 0 min and 125 ± 29 ms at 8 min, with a maximum percentage increase in t½ of 48 ± 14 % (5 cells). In addition the values of fast, slow and Rf/s at 0 and 8 min were, respectively, 143 ± 12 and 143 ± 19 ms for fast, 3430 ± 760 and 3400 ± 970 ms for slow, and 8·1 ± 2·0 and 6·8 ± 0·9 for Rf/s. Adding cadmium within 1-2 min after break-in also completely blocked this slowing (data not shown). The bath application of nickel at a concentration of 50 µM, which should selectively block the low-voltage-activated T-type voltage-gated Ca2+ channel in NG108-15 cells (Brown, Docherty & McFadzean, 1989; Kasai & Neher, 1992), was without effect (Fig. 2B). Neither cadmium nor nickel at these concentrations significantly altered either the kinetics or amplitude of the 5-HT responses at t = 0 min.
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Figure 2. Cadmium, but not nickel, prevented the slowing in the rate of 5-HT3R desensitization
A, 5-HT responses in EGTA-dialysed cells with cadmium (100 µM) in the bathing solution prior to break-in. The values of t½ at 0 and 8 min for this cell were 164 and 224 ms, respectively. B, mean values of the maximum percentage increase in t½ under control conditions (i.e. EGTA-dialysed cells), or with cadmium or nickel (50 µM) in the bathing solution prior to break-in. The number of cells are shown in parentheses. The asterisk indicates a significant difference (P < 0·05) using Student's t test.
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The rate of desensitization kinetics is dependent on membrane potential
The rate of desensitization of the 5-HT3R channel was dependent on the membrane potential. In EGTA-dialysed cells, the mean t½ values were plotted (Fig. 3) from 0 to 20 min at holding potentials of -50 and -90 mV. The value of t½ at 0 min was 536 ± 200 ms (6 cells) at a holding potential of -50 mV, which was significantly higher than the value of 177 ± 26 ms (9 cells) at a holding potential of -90 mV. At 8 min, the values of t½ had increased to 3040 ± 520 ms at -50 mV and 1070 ± 290 ms at -90 mV. The t½ values at -50 mV were significantly larger than those at -90 mV from 0-12 min; the t½ values at -70 mV were intermediate (data not shown).
Rate of desensitization is stable using perforated-patch recordings
As disruption of endogenous cytoplasmic Ca2+ buffers was most probably responsible for the change in the rate of desensitization of the 5-HT3R channel in whole-cell recording conditions, we tested whether the slowing in the rate of desensitization was observed when using the perforated-patch recording technique, as endogenous Ca2+ buffers should remain intact. Under these conditions, the rate of desensitization was more stable with time. On average there was a decrease in the value of t½ between 0 and 4 min (Fig. 4B), resulting in a negative value for the maximum percentage increase with time (Fig. 4C); after 4 min, the value of t½ stabilized.
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Figure 4. The kinetics of desensitization were stable in perforated-patch recording conditions
A, 5-HT responses at 0 and 8 min. The values of t½ at 0 and 8 min for this cell were 180 and 192 ms, respectively. B, mean value of t½ (12 cells) with time. C, the maximum percentage increase in t½ in perforated-patch (PP) versus whole-cell recording (WCR) conditions.
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Depolarization slows the desensitization rate in perforated-patch recordings
We next tested whether it was possible to overwhelm the Ca2+-buffering capacity of the cells in perforated-patch recordings by increasing [Ca2+]i and thus inducing a slowing in the rate of desensitization of the 5-HT3R channel; to do this, cells were depolarized to elicit peak Ca2+ currents to induce influx of Ca2+ through voltage-gated Ca2+ channels (data not shown). When cells were depolarized to 0 mV for 400 ms and then 5-HT was applied 200 ms after the end of the depolarizing pulse, the rate of 5-HT3R channel desensitization slowed dramatically (Fig. 5A). 5-HT was applied without the depolarizing pulse from 0 to 16 min; at between 20 and 60 min, the 5-HT application was preceded by the depolarizing pulse. The mean value of t½ increased significantly from 186 ± 30 ms (17 cells) at 20 min to 1430 ± 540 ms at 60 min (Fig. 5B); the increase in t½ was significant from 36 to 60 min. The maximum percentage increase in t½ was 1380 ± 480 % under these conditions. There was no slowing in the rate of desensitization in the absence of the depolarizing pulse (Fig. 5B; up to 60 min); the extent of slowing with the depolarizing pulse was not blocked by tetrodotoxin (1 µM; data not shown), a blocker of voltage-gated Na+ channels. Note that the depolarization to 0 mV elicited net outward (mostly K+) currents. We were not able to block these currents, to reveal net inward Ca2+ currents, with the classical K+ channel blocker TEA as this also blocks 5-HT3R channels (Kooyman, Zwart & Vijverberg, 1993).
