Address correspondence to Malcolm Slaughter, Department of Physiology and Biophysics, University at Buffalo, 124 Sherman Hall Buffalo, NY 14214. Tel.: (716) 829-3240; Fax: (716) 829-2344; E-mail: mslaught{at}buffalo.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: BK channels ryanodine receptors transient potassium current ganglion cells dihyropyridines
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Subsequently, Merriam et al. (1999) reported that Ca2+ influx through VGCCs was necessary for SMOC generation in mudpuppy parasympathetic neurons, even though SMOCs occurred in the presence of Cd2+, albeit at a lower frequency. They found that SMOC generation in these neurons involved a process of Ca2+ influx that triggered Ca2+-induced Ca2+ release (CICR), presumably analogous to the mechanism seen in cardiac cells. Although lowering Ca2+ influx into the cell lowered the frequency of SMOCs, their amplitude and other parameters were found to be largely unaffected.
As described in the preceding paper (Mitra and Slaughter, 2002, this issue), SMOCs in retinal amacrine cell neurons result from a sequence of events that includes Ca2+ influx through high voltageactivated (HVA) Ca2+ channels (including the L-type) and subsequent amplification by CICR (Mitra and Slaughter, 2002
, this issue). However, a characteristic feature of these cells is that SMOCs in normal Ringer's appear exclusively within the narrow voltage range of -60 to -40 mV. Depolarization beyond -40 mV leads to disappearance of SMOCs, along with the appearance of a transient (Ito) and a sustained (Iso) outward K+ currents. Since retinal SMOCs were shown to be K+ currents and the recordings were made in quasiphysiological K+ gradients (Ek = -95 mV), it is intriguing as to why they disappear at depolarized levels. It would be expected that depolarization, by providing a larger driving force for K+, should enhance their amplitudes.
Experiments described here indicate that Ca2+ influx serves two roles: both to generate and to eliminate discrete SMOCs. At comparatively low levels of Ca2+ influx, such as would occur at negative membrane potentials or when VGCCs were partially blocked, discrete SMOCs are generated and have large amplitudes. However, when Ca2+ influx increases then SMOCs undergo a negative modulation of their amplitudes and a decrease in their first latency. As a consequence, discrete SMOCs disappear and a fast transient outward current (Ito) appears. Preliminary descriptions of initial aspects of this work have been presented previously (Mitra and Slaughter, 2000).
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To isolate calcium currents, the cells were bathed in an extracellular solution containing (in mM) 23 NaCl, 80 TEA, 2.5 KCl, 10 CaCl2, 1 MgCl2, 10 dextrose, and 5 HEPES, buffered to pH 7.8 with NaOH, and oxygenated. The internal pipette solution for such recordings contained (in mM) 40 K-gluconate, 70 TEA, 5 NaCl, 1 MgCl2, 5 EGTA, 5 HEPES, and was adjusted to pH 7.4 with KOH. The pipette solution also contained an "ATP-regenerating cocktail" consisting of 4 mM ATP, 20 mM phosphocreatine, and 50 U/ml creatine phosphokinase. The voltage values stated have been corrected for pipette junction potential but not for series resistance. Data are expressed as mean ± SEM. Statistical differences were ascertained by Student's t test, where P < 0.05 was deemed significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SMOC Frequency
This section reports results of investigations determining the effects of depolarization and the extent of Ca2+ influx on SMOC frequency. To test the effect of Ca2+ influx on SMOC frequency, SMOCs were generated at a constant voltage of -10 mV in 6 mM Co2+ Ringer's containing increasing amounts of [Ca2+]. As mentioned in the Mitra and Slaughter (2002)(this issue), SMOCs were absent when bath Ca2+ was kept at nominal levels. However, increasing bath [Ca2+] led to their appearance and a monotonic increase in frequency within the range tested. Fig. 1 A shows representative traces of SMOCs at 0.9 mM and 4.5 mM [Ca2+]. A plot of SMOC frequency vs. [Ca2+] from the same cell is shown in Fig. 1 B. The SMOC frequency at 0.9 mM [Ca2+] was 7 ± 1.4 Hz. SMOC frequency increased with increased [Ca2+], leading to a frequency of 54.2 ± 1.6 Hz at 4.5 mM [Ca2+] (P < 0.01). In eight cells, average SMOC frequencies of 4.5 ± 0.7, 26.3 ± 2.9, and 44.4 ± 3.1 Hz were observed at 0.9, 2.7, and 4.5 mM [Ca2+], respectively. These values are significantly different from each other at P < 0.01. All experiments described in this and subsequent sections, in which the bath [Ca2+] was varied, gave results that were fully reversible.
