Address correspondence to Malcolm Slaughter, Department of Physiology and Biophysics, State University of New York 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: potassium channels calcium-induced calcium release ryanodine receptors L-type calcium channels ganglion cells
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
K+ channels can be broadly categorized based on gating into voltage- and ligand-activated subtypes (Hille, 1992). A notable group amongst the ligand-activated are those gated by intracellular Ca2+, the Ca2+-activated K+ channels (KCa), and their presence in retinal third order neurons has been documented (Lipton and Tauck, 1987
; Latorre et al., 1989
; Vergara et al., 1998
; Wang et al., 1998
). Cytoplasmic calcium ([Ca2+]i) responsible for the gating can arise either via influx through plasmalemmal channels or efflux from the ER (Zorumski et al., 1989
; Kostyuk and Verkhratsky, 1994
; Wisgirda and Dryer, 1994
; Davies et al., 1996
; Berridge, 1998
; Marrion and Tavalin, 1998
; Hurley et al., 1999
; Imaizumi et al., 1999
). The release of organelle Ca2+ is mediated primarily via two subtypes of intracellular Ca2+ channels. While both these channels are calcium sensitive, one subtype releases calcium in response to elevations in the levels of inositol 1,4,5-trisphosphate (IP3) and the other responds primarily to elevations of cytoplasmic Ca2+ by Ca2+-induced Ca2+ release (CICR)*(Fabiato, 1983
; Berridge, 1993, 1998). The latter is sensitive to the plant alkaloid ryanodine, and is thus called the ryanodine receptor (RyR; Pozzan et al., 1994
; Sutko and Airey, 1996
; Zucchi and Ronca-Testoni, 1997
). CICR is extremely well documented in cardiac muscle and also found in neurons (Kuba, 1994
; Cheng et al., 1996
; Verkhratsky and Shmigol, 1996
; Cohen et al., 1997
; Jacobs and Meyer, 1997
; Usachev and Thayer, 1997
). It is thought to amplify calcium signals, such as those originating from influx via voltage-gated calcium channels (VGCCs) (Friel and Tsien, 1992
). This amplified [Ca2+]i signal may rise to magnitudes of several µM in local regions, activating membrane proteins such as Ca2+-activated K+ or Cl- channels (Imaizumi et al., 1998
; Gordienko et al., 1999
). These CICR-generated localized elevations of [Ca2+]i, called "Ca2+ sparks," have been detected in smooth as well as cardiac muscle and there is evidence that sparks can activate clusters of large conductance KCa channels (BK subtype) (Nelson et al., 1995
; Imaizumi et al., 1998
; ZhuGe et al., 1999
). In muscle cells under voltage clamp, K+ fluxes through BK channels generated in this manner appear as random outward currents termed spontaneous transient outward currents (STOCs) (Benham and Bolton, 1986
; Bolton and Imaizumi, 1996
). STOCs are thought to govern the resting membrane potential in smooth muscle (Nelson et al., 1995
; Jaggar et al., 1998
). A similar mechanism, if existent in excitable neurons such as retinal amacrine cells, can potentially modulate neuronal excitability.
This study characterizes a subtype of retinal amacrine cell with respect to its K+ current signature. In addition to voltage-activated K+ currents, these neurons display a set of KCa currents with different temporal properties. These currents activate sequentially as the membrane potential of the neuron depolarizes. The KCa currents consist of a randomly occurring transient outward current, an early onset transient outward current, and a sustained outward current. The random outward current has properties similar to the STOCs seen in muscle cells, and in neurons are called spontaneous miniature outward currents (SMOCs) (Hartzell et al., 1977; Mathers and Barker, 1981
, 1984
; Satin and Adams, 1987
; Fletcher and Chiappinelli, 1992
; Merriam et al., 1999
; Arima et al., 2001
). This study reports the presence of SMOCs in retinal neurons and elucidates their mechanism of generation. A preliminary description of this work was presented by Mitra and Slaughter (2000)
.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experimental Setup and Data Acquisition
The plated coverslips were placed in a plexiglass recording chamber having a volume of 350 µl. One end of the chamber housed a suction tube connected to a peristaltic pump. The cells were viewed with an inverted microscope equipped with a 40x lens. Retinal cells were constantly superfused at room temperature with normal amphibian Ringer's solution bubbled with oxygen using a gravity fed perfusion system. The tubes of the gravity-fed perfusion system were connected to a common manifold, which held a wide bore patch pipette. This patch pipette was placed close to the cell during recordings, and solution exchanges could be achieved within 23 s. Control and test solutions were applied using this system. The drug effects described in this paper refer to steady-state effects usually obtained after 1030 s of drug application. Data were acquired with an Axopatch-1C patch clamp amplifier (Axon Instruments, Inc.). Analogue signals were filtered at 5 KHz and sampled at 20 KHz with the DigiData 1200 analogue-to-digital board (Axon Instruments, Inc.) using Clampex 8 software (Pclamp 8; Axon Instruments, Inc.). Data were acquired using the whole cell variant of the patch clamp technique, in both the continuous voltage and current clamp modes (Hamill et al., 1981). Patch pipettes were fashioned from borosilicate glass and had resistances of
5 M
when measured in bath solution. Data shown in this paper were corrected for pipette junction potential (
10 mV). Except for the voltage values given in Fig. 2, C and D, and associated text, where the voltage error due to series resistance (
10 M
) could have an impact on the interpretation, the voltages are uncorrected for series resistance. Since these neurons had high input resistances, leak subtraction was not necessary. This, along with the observation that at relatively negative voltages (-90 to -30 mV) the clamp currents were very small, suggested that the voltage error due to uncompensated series resistance was insignificant within this range. At more depolarized voltages the error can be significant. However, most of the arguments presented in this and the following paper do not rely on the absolute value of the voltage at these potentials. Cells were held at -80 mV except when specifically mentioned in the text.