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Figure 5. Depolarization prior to 5-HT application in perforated-patch recordings slowed desensitization
A, 5-HT responses at 20 min (the first time point where 5-HT application was preceded by depolarization to 0 mV; upper trace) and 60 min. The values of t½ at 20 and 60 min for this cell were 180 and 4000 ms, respectively. B, mean values of t½ with time without (control;  ; 5 cells) or with the depolarizing prepulse ( ; 17 cells) which began at the 20 min time point (indicated by the arrow).
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BAPTA-AM and cadmium block the depolarization-induced slowing of desensitization in perforated-patch recordings
By analogy with the whole-cell recording experiments, Ca2+ influx through voltage-gated Ca2+ channels seemed the most likely explanation for the depolarization-induced slowing of 5-HT3R channel desensitization. Pre-incubating cells with BAPTA-AM (see Methods and Fig. 6A), a membrane-permeant analogue of BAPTA, or adding cadmium (100 µM; Fig. 6B) to the bathing solution, completely blocked the depolarization-induced slowing in the rate of desensitization. On average, the value of t½ (at 60 min) and the maximum percentage increase in t½ for BAPTA-AM- treated cells was 240 ± 79 ms and 45 ± 47 % (6 cells), respectively, and for cadmium-treated cells it was 126 ± 25 ms and 34 ± 32 % (4 cells); these values were not significantly different from control values obtained without the depolarizing pulse.
Interestingly, pretreatment with BAPTA-AM significantly reduced the rate of 5-HT3R channel desensitization at t = 0 min; the value of t½ for BAPTA-AM-treated cells (0 min) was 1326 ± 329 ms versus a control value of 256 ± 41 ms. In addition, in the BAPTA-AM-treated cells the value of t½ had decreased to 472 ± 181 ms by 16 min in the absence of depolarizing pulses. We are currently exploring the basis of this effect.
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Figure 6. Depolarization-induced slowing of desensitization in perforated-patch recordings was blocked by BAPTA-AM pre-incubation and bath-applied cadmium
A, 5-HT responses at 20 and 60 min in a BAPTA-AM-pretreated cell following a depolarizing prepulse to 0 mV (upper traces); the values of t½ were 431 and 166 ms, respectively. B, 5-HT responses at 20 and 60 min with cadmium (100 µM) added to the bathing solution; the values of t½ were 104 and 96 ms, respectively. C, mean values of t½ with time with a depolarizing prepulse which began at the 20 min time point (indicated by the arrow). BAPTA-AM, 6 cells; cadmium, 4 cells.
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DISCUSSION |
The present study employed the NG108-15 cell line as a model system to address the question of whether [Ca2+]i can regulate desensitization of the 5-HT3R channel. Whole-cell and perforated-patch recording techniques provided several lines of evidence that increased levels of [Ca2+]i via the influx of Ca2+ through cadmium-sensitive voltage-gated Ca2+ channels induced a slowing in the kinetics of desensitization of the 5-HT3R channel.
In the whole-cell recording mode, increasing the [Ca2+]i buffering capacity by replacing EGTA (5 mM) and Ca2+ (0·5 mM) with BAPTA (20 mM) prevented the slowing in the rate of 5-HT3R channel desensitization. We propose that after breaking into the cell to obtain the whole-cell recording configuration, endogenous cytoplasmic Ca2+ buffers dialyse out of the cells, leading to an increase in [Ca2+]i and the activation of a Ca2+-dependent process that induces a dramatic slowing in the kinetics of 5-HT3R channel desensitization. Next we investigated the source of Ca2+ that could elevate [Ca2+]i under our whole-cell recording conditions while clamping the membrane potential to -70 mV. As mentioned above, NG108-15 cells have a variety of mechanisms for increasing [Ca2+]i. In the present study, the slowing in the rate of desensitization when using modest Ca2+-buffering conditions (whole-cell recording with EGTA to buffer [Ca2+]i) was blocked by using the voltage-gated Ca2+ channel blocker cadmium (100 µM), but not nickel (50 µM). As this concentration of nickel should have selectively blocked only the low-voltage-activated T-type voltage-gated Ca2+ channels in NG108-15 cells (Brown et al. 1989; Kasai & Neher, 1992), the block by cadmium suggests the involvement of high-voltage-activated voltage-gated Ca2+ channels in regulating the rate of desensitization of the 5-HT3R channel. Undifferentiated NG108-15 cells express primarily the L-type class of high-voltage-activated voltage-gated Ca2+ channels (Kasai & Neher, 1992). Furthermore, the rate of desensitization varied with membrane potential, being faster at more hyperpolarized versus depolarized potentials. These data are consistent with a potential role linking the influx of Ca2+ via voltage- and cadmium-sensitive channels and a slowing in the rate of desensitization of the 5-HT3R channel.