|
In control Ringer's, SMOCs occurred over a narrow voltage range from -60 to -40 mV. However, when an inorganic HVA Ca2+ channel blocker (6 mM Co2+) was present in the bathing solution, SMOCs were induced at voltages of -20 mV and above (Mitra and Slaughter, 2002, this issue). This afforded an opportunity to evaluate SMOCs over a much broader voltage range. To investigate the effects of voltage on SMOC frequency, SMOCs were generated in 6 mM Co2+/1.8 mM Ca2+ Ringer's by a protocol which stepped cell voltage from -20 to 70 mV in 10-mV increments. Fig. 1 C shows representative SMOC recordings at 70, 30, and -10 mV. SMOCs were more frequent at the intermediate voltage of 30 mV as compared with either extreme. This cell had SMOC frequencies of 4.4 ± 0.5 Hz at -10 mV, 9.7 ± 0.9 Hz at 30 mV, and 3.8 ± 0.6 Hz at 70 mV. A plot of SMOC frequency at various voltages reveals a biphasic relationship (Fig. 1 D). The average SMOC frequencies in 10 cells under such conditions were 4.6 ± 0.4 Hz at -10 mV, 9.3 ± 0.7 Hz at 30 mV, and 3.9 ± 0.4 Hz at 70 mV. The frequency at 30 mV was significantly different from either extreme (P < 0.01). Such a biphasic effect of voltage on SMOC frequency has also been noted in other neuronal types (Satin and Adams, 1987
; Merriam et al., 1999
). The simplest interpretation of this response is that Ca2+ influx through VGCCs is biphasic. Ca2+ influx is governed by the channel open probability and the probability of unblock by cobalt, both of which increase with depolarization, and the calcium driving force, which decreases with depolarization.
Peak SMOC Amplitudes
Ca2+ influx affected SMOC amplitude as well as frequency. SMOCs were generated at 30 mV in 6 mM Co2+ Ringer's solution containing increasing amounts of [Ca2+] and peak SMOC amplitudes were determined. Fig. 2 A is a plot of average SMOC amplitudes at various [Ca2+] obtained from seven cells, showing that peak amplitudes declined in a graded manner as bath [Ca2+] increased. SMOCs under these conditions had peak amplitudes of 204.9 ± 10.4 pA, 178 ± 5.4 pA, and 150.8 ± 4.3 pA in 0.9, 1.8, and 2.7 mM [Ca2+], respectively. These values are significantly different from each other, with the value at 2.7 mM being different from that obtained at 0.9 mM at P < 0.01. This suggests that Ca2+ influx causes a graded reduction in the mean peak SMOC amplitudes.
|
If increased Ca2+ influx results in a decrease in mean peak SMOC amplitudes, then reduction of Ca2+ influx should result in an increase. Experimentally, Ca2+ influx was reduced either by addition of the DHP antagonist nifedipine, or by increasing amounts of bath Co2+. Fig. 2 C shows results from a cell in which SMOCs were generated at 30 mV while increasing amounts of cobalt were added to Ringer's containing 1.8 mM Ca2+. The mean SMOC amplitude in this cell was 215 ± 12 pA when 4 mM Co2+ was present in the bath solution. Increasing the Co2+ to 8 mM raised the mean amplitude reversibly to 389 ± 29 pA (P < 0.01). In eight cells, mean SMOC amplitudes were 182.5 ± 5 pA and 244.8 ± 12.7 pA in 4 and 8 mM Co2+, respectively (P < 0.01). Reduction of Ca2+ influx by nifedipine gave similar results (n = 18). A representative data plot is shown in Fig. 2 D. The mean peak SMOC amplitude in this cell, for control SMOCs generated at -10 mV in 6 mM Co2+/1.8 mM Ca2+ Ringer, was 114 ± 3 pA. Nifedipine caused a dose-dependent increase in the mean amplitude, raising it to 168 ± 9 pA at 30 µM (P < 0.01). Average data from seven cells in which SMOCs were generated at 30 mV in 6 mM Co2+/1.8 mM Ca2+ Ringer's gave a mean amplitude of 216.5 ± 5.2 pA. Amplitude increased to 304.7 ± 11.9 pA and 332.8 ± 16.6 pA with addition of 10 and 30 µM nifedipine, respectively (P < 0.01). The effects of nifedipine described here and in subsequent sections were partially reversible on washout of the drug. The lack of reversibility seen with DHPs is perhaps due to their lipid solubility, which results in uptake into membranes (Pang and Sperelakis, 1984; Nerbonne and Gurney, 1987
), particularly when high doses were used.
These results reveal that SMOCs are negatively modulated by Ca2+, whereby an increase in its influx causes a decrease in peak SMOC amplitude. Conversely, reducing Ca2+ influx is equivalent to removal of the negative modulation, as shown by the increase in the peak amplitudes obtained either by nifedipine or elevated Co2+.