|
Solutions
Normal amphibian Ringer's contained (in mM): 111 NaCl, 2.5 KCl, 1.8 CaCl2, 1 MgCl2, 10 dextrose, and 5 HEPES, buffered to pH 7.8 with NaOH and oxygenated. Co2+ Ringer was similar to normal Ringer's except that it contained 420 mM CoCl2 and variable amounts of CaCl2 (04.5 mM). Addition or removal of CoCl2 or CaCl2 to normal Ringer was accompanied by adjustment of the NaCl concentration to maintain osmolarity. Solutions mentioned as containing "0 mM Ca2+" contain nominal unbuffered levels of calcium. Since only the bath calcium concentration was manipulated in this study, the notation "Ca2+" used throughout the text, unless indicated otherwise, refers to external calcium. High K+ Ringer's with or without the addition of Co2+ was prepared by equimolar substitution of NaCl with KCl. Recording pipettes contained (in mM): 110 K-gluconate, 5 NaCl, 1 MgCl2, 5 EGTA, 5 HEPES 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. As noted in the RESULTS, bis (o-aminophenoxy)-N, N, N', N'-tetraacetic acid (BA PTA, 10 mM) sometimes replaced EGTA in the internal solution. Ryanodine was dialyzed into the neurons by including it in the internal solution. Iberiotoxin, apamin, nifedipine, and S (-) BayK 8644 were obtained from Research Biochemicals, Inc. All other chemicals used in this study were obtained from Sigma-Aldrich. Dihydropyridines (DHPs) were prepared as 10 mM stock in ethanol and stored at -20°C. When using DHPs, care was taken to prevent exposure to light. Appropriate controls indicated that the final concentrations of ethanol used in this study did not have measurable effects. When using peptide toxins (iberiotoxin or apamin), BSA (0.1% wt/vol) was added to both control and toxin-containing solutions.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fig. 1 A shows current recordings obtained from such a cell in normal Ringer's, under the whole cell voltage clamp. The cell was held at -80 mV and depolarized from -90 to 70 mV in 10 mV increments, in pulses of 500 ms. The family of traces obtained illustrates the current signature for this cell type. An inward TTX-sensitive (500 nM) Na+ current (marked as INa in Fig. 1 A) activates at -30 mV. Large outward currents are activated in response to depolarization. These outward currents are primarily K+ fluxes, as they reverse near 0 mV when bath Ringer's contained high K+ equivalent to that in the recording pipette (n = 11; unpublished data). Since these cells have high input resistances, very small currents are seen in the voltage range between -90 and -70 mV. The first induced outward current appears at -60 mV. These currents, termed SMOCs, are spike-shaped and TTX-insensitive and occur within the narrow voltage range of -60 to -40 mV (n = 12). Fig. 1 B shows SMOCs elicited in normal Ringer's at -50 mV. SMOCs at -50 mV have a mean amplitude of 33 ± 0.5 pA, mean time to peak of 2.8 ± 0.1 ms, and a mean 95% decay time of 5.5 ± 0.1 ms (average of four cells, 1,000 events). In this set of 1,000 analyzed events, SMOCs appeared at a frequency of 9.8 ± 0.5 Hz and the highest SMOC amplitude was 92 pA.
|
Calcium-dependent Outward Currents
The voltage-induced outward currents have an extracellular Ca2+-dependent component. Fig. 2 A shows representative current recordings at 0 mV in normal Ringer's and in Ringer's containing 6 mM Co2+/0 mM Ca2+. Elimination of Ca2+ influx abolished Ito and reduced Iso at all voltages capable of eliciting these currents. The effects were fully reversible on switching back to control Ringer's. The subtracted trace at 0 mV is shown in Fig. 2 B, which is the Ca2+ influxsensitive component, and is presumably mediated via KCa channels. It includes the entire Ito and a component of Iso, referred to as Iso-Ca (n = 8). The residual current in 6 mM Co2+/0 mM Ca2+ is defined as the Ca2+-insensitive, voltage-dependent outward K+ current, Iso-v (Fig. 2 A, lower trace). Fig. 2 C shows a normalized I-V plot for the Iso (open circles) and its components, viz: Iso-v (closed diamonds) and Iso-Ca (open triangles) (average data from eight cells). As shown in Fig. 2 B, the Iso-Ca is obtained by subtraction from the mean values of the Iso and Iso-v. The voltage values in Fig. 2, C and D, have been corrected for the drop across the series resistance (10 M
). The figure shows that beyond 0 mV, both Iso and Iso-v increase in a relatively linear fashion, whereas Iso-Ca shows a reduction in its slope. Fig. 2 D is a plot of the constituent Iso-Ca (open triangles) and Iso-v (closed diamonds) at each test voltage. Both components are expressed as a percentage of the total Iso at that particular voltage. Note the decline in the percentage of the Iso-Ca component with increasing depolarization. The initial decline is due to the appearance of the Iso-v. However, its percentage continues to decrease at voltages beyond 0 mV; a range in which the Iso-v I-V plot is relatively linear (see Fig. 2 C), suggesting that the channels comprising the Iso-v are near maximal activation. This decline of the Iso-Ca fraction would be expected if it was dependent on Ca2+ influx for its activation, since depolarization would tend to reduce the driving force for Ca2+ influx into the cell. At 0 mV, Iso-Ca makes up 76.8 ± 2.9% of Iso. However, at 30 mV the Iso-Ca makes up only 59.2 ± 4.6% of Iso; a shift in favor of the voltage-dependent component. SMOCs, similar to the Ito and Iso-Ca, were eliminated at -50 mV in the presence of 6 mM Co2+/0 mM Ca2+ Ringer's, thus suggesting that they too were Ca2+ dependent (see next section).