We considered the possibility that the effect of cadmium in regulating the desensitization of the 5-HT3R channel was due to a block of a Na+-Ca2+ exchange pump, a Ca2+-ATPase, or store-operated Ca2+ entry channels. However the dependence of the rate of desensitization on membrane potential in the whole-cell configuration and the ability of depolarizing pulses to slow the rate of desensitization in the perforated-patch configuration are not consistent with the block of these proteins. Theoretically, inhibiting either the Na+-Ca2+ exchange pump or Ca2+-ATPase would be expected to increase [Ca2+]i, which according to our data should produce a slowing in the rate of desensitization of the 5-HT3R channel. In addition, in NG108-15 cells, it has previously been reported that the Na+-Ca2+ exchange pump does not regulate [Ca2+]i when the intracellular Ca2+ stores are intact (Hsu et al. 1995), and that these cells lack the store-operated Ca2+ entry pathway (Lo & Thayer, 1993).
Another possibility is that our data indicate that there is a basal entry of Ca2+ at -70 mV via voltage- and cadmium-sensitive channels. Evidence for a resting Ca2+ entry through such channels in neuronal preparations is not unprecedented. Recently, combined patch-clamp electrophysiological recording and fluorescence Ca2+ imaging techniques have been used to show that dihydropyridine (DHP)-sensitive voltage-gated Ca2+ channels contribute to the resting (or near resting) level of [Ca2+]i in rat cultured cerebellar granule cells (Marchetti, Amico & Usai, 1995) and hippocampal CA1 pyramidal neurones (Magee, Avery, Christie & Johnston, 1996).
Our hypothesis that, after break-in to obtain a whole-cell recording, the endogenous cytoplasmic Ca2+ buffers are lost is supported by the fact that in perforated-patch recording conditions, where endogenous Ca2+ buffers would be retained, the kinetics of desensitization were stable for up to 60 min. Furthermore under perforated-patch recording conditions, we could induce a dramatic slowing in the rate of 5-HT3R channel desensitization by depolarizing cells; this was done in order to open voltage-gated Ca2+ channels and increase [Ca2+]i levels. The dependence of this effect on [Ca2+]i and voltage-gated Ca2+ channels was supported by the fact that the slowing was blocked by pre-incubation with BAPTA-AM (a membrane-permeant analogue of BAPTA) or by the addition of the voltage-gated Ca2+ channel blocker cadmium to the bath. We propose that the depolarization-induced slowing observed in perforated-patch recordings is due to the fact that the influx of Ca2+ through the cadmium-sensitive voltage-gated Ca2+ channels overwhelms the cellular endogenous Ca2+ buffers and, similar to whole-cell recording conditions, an increase in [Ca2+]i is responsible for inducing the slowing in 5-HT3R channel desensitization.
There are several possible molecular mechanisms whereby [Ca2+]i could alter the function of the 5-HT3R channel. For the NMDA receptor, Ca2+ influx through the channel has been proposed to bind to and activate calmodulin. The Ca2+-calmodulin complex then binds directly to the receptor and inhibits the opening of the channel (Ehlers, Zhang, Bernhardt & Huganir, 1996). Ca2+ could also be activating a Ca2+-dependent enzymatic process that may be altering the function of the 5-HT3R channel. The activation of protein kinase C (PKC) enhanced the function of the 5-HT3R channel in N1E-115 neuroblastoma cells (Van Hooft & Vijverberg, 1995) and in Xenopus oocytes expressing the 5-HT3R channel (Zhang, Oz & Weight, 1995). In addition the Ca2+-calmodulin-regulated protein phosphatase, calcineurin, was also reported to regulate the function of the 5-HT3R channel in NG108-15 cells (Boddeke, Meigel, Boeijinga, Arbuckle & Docherty, 1996). We are currently exploring the possible involvement of these and other Ca2+-dependent enzymatic processes in the regulation of the 5-HT3R channel desensitization. Other Ca2+-dependent signalling pathways have been shown to be activated by depolarization-evoked Ca2+ entry in NG108-15 cells, such as Ca2+-calmodulin-dependent protein kinases (Enslen, Tokumitsu, Stork, Davis & Soderling, 1996; Higuchi et al. 1996), and these enzymes may also participate in the regulation of 5-HT3R channel function. Further evidence in favour of the role of a phosphorylation cascade in the slowing in the rate of 5-HT3R channel desensitization is the dependence of this process on the hydrolysis of ATP (Yakel et al. 1991).