In the same set of data that was used to study the effect of voltage on SMOC frequency, variation of SMOC amplitudes with the level of depolarization was examined. Fig. 2 E shows an I-V plot for SMOCs obtained from a representative cell. There is an increase in peak SMOC amplitude with depolarization. The mean SMOC amplitude at -10 mV is 152 ± 8 pA. The amplitude increased to 227 ± 9 pA at 30 mV and to 511 ± 43 pA at 70 mV. Average data from 10 cells gave mean SMOC amplitudes of 148.7 ± 5.4 pA, 237.1 ± 6.1 pA, and 541.1 ± 23.1 pA at -10, 30, and 70 mV respectively (values are significantly different from each other at P < 0.01). It should be noted that the gradient of the curve in Fig. 2 E is not constant along the voltage axis. Although increasing relatively linearly from -20 to 40 mV, it tends to show an increase in slope beyond 40 mV. Since SMOCs are K+ currents, a linear increase in peak amplitude with depolarization is explained as an increased driving force for K+ ions. The reason for the increase in slope above 40 mV was not determined. It may reflect a pure voltage dependence of SMOCs, or it may be due to removal of the Ca2+-induced negative modulation of SMOCs at very depolarized voltages (see DISCUSSION).
Temporal Dependence of SMOC Amplitudes
Although increased Ca2+ influx reduced peak SMOC amplitudes, this reduction was greater for SMOCs occurring in the latter half of a 500 ms depolarizing voltage step. This temporal dependence becomes significant at higher [Ca2+]. Fig. 3 A shows representative traces for SMOCs generated with 500-ms steps to 30 mV in the presence of 6 mM Co2+ Ringer's containing 0.9 or 2.7 mM Ca2+. The double-headed arrows mark the midpoint of the 500-ms voltage steps. In 2.7 mM [Ca2+], SMOCs elicited in the latter 250 ms (ß) are on the average smaller in peak amplitude as compared with those seen in the first half of the step (). Such a temporal dependence is not obvious with SMOCs generated in 0.9 mM [Ca2+]. Fig. 3 B is a plot from the same cell of mean SMOC amplitudes in the first and second 250-ms intervals of the voltage step, as ascertained under different [Ca2+]. It illustrates the temporally dependent reduction of amplitudes, with statistically significant reductions obtained for SMOCs generated in 2.7 and 3.6 mM [Ca2+] (P < 0.01). The mean SMOC amplitude in this cell elicited with 3.6 mM [Ca2+] in the first 250 ms of the pulse (
) was 291 ± 37 pA, and was reduced by
55% to 132 ± 7 pA in the latter half of the pulse (ß). Average data from seven cells yielded mean peak SMOC values of 277.8 ± 27 pA (
) and 312.8 ± 30.9 pA (ß) under 0.9 mM [Ca2+], values which are not significantly different at P < 0.05. But increasing [Ca2+] to 2.7 mM gave values of 255.9 ± 12.8 pA (
) and 129.7 ± 4.9 pA (ß); a reduction of
50% in the latter half of the pulse (P < 0.01).
|
In addition to modulation of SMOC amplitudes, SMOC decay characteristics were modulated by Ca2+ influx in a qualitatively parallel manner. Experimental conditions that permitted greater Ca2+ influx resulted in SMOCs that decayed faster (measured as the time required to decay to 50% of peak or t1/2). This reduction in decay time was graded with the extent of influx. Average data from seven cells in which SMOCs were generated at 30 mV in 6 mM Co2+ Ringer's containing varying amounts of [Ca2+] gave t1/2 values of 3.26 ± 0.09, 3.01 ± 0.06, and 2.86 ± 0.06 ms at 0.9, 1.8, and 2.7 mM [Ca2+], respectively (values are significantly different at P < 0.05 with respect to 0.9 mM [Ca2+]). Increasing Ca2+ influx by addition of the DHP agonist resulted in SMOCs that were more transient. Average data from six cells in which SMOCs were generated at -10 mV in 6 mM Co2+/1.8 mM Ca2+ Ringer's gave t1/2 values of 3.09 ± 0.08 ms (control) and 2.84 ± 0.06 ms (3 µM S [-] BayK 8644). These values are significantly different from each other at P < 0.05. Reducing Ca2+ influx with nifedipine gave opposite results. Average data from seven cells in which SMOCs were generated at 30 mV in 6 mM Co2+/1.8 mM Ca2+ Ringer's gave t1/2 values of 3.12 ± 0.09 ms (control), 3.92 ± 0.17 ms (10 µM nifedipine), and 4.42 ± 0.22 ms (30 µM nifedipine). These values are significantly different from control at P < 0.01. Reducing Ca2+ influx into the cell by depolarizing the cell to very positive potentials produced similar results. SMOCs generated at -10 mV were more transient as compared with those generated at 70 mV. Average data from 10 cells gave t1/2 values of 3.73 ± 0.12, 3.85 ± 0.1, and 6.24 ± 0.25 ms at -10, 30, and 70 mV, respectively. The value obtained at 70 mV is significantly different from -10 mV at P < 0.01. It is not possible to ascertain via our experiments whether the slowing of decay at extremely positive voltages is an inherently voltage-dependent feature, or if it is a consequence of reduction of Ca2+ influx. It is likely to be a combination of both. The biphasic nature of Ca2+ influx into the cell through VGCCs might account for the fact that the t1/2 at 30 mV is not significantly different from that at -10 mV. Similar to SMOC amplitudes, the 50% decay times of SMOCs showed a temporal dependence under conditions of increased influx. SMOCs generated in the latter part of the pulse were more transient as compared with those generated in the initial part. Average data from seven cells gave t1/2 values of 3.77 ± 0.22 ms (; 0250 ms) and 4.11 ± 0.29 ms (ß; 250500 ms) with 6 mM Co2+/0.9 mM Ca2+ Ringer's. These values are not significantly different at P < 0.05. However, in 6 mM Co2+/2.7 mM Ca2+ Ringer's, t1/2 values of 3.61 ± 0.13 ms (
) and 2.68 ± 0.12 ms (ß) were obtained (significantly different at P < 0.01). Increasing Ca2+ influx into the cell via addition of 3 µM of the DHP agonist (n = 6) introduced a temporal dependence in the decay times, whereas reduction of influx via addition of the antagonist nifedipine (15 µM; n = 5) or increasing doses of Co2+ (n = 8) eliminated it (unpublished data).