Ca2+ Influx through VGCCs Is Required for the Generation of SMOCs
Reducing Ca2+ influx through VGCCs by the addition of inorganic blockers to normal Ringer's (6 mM Co2+/1.8 mM Ca2+ Ringer's), led to the generation of SMOCs at -20 mV and above, while eliminating them at hyperpolarized voltages. Representative results are shown in shown in Fig. 3, A1 and A2 (Vcmd steps to -50 and 30 mV; n = 20). This is in contrast to the appearance of SMOCs in the narrow voltage range of -60 to -40 mV in normal Ringer's. SMOCs generated in 6 mM Co2+/1.8 mM Ca2+ Ringer's at -10 mV had a mean amplitude of 158.2 ± 2.6 pA, mean time to peak of 3.7 ± 0.1 ms and a mean 95% decay time of 6.2 ± 0.1 ms (average data from 800 events in five cells). In this set of five cells used for analysis, SMOCs appeared at a frequency of 10.7 ± 0.40 Hz. A plot from a representative cell showing SMOC frequency versus inorganic blocker dose at 30 mV is shown in Fig. 3 B. Increasing the cobalt concentration, while maintaining 1.8 mM Ca2+, reduced SMOC frequency. 20 mM cobalt eliminated SMOCs. In this cell, SMOC frequency was 21 ± 0.6 Hz at 4 mM Co2+ and was reduced to 7.2 ± 0.6 Hz at 8 mM cobalt (P < 0.01). Average data from five cells under similar conditions gave SMOC frequencies of 23.4 ± 1.2 Hz at 4 mM Co2+ and 7.9 ± 0.5 Hz under 8 mM Co2+ (P < 0.01). Addition of 100 µM Cd2+, instead of Co2+, also led to generation of SMOCs at depolarized voltages (n = 15). These effects of inorganic blockers were fully reversible on washout.
|
The dose-dependent reduction in SMOC frequency with increasing doses of inorganic blocker, and their elimination in nominal Ca2+-containing solutions suggest that Ca2+ influx through VGCCs is involved in the SMOC generation pathway. Another possible route of Ca2+ influx into the cell is via receptor-operated channels (Betz, 1990; Gilbertson et al., 1991
; Brorson et al., 1992
; Kostyuk and Verkhratsky, 1994
). In retinal third order neurons, the kainic acid (KA) receptor, a glutamate receptor subtype, has been shown to be permeable to Ca2+ (unpublished data; Otori et al., 1998
; Taschenberger and Grantyn, 1998
; Yoon et al., 1999
). Addition of 50 µM KA to cells did not alter SMOC frequency (n = 11). SMOC frequency in 6 mM Co2+/1.8 mM Ca2+ Ringer's at 30 mV was 5.3 ± 0.6 Hz. Addition of 50 µM KA resulted in a SMOC frequency of 5.2 ± 0.7 Hz (average data from three cells, difference between frequency values is not significant at P < 0.05). Increasing the driving force for KA-induced Ca2+ influx by generating SMOCs at -10 mV (n = 3) or -50 mV (n = 5) did not result in any significant change in frequency. These observations indicate that Ca2+ influx through VGCCs is necessary for SMOC generation.
SMOCs generated in the presence of cobalt were more stable, gave a better signal to noise ratio, and afforded a broader dynamic range of voltage activation. Due to these experimental advantages, it was far easier to unambiguously identify and quantitate SMOCs generated in this manner, as compared with those seen within a narrow voltage range in normal Ringer's. Therefore, the properties of SMOCs were examined in the presence of cobalt. However, equivalent studies, when feasible, were done on SMOCs generated in normal Ringer's. The results of the two paradigms were qualitatively similar.
SMOCs occurred randomly irrespective of whether they were generated in normal or Co2+-containing Ringer's. Fig. 3 D shows an interevent interval histogram for SMOCs generated in 6 mM Co2+/1.8 mM Ca2+ Ringer's. The exponential interevent interval distribution obtained suggests the random nature of their occurrence.
