1 Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112; and 2 Department of Biological Sciences, Western Michigan University, Kalamazoo, Michigan 49008
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ABSTRACT |
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In this study, we investigated the mechanism that links activation of N-methyl-D-aspartate (NMDA) receptors to inhibition of voltage-gated sodium channels in isolated catfish cone horizontal cells. NMDA channels were activated in voltage-clamped cells incubated in low-calcium saline or dialyzed with the calcium chelator BAPTA to determine that calcium influx through NMDA channels is required for sodium channel modulation. To determine whether calcium influx through NMDA channels triggers calcium-induced calcium release (CICR), cells were loaded with the calcium-sensitive dye calcium green 2 and changes in relative fluorescence were measured in response to NMDA. Responses were compared with measurements obtained when caffeine depleted stores. Voltage-clamp studies demonstrated that CICR modulated sodium channels in a manner similar to that of NMDA. Blocking NMDA receptors with AP-7, blocking CICR with ruthenium red, depleting stores with caffeine, or dialyzing cells with calmodulin antagonists W-5 or peptide 290-309 all prevented sodium channel modulation. These results support the hypothesis that NMDA modulation of voltage-gated sodium channels in horizontal cells requires CICR and activation of a calmodulin-dependent signaling pathway.
intracellular calcium stores; voltage-gated sodium channels
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INTRODUCTION |
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REGULATION OF CYTOPLASMIC free calcium concentrations is an important mechanism by which neurons modulate their excitability. Understanding how neurons regulate intracellular calcium levels can have important physiological consequences for all areas of neurobiology. Most neurons contain two nonmitochondrial intracellular calcium stores located in the smooth endoplasmic reticulum (33, 34). One is sensitive to the phosphatidylinositol 4,5-bisphosphate metabolite, inositol 1,4,5-trisphosphate (IP3). IP3 is generated when membrane-bound enzyme phospholipase C is activated by a ligand binding to G protein-coupled receptors. The binding of IP3 receptors on the surface of the endoplasmic reticulum is coupled to calcium release into the cytoplasm (4).
The other type of nonmitochondrial calcium store found in most neurons is sensitive to increased cytoplasmic calcium and agents such as caffeine and ryanodine (33, 48). Typically, increased cytoplasmic calcium occurs through activation of ligand-gated receptors and/or by opening of voltage-gated calcium channels. The resulting increase in intracellular free calcium concentration ([Ca2+]i) causes release of calcium from the ryanodine-sensitive intracellular store. This mechanism, called calcium-induced calcium release (CICR), has been shown to increase cytoplasmic calcium concentrations from resting levels of ~100 nM to over 1 µM (53, 63). Calcium entry through ligand-gated receptors and voltage-gated channels results in short increases in cytoplasmic calcium concentration and brief changes in neuronal membrane potential (4). Increases in intracellular calcium through CICR result in a larger and more persistent change in intracellular calcium levels, which can activate various signal transduction pathways and affect greater changes in cellular physiology such as modulation of neuronal excitability, transmitter release, and gene expression. Both IP3- and ryanodine-sensitive intracellular stores have been demonstrated in catfish cone horizontal cells (30-32).
Catfish cone horizontal cells are second-order neurons in the retina
that receive synaptic input from photoreceptors and interplexiform cells (62, 68) and make synaptic connections onto bipolar cells and photoreceptors (2, 68). Horizontal cells are
electrically coupled by gap junctions (43) and are
responsible for the lateral spread of information in the distal retina
(42, 68) and the surround inhibition recorded from bipolar
cells and retinal ganglion cells (2, 68) to affect
contrast detection and visual acuity in the retina. Voltage-clamp
studies have identified several categories of voltage-dependent
channels (58, 59) and numerous types of
neurotransmitter-gated receptors (23, 47, 48, 51) on catfish cone horizontal cells. The voltage-gated ion channels include
three types of channels that are activated in the physiological operating range of the horizontal cells. These include the classic delayed rectifier, the transient tetrodotoxin (TTX)-sensitive sodium
channel (26, 58, 59), and a long-lasting, sustained dihydropyridine-sensitive (L-type) calcium channel (58,
59). All of these channels activate between 70 and
20 mV,
although in the catfish the classic delayed rectifier is unusually
small in this range of membrane potentials. It has been proposed that the other two voltage-gated channels activated between
70 and
20
mV, the voltage-gated sodium and calcium channels, contribute to
shaping the physiological response to light and help maintain the
cell's membrane potential in the dark (1, 59, 60, 71). In
addition, modulation of these channels would have considerable physiological implications as to the type of information conveyed through the retina.