First Latency to SMOC Occurrence
Another parameter examined for its Ca2+ and voltage dependence was the first latency to SMOC occurrence. It is defined as the average time that elapses after the onset of the voltage clamp step, before detection of the first SMOC. SMOCs were generated at -10 mV in 6 mM Co2+ Ringer's containing varying amounts of [Ca2+], and the mean first latency to their occurrence was measured. Fig. 4 A shows representative traces generated at 0.9, 1.8, and 2.7 mM [Ca2+]. The mean first latencies from this cell are plotted in Fig. 4 B as a function of [Ca2+]. There is a graded decrease in the mean first latency to SMOC occurrence as [Ca2+] increased. The mean first latency in this cell is 189 ± 37.3 ms at 0.9 mM [Ca2+], and it decreases to 21.3 ± 4.6 ms at 2.7 mM [Ca2+] (P < 0.01). Further increase to 3.6 mM [Ca2+] leads to a value of 7.4 ± 1.3 ms, suggesting that at these high levels of influx, the delay to appearance of the first SMOC is extremely small. Average data from six cells gave a mean first latency value of 204.2 ± 24.5 ms at 0.9 mM [Ca2+], which decreased to 52.3 ± 9.3 ms at 2.7 mM [Ca2+] (P < 0.01).
|
First latency values were found to be biphasic with voltage when SMOCs were generated at different voltage levels with a constant [Ca2+]. SMOCs were generated in 6 mM Co2+/1.8 mM Ca2+ Ringer's by a protocol in which the voltage was stepped to -10, 30, and 60 mV. Average first latency values obtained from 10 cells are shown in Fig. 4 E. SMOCs at -10 mV appeared with a mean first latency of 225.2 ± 17 ms. Subsequent depolarization to 30 mV decreased mean first latency to 99.3 ± 11.3 ms. However, further depolarization to 60 mV lead to an increase in the mean first latency (167.4 ± 18.6 ms). The values at -10 and 60 mV were significantly different from the first latency observed at 30 mV (P < 0.01).
The Ca2+-induced Transition of SMOCs into the Ito
The results presented thus far suggest a Ca2+ influx dependence of SMOCs, whereby increasing influx lead to a temporally dependent reduction in peak amplitude and first latency of SMOC occurrence. An extreme consequence of these effects is that at high levels of Ca2+ influx individual SMOCs disappear, with the concomitant appearance of the transient outward K+ current (Ito). This occurs in a gradual fashion as influx increased (Fig. 5). Cells were bathed in 6 mM Co2+ Ringer's containing varying amounts of [Ca2+] (0.9, 1.8, 2.7, 3.6, and 4.5 mM) and pulsed to 30 mV. Fig. 5 A is a trace from a representative cell in 0.9 mM [Ca2+]. Individual SMOCs are clearly evident. However, increasing bath [Ca2+] caused a gradual disappearance of individual SMOCs along with the appearance of the Ito. In this cell, at 3.6 mM [Ca2+], individual SMOCs completely disappear, leaving the Ito (Fig. 5 D). In a total of eight cells studied at 30 mV, three cells displayed a total disappearance of SMOCs and generated the Ito by 3.6 mM [Ca2+], whereas in the rest SMOCs disappeared at 4.5 mM [Ca2+]. Thus, increasing levels of Ca2+ influx into the cells causes a gradual disappearance of discrete SMOCs along with the appearance of the Ito.