Ca2+ Influx through High Voltageactivated (HVA) Ca2+ Channels Is Responsible for SMOC Generation
Since VGCCs are important for SMOC generation, the type of calcium channel was examined. SMOCs recorded in normal Ringer's are generated within a relatively hyperpolarized voltage range of -60 to -40 mV. It is possible that Ca2+ influx through low voltageactivated Ca2+ channels (T-type) might be involved. Lacking a pharmacological method of blocking this channel, a common biophysical approach is to inactivate T-type channels by holding the cells at depolarized voltages (Narahashi et al., 1987; Bean, 1989
). With the holding potential at -80 mV, SMOCs generated at -50 mV in normal Ringer's had a frequency of 15.3 ± 1.6 Hz. Maintaining the holding potential at -50 mV resulted in a SMOC frequency of 13 ± 1.4 Hz (average from four cells; difference in frequencies was not significant at P < 0.05). Similarly, SMOCs generated in 6 mM Co2+/1.8 mM Ca2+ by a step to -10 mV also failed to be eliminated when the holding voltage was -40 mV (n = 3; unpublished data). These results suggest that T-type channels are not involved in SMOC generation.
Fig. 4 A shows representative SMOC recordings obtained after a depolarization to -10 mV in 6 mM Co2+/1.8 mM Ca2+ Ringer's in the absence (A1) and presence (A2) of 10 µM nifedipine, a blocker of L-type Ca2+ channels. Fig. 4 B shows that nifedipine produces a graded, dose-dependent reduction in SMOC frequency. In this cell, nifedipine reduced SMOC frequency from a control value of 54.6 ± 2.9 Hz to 22 ± 1.6 Hz at 10 µM (P < 0.01). However, even at doses of 60 µM, nifedipine was unable to totally block SMOC generation. Average data from seven cells in which SMOCs were recorded under 6 mM Co2+/1.8 mM Ca2+ Ringer's at 30 mV gave control frequencies of 20 ± 1.6 Hz, but 8.6 ± 0.6 Hz in 10 µM nifedipine (P < 0.01). This effect of nifedipine was partially reversible and was qualitatively confirmed in a total of 18 cells. Addition of S (-) BayK 8644 (3 µM), an L-type Ca2+ channel agonist, had the opposite effect. Traces from a representative cell are shown in Fig. 4, C1 (control) and C2 (agonist), for SMOCs generated in 6 mM Co2+/1.8 mM Ca2+ Ringer's with a voltage step to -10 mV. Control SMOC frequency in this cell was 4.5 ± 0.6 Hz, and it increased to 14.8 ± 0.7 Hz (P < 0.01) on addition of the agonist (Fig. 4 D). Average data from six cells in which SMOCs were recorded under similar conditions gave control frequencies of 7.6 ± 0.8 Hz, but 15.6 ± 1 Hz in S (-) BayK 8644 (3 µM) (P < 0.01). Qualitatively similar data were recorded from a total of 10 cells. S (-) BayK 8644 at the same dose also enhanced the frequency of SMOCs generated in normal Ringer's at -50 mV (n = 3; unpublished data). The effect of the agonist was not reversible. The lack of reversibility of DHP agents has been noted in earlier studies and is probably due to their lipid solubility and uptake into membranes (Pang and Sperelakis, 1984; Nerbonne and Gurney, 1987
). The reduction of SMOC frequency by nifedipine and enhancement by S (-) Bay K 8644 strongly suggest that L-type HVA Ca2+ channels play a role in SMOC generation. However, the lack of a complete block by high doses of nifedipine leaves open the question of whether this is the only subtype of HVA Ca2+ channels involved. Although there is a possibility that other VGCC subtypes, such as N, P, Q, and R, may participate in this process (Bean, 1989
; Zhang et al., 1993
; Dunlap et al., 1995
), a pharmacological examination of the influence of these other Ca2+ channels on SMOC frequency was not performed.
|
|
SMOCs Are K+ Currents: Pharmacological Block
SMOCs recorded in Co2+-containing solutions were blocked completely by the K+ channel blocker, TEA (10 mM; n = 10). Representative traces showing TEA block of SMOCs generated at -10 mV in 6 mM Co2+/1.8 mM Ca2+ Ringer's are shown in Fig. 6 A. The TEA block was fully reversible. Increasing concentrations of TEA produced a graded reduction of SMOC amplitude at -10 mV in 6 mM Co2+/1.8 mM Ca2+ Ringer's (Fig. 6 B). In this cell, SMOCs had a mean amplitude of 159 ± 3 pA under control conditions and 91 ± 2 pA in the presence of 0.5 mM TEA. Average SMOC data from four cells under similar conditions gave control amplitudes of 204.9 ± 6.4 pA, and 108.4 ± 3.1 pA in 500 µM TEA (a reduction of 47%; P < 0.01). TEA also produced a dose-dependent decline in SMOC frequency (unpublished data). This is probably due to reduction in amplitude by TEA, causing SMOCs of smaller amplitude to merge with the baseline noise and escape detection. SMOCs generated in normal Ringer's at -50 mV were also blocked by 10 mM TEA (n = 4). SMOCs were not blocked by 5 mM 4-aminopyridine (4-AP; n = 9). Prolonged exposure (>2 min, as compared with a perfusion equilibration time with an upper limit of 10 s) to 4-AP resulted in a gradual, irreversible decline in SMOC frequency and subsequent elimination of SMOCs. This was not reversible on returning to control. However, SMOCs could be reinstated after pulsing to depolarized levels in normal Ringer's, a process that presumably permits Ca2+ influx into the cell and replenishes stores. This effect of prolonged 4-AP application may be nonspecific. It is reminiscent of a gradual rundown/depletion phenomena, probably arising from effects of 4-AP on other molecules, such as the Ca2+ ATPases (Ishida and Honda, 1993
). The significant blockade of SMOCs by submillimolar amounts of TEA further substantiates that SMOCs are currents generated via fluxes through K+ channels.
|
Since intracellularly stored Ca2+ is involved in the SMOC generation pathway, care was taken to ensure that the slow time course and irreversibility of iberiotoxin block was not mistaken for rundown/depletion. Control recordings for 2 min did not show a significant change in SMOC frequency or amplitude. However, addition of 100 nM iberiotoxin led to a total block of SMOCs in the subsequent 2 min. Moreover, contrary to what would be expected for depletion-induced phenomena, refilling of stores by pulsing to depolarized levels in normal Ringer's failed to reinstate SMOCs. This suggested a persistence of iberiotoxin block independent of depletion of the Ca2+ store.