Glutamate is the major excitatory neurotransmitter released from teleost photoreceptors onto the second-order horizontal cells (18, 37, 50). Glutamate receptors have been classified into two major classes, metabotropic and ionotropic. Metabotropic glutamate receptors are G protein-coupled receptors linked to a variety of signal transduction pathways, whereas ionotropic glutamate receptors directly couple glutamate binding to opening of a nonspecific cation channel. Ionotropic glutamate receptors have been further subdivided into N-methyl-D-aspartate (NMDA) and non-NMDA receptors on the basis of their pharmacological and electrophysiological characteristics. The NMDA receptor is characterized by its voltage dependence due to a magnesium block and its high permeability to calcium (36, 38). The presence of the NMDA-type glutamate receptor has been pharmacologically and electrophysiolgically characterized in isolated catfish cone horizontal cells (47, 48). However, the function of the NMDA receptor in the outer retina is unclear.
In our recently published paper (11), we described how NMDA receptor activation modulates both the voltage-gated sodium and calcium channels in catfish cone horizontal cells and provided the first step toward understanding the physiological implications of NMDA's effects on visual processing in the distal retina. Previous studies demonstrated that an increase of intracellular calcium through calcium-permeable channels induces CICR from calcium-sensitive intracellular stores and modulates the activity of voltage-gated calcium currents (31, 32). However, the mechanism by which NMDA receptor activation leads to modulation of voltage-gated sodium channels has yet to be described. In the present study, we explore this mechanism and provide evidence that NMDA modulation of voltage-gated ion channel activity in catfish cone horizontal cells involves CICR and a calmodulin-dependent signaling pathway.
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MATERIALS AND METHODS |
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Animals. Channel catfish (Ictalurus punctatus) were obtained from a local catfish farm. Experimental protocols used in this study were approved by the Institutional Animal Care and Use Committee in accordance with Public Health Service Policy on Humane Care and Use of Laboratory Animals.
Experimental solutions.
All voltage-clamp experiments in which the voltage-gated sodium current
was studied were performed in catfish saline containing (in mM) 126 NaCl, 4 KCl, 3 CaCl2, 15 dextrose, 2 HEPES, and 10 4-aminopyridine (4-AP), to block the outwardly rectifying potassium current, with the L-type calcium channel dihydropyridine antagonist nitrendipine (10 µM), to block the L-type calcium channel. Magnesium blocks the pore of the NMDA receptor at membrane potentials
hyperpolarized from 30 mV (38) and was therefore omitted
from all extracellular solutions.