|
Individual SMOCs and the Ito Show Similar Pharmacological Sensitivities
The results indicate that Ito is formed from SMOCs. It was shown in the previous paper that both SMOCs and the Ito were Ca2+ influxsensitive. SMOCs are K+ currents through BK channels and are insensitive to apamin but blocked by TEA and iberiotoxin. They are also dependent on CICR from caffeine- and ryanodine-sensitive stores (Mitra and Slaughter, 2002, this issue). If SMOCs lead to the Ito, it follows they should have similar pharmacological sensitivities. Fig. 6 presents data supporting this hypothesis. Similar to SMOCs, the Ito was eliminated on depletion of intracellular stores with 10 mM caffeine (n = 10). Fig. 6 A1 shows recordings in normal Ringer's from a representative cell with a voltage step to 10 mV. Addition of 10 mM caffeine results in a selective elimination of the Ito (A2). The effects of caffeine were fully reversible on washout of the drug. 10 mM caffeine selectively blocked the Ito generated by depolarizations up to 10 mV while leaving the sustained outward current, Iso, unchanged. However, at more depolarized levels such as 50 or 70 mV, 10 mM caffeine did reduce the Iso in addition to eliminating the Ito. This is presumably because caffeine at these doses is known to block the delayed rectifier K+ current, the inhibition being greater at more depolarized voltages (Reiser et al., 1996
). Recordings in normal Ringer's with 200 µM ryanodine in the internal pipette solution also led to an elimination of the Ito at all voltages at which it appears (n = 6; unpublished data). This finding, combined with the effect of store depletion by caffeine, suggests that CICR generates Ito as well as SMOCs.
|
The Ca2+ Influx Pathway Is not a Likely Site for the Negative Modulation of SMOCs
The earlier sections showed that increasing Ca2+ influx caused a temporally dependent, graded reduction in peak SMOC amplitudes. This constitutes a Ca2+ influx induced negative modulation of SMOCs. The disappearance of SMOCs and the concomitant appearance of the Ito results from a combination of increased levels of negative modulation, combined with the decreased first latency to SMOC appearance seen with increasing Ca2+ influx. Based on the SMOC generation pathway elucidated in the preceding paper (Mitra and Slaughter, 2002, this issue), there are three potential sites where Ca2+-induced negative modulation could occur. The first candidate is the Ca2+ influx pathway (HVA Ca2+ channels), since CICR is graded with the amount of Ca2+ influx in a number of other cell types (Berridge, 1997
). A smaller Ca2+ release from the stores would give rise to lower amplitude SMOCs (Bolton and Imaizumi, 1996
).
HVA Ca2+ channels, especially the L-type, are known to exhibit voltage and Ca2+-dependent inactivation (see DISCUSSION). Thus, increased influx might inactivate the calcium channel, reduce CICR, and result in the calcium- and time-dependent reduction in SMOC frequency and amplitude, eventually leading to only Ito. If true, there should be a correspondence between the temporal characteristics of the Ca2+ current and Ito. Fig. 7 A shows recordings of Ito in normal Ringer's at voltages of -40, -10, 10, and 30 mV. The Ito is least transient at -40 mV. However, in all cells examined, it decays completely within 15 ms. Thus, if this transient feature arises due to Ca2+-induced inactivation of the HVA Ca2+ current, it follows that the Ca2+ current should show a significant degree of inactivation within this time frame. To test this possibility, recordings of HVA Ca2+ currents were made from cells displaying SMOCs. These currents were recorded using 10 mM [Ca2+] as the charge carrier (n = 6). This would provide an influx level such that the degree of Ca2+-dependent inactivation is exaggerated. Fig. 7 B shows whole cell Ca2+ current recordings from a representative cell with a 30 ms pulse to 10 mV. These currents do not show any significant degree of inactivation. Since it was shown in the previous paper that DHP-sensitive L-type Ca2+ channels are involved in the SMOC generation (Mitra and Slaughter, 2002, this issue), a dissection of the DHP-sensitive component was done. Addition of 30 µM nifedipine resulted in a parallel reduction of the control current. Nifedipine (30 µM) blocked 32 ± 3% of the control current and the effects of nifedipine were partially reversible on return to control solutions (value is an average of five cells). Since Ca2+ channels run down during recordings with high concentrations of Ca2+, the percentage of block seen with nifedipine is probably an overestimate. Thus, the Ca2+ influx pathway is in all probability not the site for the Ca2+-induced negative modulation of SMOCs. The other two candidates, namely the ryanodine receptors and BK channels, are also known to exhibit Ca2+-induced negative feedback. However, their role was not tested in this study.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Calcium-dependence of SMOC Appearance and Disappearance
In cultured mouse dorsal root ganglion cells, Mathers and Barker (1984) reported that the current variance of cells displaying SMOCs increased markedly on moving the clamp potential from -60 to -30 mV, but declined above this voltage. Thus, the authors suggested that the membrane process responsible for SMOCs is either fully activated or completely inactivated at membrane potentials positive to -20 mV. Similar to their findings, it was shown by Mitra and Slaughter (2002)
(this issue) that recordings in normal Ringer's from an amacrine cell subtype revealed SMOC activity within the narrow voltage window of -60 to -40 mV. Depolarizations beyond these values led to the disappearance of SMOCs. This was intriguing, since the potassium driving force should increase with depolarization, enhancing SMOC amplitudes.