Apamin (100 nM), a blocker of small conductance Ca2+-activated K+ channels (SK channels), did not eliminate SMOCs generated at depolarized levels in 6 mM Co2+/1.8 mM Ca2+ Ringer's nor affect their characteristics (n = 7; unpublished data). SMOCs generated in normal Ringer's at -50 mV were blocked by 100 nM iberiotoxin, but not by 100 nM apamin (n = 8; unpublished data). These results suggest that SMOCs are K+ fluxes through the BK subtype of KCa channels.
CICR Mediates SMOC Generation
Although the earlier sections showed that Ca2+ influx through HVA Ca2+ channels plays a role in the generation of SMOCs, there are reports in other neuronal preparations of SMOCs being activated by CICR triggered by Ca2+ influx. Ca2+ release from internal stores also mediates the generation of STOCs in muscle cells (see INTRODUCTION). Moreover, SMOCs were induced under conditions of inorganic Ca2+ channel blockade, where Ca2+ influx was severely reduced. Under such conditions an internal amplification mechanism is likely to exist. To test this possibility, we internally dialyzed neurons with ryanodine, a plant alkaloid that blocks RyRs at high doses (Zucchi and Ronca-Testoni, 1997). Internal dialysis of ryanodine (200 µM) abolished SMOCs generated either in normal Ringer's or in Co2+-containing solutions (n = 7). Cells were identified as having the requisite current signature immediately on achieving the whole cell configuration. Control SMOCs were immediately recorded, before sufficient dialysis took place. Fig. 7 A1 shows an example in which control SMOCs were recorded 17 s after achieving the whole cell configuration. These SMOCs were recorded in 6 mM Co2+/1.8 mM Ca2+ Ringer's at 30 mV with 200 µM ryanodine added to the internal pipette solution. Dialysis of the neuron for a further 35 s led to the total elimination of SMOCs (Fig. 7 A2).
|
Although prolonged exposure to high doses of caffeine causes store depletion, caffeine should initially be able to stimulate CICR and augment any process dependent on it. This was verified by exposing cells transiently to low doses of caffeine (13 mM). In a representative cell, control SMOCs were generated in 6 mM Co2+/0.9 mM Ca2+ Ringer's at -10 mV (Fig. 7 C1). Exposure to 3 mM caffeine increased SMOC frequency (Fig. 7 C2). In a set of four cells, mean SMOC frequency under control conditions was 2.4 ± 0.4 Hz, but on exposure to 3 mM caffeine SMOC frequency increased to 7.4 ± 0.7 Hz (Fig. 7 D, P < 0.01).
A comparison of Fig. 7 C2 with C1 shows that 3 mM caffeine also increased SMOC decay time. The mean 50% decay time (t1/2) of control SMOCs in 6 mM Co2+/0.9 mM Ca2+ Ringer's at -10 mV was 3.2 ± 0.2 ms, whereas caffeine-induced SMOCs had a t1/2 of 8.7 ± 0.4 ms (four cells, P < 0.01). Combined with the earlier finding that Ca2+ influx is a prerequisite for SMOC generation, these experiments with caffeine and ryanodine argue in favor of a RyR-mediated CICR in the process of SMOC generation.
SMOC Activity Is Eliminated by Intracellular Ca2+ Chelation with BAPTA but not by EGTA
As expected for any [Ca2+]i-dependent process, intracellular Ca2+ chelators have been shown to eliminate both STOCs and SMOCs (Benham and Bolton, 1986; Bychkov et al., 1997
; Merriam et al., 1999
). Since CICR was shown to be involved in the SMOC generation pathway in retinal neurons, the effect of internal Ca2+ chelators was tested. The affinities of both BAPTA and EGTA for Ca2+ are similar, although BAPTA is known to have an association rate that is
150 times greater as compared with EGTA (Naraghi, 1997
). Thus, contrasting the effects of these two chelators would allow distinction between fast and slow Ca2+-stimulated events over short distances. BAPTA eliminated SMOCs generated either in normal Ringer's or Co2+-containing solutions. An example is shown in Fig. 8 A, in which SMOCs were recorded 30 s after achieving the whole cell configuration at 30 mV in 6 mM Co2+/1.8 mM Ca2+ Ringer's with 10 mM BAPTA in the internal solution. Dialysis of the neuron for a subsequent 46 s totally eliminated SMOCs (Fig. 8 A2). This result was confirmed in 13 cells. EGTA did not have any effect on SMOC activity even on prolonged dialysis (n = 5). Data from a representative cell is shown in Fig. 8 B, where control SMOCs were recorded 2 min after achieving the whole cell configuration (Fig. 8 B1). Continued dialysis for another 5 min had no effect on SMOC activity (Fig. 8 B2). These results suggest a local, rapid increase in [Ca2+]i is responsible for generation of SMOCs.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SMOCs observed in these experiments, whether in Ringer's or Co2+-containing solutions, were induced with voltage pulses and thus strictly speaking were not "spontaneous." However, the term "SMOCs" has been employed to maintain consistency with literature on similar phenomena in other neuronal types. Moreover, the isolated cell preparation does not allow determination of the normal resting potential. In situ, these amacrine cells may rest within the voltage range where SMOCs occur. Alternatively, these neurons may rest at more negative potentials but SMOCs could be "evoked" by small depolarizing shifts caused by spontaneous miniature excitatory postsynaptic currents (sEPSCs).