Horizontal cell isolation. Isolated catfish cone horizontal cells were prepared as previously described (32). Briefly, dark-adapted channel catfish were anesthetized with tricaine methanosulfonate (10 mg/ml). When the animal no longer responded to tactile stimulation, it was pithed and both eyes were removed under dim red light. After removal of the cornea and lens, the remaining eyecups were placed in magnesium-free, low-calcium catfish saline (0.3 mM calcium compared with 3 mM in control saline) containing hyaluronidase (0.1 mg/ml) for 4 min to digest the vitreous humor (pH adjusted to 7.4 with NaOH). The eyecups were then transferred to fresh low-calcium catfish saline containing cysteine-activated papain (0.7 mg/ml) for another 4 min. The papain-treated tissue was rinsed, and the retina was manually peeled off the eyecup. Retinas were placed in fresh papain containing low-calcium catfish saline for another 4 min, rinsed well, and cut into 8-10 pieces. Retinal pieces were stored in normal catfish saline containing 1 mg/ml bovine serum albumin. Consistent recordings can be obtained from these retinal pieces for 48 h (48). Before recording, a piece of retina was further dissociated to yield isolated cone horizontal cells. This was accomplished by manual trituration of the retina through a series of progressively smaller-tipped Pasteur pipettes. Once retinal pieces were broken down into isolated cells, a sample of cells was transferred to a recording chamber mounted on the stage on an inverted Nikon Diaphot 300 microscope. Cells were allowed to settle for 5 min before an experiment began. Individual cells were viewed with Hoffman contrast and were easily identified on the basis of characteristic morphology (41).
Electrophysiology.
Cells were voltage clamped as described by Hamill et al.
(19). Patch pipettes were pulled from borosilicate glass
by a Narishige (Tokyo, Japan) vertical microelectrode puller.
Electrodes used in these experiments were uncoated and unpolished and
contained (in mM) 1 CaCl2, 10 HEPES, 140 potassium
gluconate or 140 cesium chloride, 2 MgCl2, and 11 EGTA.
Electrode resistance was measured in normal catfish saline. Electrodes
with resistances measuring between 3 and 8 M were used in this study.
Calcium imaging. Isolated catfish cone horizontal cells were loaded with the membrane-permeant, calcium-sensitive dye calcium green 2 (Molecular Probes; Eugene, OR). Calcium green 2 has a high calcium affinity (Kd = 190 nM) and is useful for detecting low calcium signals. After cells were loaded for 20 min at room temperature, they were washed and transferred to the stage of an inverted microscope (Nikon Diaphot) equipped for fluorescence measurements in magnesium-free saline containing the L-type calcium channel dihydropyridine antagonist nitrendipine (10 µM), to block the L-type calcium channel, and 10 mM 4-AP to block the outwardly rectifying potassium current. Loaded cells were imaged with the Noran Instruments Intervision Odyssey system with fluorescence channels. A laser was used to excite the calcium green at 488 nm, and a 520-nm barrier filter was used to isolate the fluorescent emission. Recordings of fluorescence data were collected with the systems software driven by a SGI R4000SC workstation. Changes in relative fluorescence were monitored before and after agonist and antagonist applications.
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RESULTS |
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NMDA receptor activation linked to modulation of voltage-gated
sodium channels.
Figure 1 demonstrates the effect of NMDA
on the I-V relationship recorded from a voltage-clamped
catfish cone horizontal cell in saline containing pharmacological
blockers of the voltage-gated sodium and calcium currents. Current
traces were obtained under control conditions, 1 min after application
of 100 µM NMDA, and 2 min after NMDA was washed out (recovery). NMDA
at 100 µM induced an inward current from 60 mV to 0 mV and an
outward current when the cell was depolarized beyond 0 mV. Besides
inducing current that reversed near 0 mV, activation of NMDA receptors
in isolated catfish cone horizontal cells also modulated voltage-gated
sodium channels by reducing the peak amplitude of the induced
voltage-gated sodium current (Fig. 2). As
shown in Fig. 2, A and B, peak voltage-gated sodium current was elicited under control conditions and 1 min after
NMDA was applied to a voltage-clamped catfish cone horizontal cell.
Peak sodium current activity was elicited with the stepwise stimulus
paradigm and superimposed for comparison. In the presence of NMDA, the
amplitude of the voltage-gated sodium current decreased by 75%
compared with control current amplitude (Fig. 2A); however, 1 min after NMDA application the NMDA-induced current was still apparent. Therefore, the resulting decrease of input resistance and
opening of agonist-gated cation channels could be sufficient to explain
the decrease of sodium channel activity. However, in our previous paper
(11), we provided evidence that modulation of the
voltage-gated sodium and calcium channels in catfish cone horizontal
cell is not solely due to the NMDA-induced change of input resistance.