Since suppression of calcium channels permitted SMOCs to appear at more positive voltages, it suggested that a Ca2+-dependent factor governs not only the appearance, but also the disappearance of SMOCs. Data obtained in this and Mitra and Slaughter (2002)(this issue) show that increasing Ca2+ influx through HVA Ca2+ channels increases SMOC frequency. Thus, Ca2+ influx up to levels that do not result in a significant degree of negative modulation are stimulatory to SMOC appearance. However, the data presented in this paper show two other notable effects of increased Ca2+ influx on SMOCs: both their latency and amplitudes were reduced. Extrapolating the data from experiments in the presence of cobalt to normal conditions indicates that high Ca2+ influx would not only reduce SMOC amplitudes, but also cause them to appear earlier, the combination of which leads to the generation of the transient outward current (Ito).
The mean amplitude of SMOCs was reduced in a graded manner with increasing Ca2+ influx. The method of analysis employed does not disclose the exact nature of this reduction. It could arise from either a lowering in amplitude of all individual SMOCs or a relatively increased appearance of small amplitude SMOCs. At high Ca2+ influx levels, this reduction was found to show a temporal dependence; with SMOCs generated in the latter part of the pulse displaying lower amplitudes relative to those present in the earlier part of the pulse. Since this phenomenon can be manipulated by varying extracellular calcium or by modulating the calcium channel, it is likely to represent the effects of calcium influx. The modulation of SMOC amplitude constitutes a temporally dependent Ca2+ influxinduced negative modulation of SMOCs.
Modulation of the Ca2+ influx pathway by the DHP agonist S (-) BayK 8644, or the antagonist nifedipine, gave results consistent with those obtained by manipulating [Ca2+]. Nifedipine, though considered to be relatively selective for the L-type Ca2+ channel at low doses, is known to inhibit K+ channels, especially at the high end of the range of doses used in this study (Fagni et al., 1994; Avdonin et al., 1997
; Zhang et al., 1997
). Since SMOCs are K+ currents, there was a possibility that the effects of nifedipine reflected a blockade of K+ channels rather than a reduction of Ca2+ influx. However, contrasting the nifedipine effect with other K+ channel blockers such as TEA and iberiotoxin argued against this possibility. It was shown in Mitra and Slaughter (2002)
(this issue) that both TEA and iberiotoxin caused a graded reduction in SMOC amplitude. Thus, K+ channel block reduces SMOC amplitude. Nifedipine, on the other hand, enhanced SMOC amplitude, suggesting an effect independent of K+ channel block. Moreover, the reverse effect of S (-) BayK 8644 would tend to support a specific effect of these DHPs on the Ca2+ influx pathway.
In addition, elevated Ca2+ influx produced faster SMOC decay times. Although it is possible that this faster decay caused a reduction in SMOC amplitude, this may be an artifact of the measurement technique, since SMOC amplitudes were not constant under conditions of different Ca2+ influx. The peak-to-peak magnitude of the baseline noise could affect the measurement of SMOC parameters, such as amplitude and decay time. Although the absolute magnitude of the error in measuring amplitudes is constant for SMOCs of all amplitudes, this is not the case with the measurement of decay times. Decay time measurements of smaller SMOCs are more prone to errors that would underestimate the decay time. Since our analysis showed that smaller SMOCs were more transient, it was not possible to ensure that measurement error did not contribute to this result.
Coupled with the change in SMOC amplitude, the mean first latency to SMOC occurrence decreased as the amount of Ca2+ influx increased. A combination of these two Ca2+-dependent phenomena resulted in SMOCs appearing sooner in response to a voltage pulse and being bigger in peak amplitude in the earlier part, while disappearing in the latter part. At relatively high levels of Ca2+ influx (3.6 or 4.5 mM [Ca2+] at 30 mV in 6 mM Co2+ Ringer's), an extreme consequence of these two effects was the disappearance of individual SMOCs and the appearance of the transient outward current, Ito.
These observations explain the appearance of SMOCs within the narrow voltage range of -60 to -40 mV in normal Ringer's. The open probability (Po) of HVA Ca2+ channels is relatively low in this hyperpolarized range (Bean, 1989). Consequently, the Ca2+ influx into the cell is low, a condition conducive to the appearance of discrete SMOCs. Depolarizations beyond this range would cause a greater activation of HVA Ca2+ channels, a greater Ca2+ influx, and the disappearance of discrete SMOCs, and also the concomitant appearance of the Ito.