In these amacrine cells, depolarizing shifts in voltage elicit a successive series of outward currents: SMOCs, Ito, and Iso. The Iso can be further decomposed into Iso-Ca and a purely voltage-dependent Iso-v. Except for the Iso-v, the other three outward currents are Ca2+-sensitive. It is shown in the following paper that iberiotoxin not only eliminates SMOCs and the Ito, it also reduces the Iso. However, depletion of the internal stores with caffeine eliminates SMOCs and the Ito, but does not affect the Iso (Mitra and Slaughter, 2002, this issue). Thus, all three outward currents have components generated by BK channels, the distinction being that Iso-Ca is not activated by CICR.
Properties of the Sustained Outward Current
The Iso appeared at -30 mV and increased linearly with depolarization. It consisted of calcium-dependent (Iso-Ca) and calcium-independent (Iso-v) components. Iso-v activated and then increased linearly with depolarization. Iso-Ca activated at slightly more negative potentials, but showed a decline at positive voltages. When the contribution of Iso-Ca and Iso-V to total Iso is considered, the percentage of Iso-v increased with depolarizations and that of the Iso-Ca decreased. This decline of the Iso-Ca fraction may be due to the decline in Ca2+ influx with depolarization. It is known that KCa channels (BK subtype) are activated by concerted influences of both [Ca2+]i and voltage. Their sensitivity to Ca2+ increases with membrane depolarization (Barrett et al., 1982; Vergara et al., 1998
). Therefore, the decline of the Iso-Ca fraction with depolarization reflects the interaction between the activation-favoring effects of depolarized potentials and the activation-reducing effects of lowered Ca2+ influx at those voltages.
SMOC Properties
Our results indicate that SMOCs are transient K+ fluxes through plasmalemmal BK channels and originate from Ca2+ influx through VGCCs; this influx being amplified by CICR from caffeine- and ryanodine-sensitive intracellular stores. Earlier studies in smooth muscle reported STOCs, which are similar to SMOCs (Benham and Bolton, 1986; Bolton and Imaizumi, 1996
). Although similar, neuronal SMOCs are more transient than smooth muscle STOCs (Merriam et al., 1999
). Retinal SMOCs show an interevent interval distribution that is exponential, characteristic of stochastic events (Fatt and Katz, 1952
; Hubbard et al., 1969
). This is in agreement with reports of randomly occurring SMOCs in cultured bullfrog neurons (Satin and Adams, 1987
).
In smooth muscle, spontaneous activation of potassium or chloride channels produce currents of opposite polarity. Thus, smooth muscle exhibits STOCs when discrete release of Ca2+ from internal stores activates potassium channels, and STICs when the same processes activate chloride channels (Wang et al., 1992; Janssen and Sims, 1994
; Henmi et al., 1996
). But in neurons, either conductance will produce an outward current. Retinal SMOCs are K+ currents, since their mean amplitude varied with the [K+]ext in a Nernstian manner. Since they are blocked by iberiotoxin and insensitive to apamin, they likely represent openings of clusters of BK channels (McManus, 1991
; Kaczorowski et al., 1996
). Extracellular TEA, which decreases the amplitude of single channel currents through BK channels in a concentration-dependent manner (Saunders and Farley, 1992
), reduced retinal SMOC amplitudes in a dose-dependent manner, eliminating them at 10 mM. These findings concur with published data on both STOCs and SMOCs in other cell types, which show them to be coordinated openings of KCa channels of the BK subtype (Bolton and Imaizumi, 1996
; Merriam et al., 1999
). However, small conductance KCa (SK subtype) channels can also generate SMOCs, as shown in rat Meynert neurons (Arima et al., 2001
).
Retinal SMOCs generated at -50 mV in normal Ringer's displayed a mean amplitude of 33 ± 0.5 pA. BK channels have been reported to have conductances in the range of 115118 pS in third order neurons in mammalian retina (Lipton and Tauck, 1987; Wang et al., 1998
). Assuming a similar conductance in amphibian neurons, it is estimated that retinal SMOCs generated in normal Ringer's represent an average opening of
6 BK channels at their peak. The maximum SMOC height seen in normal Ringer's at this voltage was 92 pA, which would represent the opening of
18 BK channels. SMOCs generated in 6 mM Co2+/1.8 mM Ca2+ Ringer's at -10 mV had a mean amplitude of 158 ± 3 pA corresponding to
16 BK channels. The slope of the fitted data shown in Fig. 5 B gives a similar estimate. Fletcher and Chiappinelli (1992)
examined spontaneous miniature hyperpolarizations (SMHs; the voltage equivalent of SMOCs) in chick ciliary ganglion neurons and reported
1560 channels in each SMH, whereas Scornik et al. (2001)
in mudpuppy cardiac neurons arrived at a value of 1823 channels in the average SMOC. SMOCs in retinal neurons are similar to these reports, rather than bullfrog sympathetic neurons wherein each SMOC was reported to constitute activation of 105,000 channels (Satin and Adams, 1987
). In spite of the small number of channels, single channel currents were not observed. Perhaps the recording bandwidth led to the filtering of such single steps.