This evidence is based on the finding that NMDA-induced modulation of
the voltage-gated sodium and calcium channels persisted significantly
longer than the NMDA-induced current and the return of input resistance
to control conditions. Combined with evidence that NMDA does not modify
the delayed rectifier in catfish cone horizontal cells and that
voltage-clamp conditions were optimal, these results suggested
that NMDA's modulatory effects on voltage-gated sodium channel
activity may be processed through a second messenger system in catfish
cone horizontal cells. The evidence provided here supports the
hypothesis that the modulatory effect of NMDA on voltage-gated sodium
channels is likely due to activation of intracellular signaling
pathways that alter ion channel activity.
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NMDA-induced changes of intracellular calcium.
The finding that NMDA's effect on voltage-gated channel activity
lasted significantly longer than the NMDA-induced current suggests that
NMDA's modulation of channel activity is not directly due to current
through the NMDA receptor. Because NMDA receptor channels are highly
permeable to calcium influx (36, 38) and calcium
permeation through agonist-gated channels was previously found to be
involved in modulation of voltage-gated calcium currents (31,
32), we tested the hypothesis that calcium ions act as a second
messenger to link activation of NMDA receptors and modulation of the
voltage-gated ion sodium channels in catfish cone horizontal cells. To
determine whether changes in [Ca2+]i are
associated with activation of the NMDA receptor, isolated cells were
loaded with the calcium-sensitive dye calcium green 2 in saline
containing the dihydropyridine antagonist nitrendipine, to block
activity of the L-type voltage-gated calcium channels. Changes in
relative fluorescence, demonstrating the fluctuations in
[Ca2+]i, were monitored before and after NMDA
application (Fig. 3A). Although concentration changes could not be measured directly with
calcium green 2, an increase of the relative fluorescence signal from
baseline represents an increase of intracellular calcium. Application
of 100 µM NMDA to a calcium green 2-loaded catfish cone horizontal
cell elicited a relative increase of [Ca2+]i
persisting until NMDA was washed out. This response could be repeated
at intervals of 1 min after NMDA washout, which likely corresponds to
the time required for intracellular stores to refill. Similar changes
of fluorescence were obtained from 10 other catfish cone horizontal
cells loaded with calcium green 2 in the presence of 100 µM NMDA.
When NMDA was applied in the presence of the competitive NMDA
antagonist AP-7 (100 µM), NMDA failed to elicit any increase of
relative fluorescence (Fig. 3B, n = 6). AP-7
at 100 µM blocked the effect of NMDA by 100%. To demonstrate that
the NMDA-induced increase of [Ca2+]i was
initiated by extracellular calcium permeating the NMDA channel,
experiments were repeated in saline containing low (0.3 mM)
extracellular calcium (Fig. 3C) and 10 µM nitrendipine. In low-calcium saline, NMDA's fluorescence signal was reduced by 88%
compared with the fluorescence signal obtained with NMDA under control
conditions. This significant difference was recorded in seven other
cone horizontal cells, in which low calcium decreased the relative
fluorescence signal by a mean of 85% (±5.2%; P < 0.01). Likewise, when loaded cells were incubated in the
membrane-permeant calcium chelator BAPTA-AM (5 mM), the effect of NMDA
on relative fluorescence was reduced by a mean of 78% (±3.5%)
compared with control relative fluorescence signals and represents a
significant decrease (n = 5; P < 0.01). These results support the hypothesis that calcium
permeation through the NMDA receptor causes a significant increase in [Ca 2+]i, which may link receptor
activation to calcium-dependent signaling pathways and subsequent
modulation of ion channel proteins.