Further support for this explanation comes from the pharmacological similarity between SMOCs and the Ito. It was shown in the preceding paper that SMOCs are Ca2+ influx sensitive, outward currents that are blocked by TEA and iberiotoxin. They are generated by CICR, so high doses of ryanodine or caffeine lead to their elimination (Mitra and Slaughter, 2002, this issue). Similarly, Ito is blocked by TEA, iberiotoxin, and high doses of ryanodine or caffeine. Thus, the Ito, similar to SMOCs, is generated by a process of CICR and represents K+ fluxes through BK channels.
Site of SMOC-negative Modulation
Increasing Ca2+ influx leads to a temporally dependent reduction of SMOC amplitudes; this reduction constituting a Ca2+ influxinduced negative modulation of SMOCs. Based on the SMOC generation pathway, all three nodes (namely, the HVA Ca2+ channel, ryanodine receptor, or the BK channel) are potential sites, either individually or in concert, for this negative modulation.
HVA Ca2+ channels, such as the L-type, exhibit Ca2+-dependent inactivation, in addition to pure voltagedependent inactivation (Kass and Sanguinetti, 1984; Hadley and Hume, 1987
; Kalman et al., 1988
; de Leon et al., 1995
). However, our experiments suggest that inactivation of the Ca2+ current is not a likely candidate for this negative modulation. Ca2+ currents were recorded under high [Ca2+] (10 mM) at depolarized voltages which elicit the Ito. These are conditions that should accentuate Ca2+-induced inactivation. Recordings of Ca2+ currents with 30-ms pulses (a duration in which the Ito is known to have fully activated and decayed) from cells showing SMOCs and the Ito, did not reveal significant time-dependent inactivation. Even though the L-type HVA Ca2+ channel is involved in SMOC generation, pharmacological isolation of the nifedipine-sensitive Ca2+ current did not disclose significant inactivation during this time course.
The other two potential sites are the ryanodine receptor and the BK channel. The ryanodine receptor Ca2+ release channel can be negatively modulated by Ca2+. Biophysically, there are two distinct negative modulations of this receptor, namely Ca2+-induced inactivation (Ma et al., 1988; Bezprozvanny et al., 1991
; Simon et al., 1991
; Percival et al., 1994
; Jong et al., 1995
) and adaptation of the receptor to pulses of Ca2+ (Gyorke and Fill, 1993
; Gyorke and Palade, 1994
; Gyorke et al., 1994
; Yasui et al., 1994
; Valdivia et al., 1995
; Velez et al., 1997
). Both mechanisms could potentially lead to negative modulation of SMOCs. The final potential site is the BK channel, some subtypes of which are inactivating (Pallotta, 1985
; Solaro and Lingle, 1992
; McLarnon, 1995
; Solaro et al., 1995
; Hicks and Marrion, 1998
; Smith and Ashford, 2000
). Solaro and Lingle (1992)
reported a BK channel in rat adrenal chromaffin cells which undergoes rapid and complete inactivation. The inactivation is trypsin-sensitive and is enhanced by increased intracellular Ca2+ and depolarization. An examination of the above mentioned nodes might provide insights into the exact mechanism underlying the Ca2+ influxinduced negative modulation observed in this study.
High levels of Ca2+ influx might directly activate BK channels that produce the Iso-Ca. This would reduce the number of BK channels available to generate SMOCs, thus leading to a reduction in their amplitudes. However, such a mechanism would necessitate a time-dependent increase of the baseline current, since reduction of SMOC amplitudes is temporally dependent. An increase in baseline current was not observed. Thus, such a mechanism may contribute to, but not be solely responsible for, the Ca2+ influxinduced negative modulation of SMOCs.
Use of Inorganic Blockers to Evaluate the Voltage Dependence of SMOCs
To assess the effect of voltage on SMOC characteristics, SMOCs were generated at depolarized levels in Co2+ Ringer. HVA Ca2+ channel block by inorganic blockers such as Cd2+, Co2+, La3+, and Mg2+ is concentration- and voltage-dependent, with these ions acting as pore blockers (Lansman et al., 1986; Huang et al., 1989
; Winegar et al., 1991
). For a positive ion sensing the membrane electric field, hyperpolarization would pull the blocker into the cell, whereas depolarizations would tend to force it out of the channel into the external solution (Woodhull, 1973
; Fukushima and Hagiwara, 1985
; Rosenberg and Chen, 1991
; Carbone et al., 1997
). Depolarization activates the HVA Ca2+ channel (Bean, 1989
) and would also relieve the block (Thevenod and Jones, 1992
; Wakamori et al., 1998
; but see Byerly et al., 1984
; Swandulla and Armstrong, 1989
; Chow, 1991
). Membrane surface charge screening by divalent ions would produce a depolarizing shift of calcium channel activation (Frankenhaeuser and Hodgkin, 1957
; Hille, 1992
). Both mechanisms might be operative when divalent cations are added to reduce flux through the HVA Ca2+ channel. For either mechanism, a depolarizing voltage shift would result in an increase in the apparent (activated and unblocked) whole cell HVA Ca2+ channel conductance. The extent of Ca2+ influx would be governed by the product of the apparent conductance and the driving force for Ca2+ ions at that particular voltage.