The kinetics of desensitization of the ligand-gated ion channels gated by glutamate (NMDA receptors; Legendre et al. 1993; Vyklick´y, 1993; Medina et al. 1995; Kyrozis et al. 1996), ATP (P2X receptors; Khiroug et al. 1997b), ACh (nicotinic receptors; Khiroug et al. 1997a) and capsaicin (Koplas et al. 1997) are regulated by increases in [Ca2+]i, the source of which is thought to be an influx of Ca2+ through these highly Ca2+-permeable channels. However ligand-gated ion channel activity is also known to be regulated by Ca2+ influx through other ion channels. For example, NMDA receptor channel desensitization kinetics are regulated by Ca2+ influx through AMPA receptors (Kyrozis et al. 1995) and voltage-gated Ca2+ channels (Legendre et al. 1993; Kyrozis et al. 1996), and GABAA receptors are inhibited by Ca2+ influx through nicotinic ACh receptors (Mulle, Choquet, Korn & Changeux, 1992). The issue of whether native and/or expressed 5-HT3R channels are Ca2+ permeable is controversial (see review by Jackson & Yakel, 1995; Gilon & Yakel, 1995). Initially, based on electrophysiological reversal potential measurements, the Ca2+ permeability of native 5-HT3Rs was thought to be very low. However, in the N18 neuroblastoma cell line (Yang, 1990) and in rat superior cervical ganglion (SCG) neurones (Yang, Mathie & Hille, 1992), the relative Ca2+ permeability of the 5-HT3R was reported to be substantial. Using Ca2+ imaging techniques to record changes in [Ca2+]i signals due to activation of native 5-HT3Rs in the N1E-115 neuroblastoma cells or expressed 5-HT3Rs in HEK 293 cells, Hargreaves, Lummis & Taylor (1994) also reported a relatively high Ca2+ permeability. However, in this and the previous studies (Yang, 1990; Yang et al. 1992), the high Ca2+ permeability of the 5-HT3R was associated with a very low external bath concentration of Na+ and K+; Hargreaves et al. (1994) showed that the Ca2+ permeability of the 5-HT3R was much lower in physiological ionic solutions, i.e. solutions high in Na+. Thus we conclude that under our present recording conditions, the Ca2+ permeability of the native 5-HT3R channels in NG108-15 cells is very low or non-detectable. Using laser-scanning confocal microscopic techniques to measure [Ca2+]i signals due to the activation of native 5-HT3Rs in NG108-15 cells, Rondé & Nichols (1997) recently published data consistent with this suggestion.
The Ca2+-dependent regulation of 5-HT3R channel desensitization may be an important physiological phenomenon during synaptic transmission. Although the precise role that the desensitization of 5-HT3Rs plays in regulating synaptic activity in the nervous system is unknown, the onset and recovery of desensitization for ligand-gated ion channels in general can have profound effects on neuronal signal processing and integration (Jones & Westbrook, 1996). There are many ways in which [Ca2+]i levels may be regulated by 5-HT receptors in the nervous system, including the activation of G protein-/phospholipase C-coupled 5-HT receptors and the opening of voltage-gated Ca2+ channels in response to the 5-HT3R-induced depolarization of neurones, all of which could contribute to the regulation of the 5-HT3R channel via a mechanism similar to that described in this report. Recently 5-HT3Rs have been shown to play an important role in mediating the induction and maintenance of long-term potentiation in the rat superior cervical ganglion (Alkadhi, Salgado-Commissariat, Hogan & Akpaudo, 1996). Although the molecular details of how this action occurs are presently unknown, a role for the regulation of the 5-HT3R by [Ca2+]i, such as that described in the present paper, could be involved.
In conclusion, we have provided strong evidence for cross-talk between the voltage-gated Ca2+ channels and the 5-HT3Rs in NG108-15 clonal cells, and we propose that Ca2+ entry through voltage-gated Ca2+ channels provides an important mechanism for the Ca2+-dependent regulation of the 5-HT3R channel in the nervous system.
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Acknowledgements
We would like to thank David L. Armstrong, Sterling Sudweeks and Meyer Jackson for critical reading of this manuscript.
Corresponding author
J. L. Yakel: NIEHS, F2-08, PO Box 12233, 104 T. W. Alexander Drive, Research Triangle Park, NC 27709, USA.
Email: yakel{at}niehs.nih.gov
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