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Release of calcium from intracellular stores. Increased intracellular calcium can link receptor activation to modulation of channel activity in several different ways. In one scenario, calcium could directly affect channel activity. A rise in free calcium concentration could also activate a multitude of calcium-dependent enzymes, which may ultimately affect channel activity. The question remains as to whether the influx of calcium through the NMDA receptor is enough to trigger these effects or whether this calcium signal may be amplified through the process of CICR. The existence of CICR from intracellular calcium stores in catfish cone horizontal cells has been well documented. (31, 32). Previous studies demonstrated that these intracellular stores release calcium in response to calcium and agents such as 10 mM caffeine (44) or 10 µM ryanodine. In addition, release of calcium from these stores can be depleted with caffeine and directly inhibited with 2 µM ruthenium red (31, 32).
Figure 4 illustrates relative fluorescence measurements obtained from a voltage-clamped catfish cone horizontal cell before, during, and after application of NMDA. The cell was held at its resting membrane potential in saline containing zero magnesium and 10 µM nitrendipine. Caffeine (10 mM) and NMDA (100 µM) each elicited a large increase in fluorescence signal. Similar changes of relative fluorescence were obtained from eight other catfish cone horizontal cells. However, when ruthenium red was allowed to dialyze into the cell before application of caffeine or NMDA, the increase in fluorescence signal was diminished between 70% and 90% compared with fluorescence signals obtained under control conditions (Fig. 4, B and D; n = 5). Together, these results suggest that a majority of the caffeine-induced and the NMDA-induced increase of fluorescence signal is the result of calcium release from intracellular stores.
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Evidence for CICR.
Figure 5 provides evidence for
NMDA-induced CICR. As shown in Fig. 5A, an increase in
relative fluorescence was measured after application of 100 µM NMDA
to a calcium green 2-loaded catfish cone horizontal cell. After a
prolonged washout, application of 10 mM caffeine produced a similar
increase in relative fluorescence in the cell. After another prolonged
washout, 100 µM NMDA was applied again, immediately followed by a
second dose of 10 mM caffeine. There was no prolonged washout between
the second application of NMDA and the second application of caffeine.
The subsequent application of caffeine elicited a response in relative
fluorescence that was 80% smaller than the first application. This
suggests that NMDA receptor activation caused near-depletion of an
intracellular calcium store and that both NMDA and caffeine affect the
same intracellular store. This was the typical response recorded from a
total of six isolated catfish cone horizontal cells loaded with calcium
green 2 when caffeine was applied to a loaded voltage-clamped catfish
cone horizontal cell immediately after application of NMDA. Under these
conditions, the second caffeine fluorescence response was reduced by an
average of 78% (±5.1%) compared with the first control caffeine
response (P < 0.01).
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NMDA modulation of voltage-gated ion channels is mediated by CICR.
If CICR is involved in linking NMDA receptor activation to modulation
of voltage-gated ion channels in catfish cone horizontal cells,
caffeine should mimic NMDA's effect on these channels. Figure
6 illustrates a side-by-side comparison
of the effect of NMDA and caffeine on the voltage-gated sodium channel.
Voltage-gated sodium currents were elicited with the stepwise stimulus
paradigm before and after NMDA application (Fig. 6A) and
caffeine application (Fig. 6B). Caffeine reduced the peak
amplitude of the sodium current INa in a manner
that mimicked NMDA's effect. As can be seen in Fig. 6A NMDA
reduced the peak amplitude of INa by 35%,
whereas as shown in Fig. 6B caffeine reduced the peak
amplitude of INa by 58%. These results were
replicated in eight other voltage-clamped cells, in which caffeine
reduced the peak amplitude of INa by a mean of
56% (±5.1%) compared with control conditions. Caffeine always
produced a larger decrease of INa amplitude. We
propose that this is due to the extent of calcium release from
intracellular stores caused by caffeine compared with release of
calcium caused by 100 µM NMDA. As evident from data obtained in other
studies (32), 10 mM caffeine produces near-complete
depletion of calcium from intracellular stores in catfish cone
horizontal cells for a specified period of time. However, 100 µM NMDA
does not deplete stores to the same degree that 10 mM caffeine does.