Depolarization produces a biphasic effect on both SMOC frequency and first latency. These findings are easily explained by the underlying Ca2+ dependence of these parameters. Depolarization would increase the number of activated/unblocked Ca2+ channels, leading to an increased Ca2+ influx. The greater Ca2+ influx presumably leads to a greater success rate in triggering CICR events. This would increase SMOC frequency and decrease first latency. Further depolarization decreases the frequency and increases the first latency, presumably because the Ca2+ influx decreases due to reduction in the driving force for Ca2+ influx.
Depolarization also increased peak SMOC amplitude. As expected, the amount of K+ current flowing through the channels should increase with depolarization. However, the gradient of the SMOC I-V profile was not linear as would be expected for a solely passive increase in current. The I-V curve had a greater slope conductance above 40 mV. A reduced Ca2+ influx into the cell would cause less negative modulation, and this could account for the increased slope of the SMOC I-V curve at above 40 mV. However, our experiments do not rule out the possibility of a purely voltage-dependent factor, which would increase the slope of the SMOC I-V curve at these depolarized levels.
Comparison with Muscle STOCs
Spontaneous transient outward currents (STOCs) seen in muscle cells are apparently similar to SMOCs in neurons. They too are generated by Ca2+ release from ryanodine-sensitive internal stores and are simultaneous activation of clusters of BK channels. There is evidence to show that the causative factor for STOCs are highly localized elevations of internal calcium ([Ca2+]i), called "calcium sparks" (Benham and Bolton, 1986; Nelson et al., 1995
; Perez et al., 1999
; ZhuGe et al., 1999
). If spark-like signals were the causative factor for SMOCs, then one would expect a degree of parallelism between their properties. This may be the case, since it was shown in the previous paper that retinal SMOCs are eliminated on dialysis with 10 mM BAPTA, but not by 10 mM EGTA (Mitra and Slaughter, 2002
, this issue). Merriam et al. (1999)
reported a similar result in their study of SMOCs in mudpuppy parasympathetic neurons. In cardiac cells, where Ca2+ influx through L-type channels generates sparks via CICR, sparks have a bell-shaped frequency dependence with clamp potential. Their latency to occurrence is biphasic: decreasing initially with depolarization as the channel activated and then increasing as the current through the L-type channel decreased due to a reduction in Ca2+ driving force (Lopez-Lopez et al., 1995
). These results are very similar to SMOCs in retinal neurons, and suggest that there may be a close analogy of underlying mechanisms in nerve and muscle cells.
Possible Physiological Significance of SMOCs
Although the physiological role of SMOCs is not clear, it may produce the slow after-hyperpolarization that follows bouts of neuronal activity (Berridge, 1998) or govern the resting membrane potential as it does in smooth muscle cells (Nelson et al., 1995
). Third order neurons, amacrine and ganglion cells, are the only spiking cells in the vertebrate retina (Werblin and Dowling, 1969
). These neurons exhibit spontaneous excitatory postsynaptic potentials (sEPSPs; Awatramani and Slaughter, 2001
) which may reach spike threshold. This would lead to action potential propagation and transmission of information despite the absence of an external stimulus, producing a form of synaptic noise.
SMOCs result in discrete hyperpolarizations in some neurons, called SMHs (Hartzell et al., 1977; Mathers and Barker, 1981
; Fletcher and Chiappinelli, 1992
). Provided there is synchrony, SMHs could negate the sEPSP. Small sEPSPs, resulting in postsynaptic depolarizations of only a few millivolts, might result in SMOCs occurring with a long latency, poorly timed to counteract the sEPSPs. However, larger sEPSPs, which could potentially make the third order neuron reach action potential threshold, would trigger short latency SMOCs. The resulting SMH could potentially cancel out the sEPSP and prevent spiking. This would imply that the bigger the sEPSP, the greater the chance of an SMH being triggered in synchrony with the sEPSP. Thus, retinal SMOCs can putatively function as synaptic noise suppressors within a quiescent glutamatergic synapse. Such a noise suppression would be redundant with coordinated synaptic activity, which would also result in greater postsynaptic calcium influx; thus, the conversion of SMOCs into the Ito.
![]() |
FOOTNOTES |
---|
* Abbreviations used in this paper: CICR, Ca2+-induced Ca2+ release; DHP, dihydropyridine; HVA, high voltageactivated; SMH, spontaneous miniature hyperpolarization; sEPSP, spontaneous excitatory postsynaptic potential; SMOC, spontaneous miniature outward currents; STOC, spontaneous transient outward current; VGCC, voltage-gated Ca2+ channel.
![]() |
ACKNOWLEDGMENTS |
---|
Submitted: 30 July 2001
Revised: 18 March 2002
Accepted: 19 March 2002
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|