Voltage-gated Calcium Channels Initiate SMOCs
The Ca2+ ions necessary for the activation of KCa channels can be provided either via extracellular influx or release from intracellular stores (Zorumski et al., 1989; Wisgirda and Dryer, 1994
; Davies et al., 1996
; Marrion and Tavalin, 1998
; Hurley et al., 1999
; Imaizumi et al., 1999
). Satin and Adams (1987)
, in their study of SMOCs in bullfrog sympathetic neurons, reported that Ca2+ influx through surface membrane Ca2+ channels was not essential for SMOC generation. They suggested that neuronal depolarization directly coupled to Ca2+ release from internal stores. Their conclusions were based on the finding that 100 µM Cd2+ did not block SMOCs. Fletcher and Chiappinelli (1992)
found a similar insensitivity to external Ca2+ in chick ciliary ganglion neurons. Arima et al. (2001)
suggest that Ca2+ influx contributes to, but is not essential, for SMOC generation in rat Meynert neurons. However, other investigators found Ca2+ influx was required for SMOC generation (Mathers and Barker, 1981
, 1984
; Merriam et al., 1999
). Retinal SMOCs were instantly eliminated when cells were exposed to nominal [Ca2+] containing solutions, and immediately recovered when bath Ca2+ was reinstated.
Retinal SMOCs were initiated by Ca2+ influx through plasmalemmal VGCCs. SMOCs are seen in normal Ringer's at membrane potentials between -60 to -40 mV. Addition of inorganic VGCC blockers such as Co2+ or Cd2+ eliminated SMOCs at these hyperpolarized levels, although they reappeared at more depolarized voltages, at or above -20 mV. It is generally thought that these divalent cations are VGCC pore blockers (Lansman et al., 1986; Winegar et al., 1991
). Therefore, depolarization would tend to push the blocker out of the channel and thus relieve block (Woodhull, 1973
; Fukushima and Hagiwara, 1985
; Thevenod and Jones, 1992
; Carbone et al., 1997
; Wakamori et al., 1998
). Increasing the dose of the inorganic blocker resulted in a graded reduction in retinal SMOC frequency. Stimulating non-VGCCmediated Ca2+ influx by activation of kainic acid receptors did not affect SMOC frequency. These observations suggested that Ca2+ influx through VGCCs is necessary for SMOC generation.
DHPs are a group of Ca2+ channel ligands that are thought to be selective for the L-type HVA Ca2+ channel (Bean, 1984; Nowycky et al., 1985
; Fox et al., 1987
; Rane et al., 1987
). L-type HVA Ca2+ channels are known to be in close proximity to the RyRs in cardiac muscle, and it is presumably this anatomical arrangement that enables the occurrence of discrete Ca2+ release events called "sparks" (Carl et al., 1995
; Sun et al., 1995
). In smooth muscle cells, sparks have been implicated as the underlying event giving rise to STOCs (Nelson et al., 1995
; Mironneau et al., 1996
; Perez et al., 1999
; ZhuGe et al., 1999
). A similar anatomical relationship may also exist in smooth muscle cells (Jaggar et al., 1998
). Our experiments revealed the involvement of L-type HVA Ca2+ channels in retinal SMOC generation. The DHP blocker, nifedipine, reduced SMOC frequency in a dose-dependent manner and the agonist S (-) BayK 8644 increased it. Although nifedipine reduced SMOC frequency, it never completely eliminated SMOCs, even at concentrations up to 60 µM at relatively depolarized voltage of 30 mV. This result may be interpreted in two ways. One is that other subtypes of HVA calcium channel, such as N, P, Q, and R, may be involved (Bean, 1989
; Zhang et al., 1993
; Dunlap et al., 1995
), and that the system does not show any specificity as regards the type of HVA Ca2+ channel that generates SMOCs. This was the conclusion arrived at by Merriam et al. (1999)
in their study of SMOCs in mudpuppy parasympathetic cardiac neurons. The involvement of other subtypes of HVA Ca2+ channels was not examined in this present study. The alternative explanation is that the L-type VGCCs are not totally blocked, even with high doses of nifedipine, and the consequent high amplification of the trigger Ca2+ signal by CICR leads to a SMOC. Blockade by DHP antagonists is complex since cell voltage affects it. These blockers have a higher affinity for the inactivated state of the channel, the population of which increases as the cell is depolarized (Bean, 1984
; Sanguinetti and Kass, 1984
). It may be that this voltage dependence of nifedipine block does not allow a 100% block even at depolarized voltages, permitting a stochastic unblock of a few Ca2+ channels. It has been shown in heart muscle that influx through one Ca2+ channel is enough to activate CICR (Santana et al., 1996
). This would be all the more likely if the intracellular stores are filled with Ca2+, since it has been shown that store filling increases the sensitivity of the stores to generate CICR, whereas store depletion has the opposite effect (Friel and Tsien, 1992
; Jaffe and Brown, 1994
; Shmigol et al., 1996
; Hernandez-Cruz et al., 1997
; Usachev and Thayer, 1997
). Although demonstrating the involvement of L-type Ca2+ channels in the SMOC generation pathway, this study does not eliminate the involvement of other HVA types.
Calcium-induced Calcium Release Produces SMOCS
Ca2+ influx through VGCCs can either directly activate the KCa channels responsible for SMOCs, or trigger CICR (Sah and McLachlan, 1991; Sah, 1992
; Jobling et al., 1993
; Berridge, 1998
). CICR-triggered via Ca2+ influx through VGCCs is the accepted mechanism of Ca2+ spark generation in cardiac muscle (Imaizumi et al., 1999
). In neurons, CICR has been shown to be an intermediate step for SMOC generation (Mathers and Barker, 1981
, 1984
; Merriam et al., 1999
). In retinal neurons, the effects of both caffeine and ryanodine indicate that CICR is involved in SMOC generation. Low concentrations of ryanodine (<10 µM) lock the release channel in a subconductance state, which is 4060% of the normal conductance. Higher concentrations (>100 µM) of ryanodine block the release channel in a use-dependent manner (Sutko and Airey, 1996
; Zucchi and Ronca-Testoni, 1997
). Caffeine, on the other hand, increases the sensitivity of the channel to trigger Ca2+ release, causing significant release even at resting levels of [Ca2+]i (Verkhratsky and Shmigol, 1996
). It increases the open probability of the release channel by generating more frequent openings, whereas the mean open time and conductance is not altered (Rousseau et al., 1988
; Rousseau and Meissner, 1989
; Sitsapesan and Williams, 1990
; Hernandez-Cruz et al., 1995
). High doses of caffeine (10 mM) deplete the internal Ca2+ stores and thus eliminate CICR (Friel and Tsien, 1992
). Low doses of caffeine (13 mM) have been shown to augment CICR-dependent process by causing additional Ca2+ release in response to the trigger signal. Internal dialysis with 200 µM ryanodine completely eliminated retinal SMOCs. Exposure to 10 mM caffeine eliminated retinal SMOCs, which recovered only after store refilling. Caffeine at low doses (13 mM) increased SMOC frequency. Similar effects of low doses of caffeine have been observed on both STOCs and SMOCs (Bolton and Imaizumi, 1996
; Merriam et al., 1999
; Arima et al., 2001
). Retinal SMOCs induced by 13 mM caffeine showed a prolonged decay time. Merriam et al. (1999)
made a similar observation in mudpuppy parasympathetic neurons, suggesting this reflected caffeine's ability to cause the release channel to open more frequently. As a consequence, there are multiple activations of the BK channels in the vicinity of the release channel, slowing the rate of decay.
Internal BAPTA eliminated SMOC activity, whereas EGTA did not. BAPTA and EGTA have similar affinities for Ca2+, but the former has faster association kinetics (Naraghi, 1997). Consequently, BAPTA can eliminate rises of [Ca2+]i within more restricted microdomains (Deisseroth et al., 1996
; Sham, 1997
; Neher, 1998
). This differential effect of the two buffers suggests that the [Ca2+]i rise responsible for activating the BK channels is a highly localized event. A similar observation was reported by Merriam et al. (1999)
in mudpuppy cardiac neurons. Such localized [Ca2+]i elevations, "Ca2+ sparks," have been noted in cardiac and smooth muscle (Imaizumi et al., 1999
). Based on these findings and the mechanism known to exist in smooth muscle, it would be a reasonable conjecture that a "Ca2+ spark" underlies SMOC generation in retinal neurons.
In summary, our experiments show that, in a subset of retinal amacrine cells, Ca2+ influx through L-type HVA Ca2+ channels triggers CICR from caffeine and ryanodine-sensitive stores. This amplified Ca2+ signal subsequently activates clusters of BK channels leading to outward K+ current fluxes which appear as SMOCs. However, there is a complex relationship between Ca2+ influx and SMOC appearance. Although Ca2+ influx was found to be a prerequisite for SMOC generation, reducing Ca2+ influx caused SMOCs to appear at depolarized voltages while eliminating them at hyperpolarized levels. This would suggest that large amounts of Ca2+ influx probably has a negative modulatory effect on SMOCs, since the open probability of VGCCs is usually higher at depolarized voltages. This is addressed in the subsequent paper, which characterizes the biophysical properties of SMOCs, including their Ca2+ dependence, and thus provides an explanation for their disappearance at depolarized voltage levels in normal Ringer's. These findings suggest that SMOCs putatively serve to suppress spontaneous excitatory synaptic events within the retinal network (Mitra and Slaughter, 2002, this issue).
![]() |
FOOTNOTES |
---|
* Abbreviations used in this paper: CICR, Ca2+-induced Ca2+ release; DHP; dihydropyridine; KA, kainic acid; RyR, ryanodine receptor; SMOC, spontaneous miniature outward current; STOC, spontaneous transient outward current; TTX, tetrodotoxin; VGCC, voltage-gated calcium channel.
![]() |
ACKNOWLEDGMENTS |
---|
This work was supported by NEI grant EY05725. P. Mitra was a recipient of the Mark Diamond Research Fund award for the year 19992000.
Submitted: 30 July 2001
Revised: 18 March 2002
Accepted: 19 March 2002
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|