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NMDA modulation of voltage-gated ion channels is mediated by a calmodulin-dependent pathway. The results presented above demonstrate that the effect of NMDA on voltage-gated sodium and calcium channels of catfish retinal horizontal cells is mediated by CICR from ryanodine-sensitive stores. The question of how this rise in intracellular calcium results in a modulation of ion channels remains. One possibility involves calcium acting directly on ion channel proteins (20). Calcium has been shown to modulate neuronal activity through modulation of voltage-gated ion channels (8, 57). However, the high permeability of the NMDA receptor to calcium and its ability to initiate CICR make it an ideal activator of other calcium-dependent signaling pathways. One possible pathway involves the ubiquitous calcium-binding protein calmodulin (CaM). Calcium has been shown to bind CaM, leading to activation of Ca2+/CaM-dependent protein kinases such as CaM kinase II, and can cause phosphorylation of ion channel proteins (61, 72), thereby altering their activity. The modulation of voltage-gated ion channels by NMDA and non-NMDA receptor activation via a CaM-dependent signal transduction pathway was demonstrated previously in other systems (31, 73). Therefore, we tested the hypothesis that modulation of voltage-gated sodium channels in catfish cone horizontal cells by NMDA is CaM dependent.
The effect of NMDA on the voltage-gated sodium channels was measured in the presence of one of two CaM antagonists, W-5 and peptide 290-309. Each of these agents was allowed to dialyze into voltage-clamped cells through the recording pipette solution. Both CaM antagonists significantly reduced NMDA's effect on voltage-gated sodium current amplitude. W-5 (50 µM) reduced the NMDA-induced reduction of peak calcium current amplitude by only 8% (±1.0%; n = 5) compared with the 35% reduction obtained under control conditions (Fig. 7). Peptide 290-309 (20 µM) also reduced NMDA's effect to an average reduction of only 3% (±0.9%; n = 5) compared with control conditions (Fig. 7). Both of these CaM antagonists significantly affected NMDA's effect on channel activity (P < 0.01). These results suggest that NMDA receptor modulation of voltage-gated sodium channels in catfish cone horizontal cells may be dependent on CaM activation. Previous electrophysiological and calcium-imaging studies have identified non-NMDA as well as NMDA channels on catfish cone horizontal cells. The non-NMDA channels have been pharmacologically classified as dl-
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DISCUSSION |
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In our earlier report (11), we described modulation of voltage-gated sodium and calcium conductances by NMDA receptor activation in retinal horizontal cells of the catfish. Evidence from the current paper demonstrates that voltage-clamped sodium and calcium currents were dramatically reduced for a relatively long period of time after application of NMDA. Both channels have activation ranges well within the physiological operating range of the horizontal cell, and evidence was presented that both voltage-gated channels could contribute to help shape the physiological response to light and that the L-type sustained calcium current could help maintain the membrane potential in the dark. The fact that these currents could play a role in determining neuronal excitability and maintaining membrane potential means that their modulation would have considerable physiological implications as to the type of information conveyed throughout the retina in terms of the on- and off-center receptive fields.
Previous studies in this laboratory (31, 32) have focused on the mechanism of voltage-gated L-type calcium channel modulation in catfish cone horizontal cells. These studies demonstrated that an increase of intracellular calcium through agonist-induced CICR linked activation of glutamate receptor subtypes to modulation of the voltage-gated calcium currents recorded in these cells. In the present study, we determined that a similar mechanism links NMDA receptor activation to modulation of voltage-gated sodium channels via a calmodulin-dependent pathway. This mechanism acts to provide a prolonged reduction of the voltage-gated sodium current.
Calcium ions were explored as a possible second messenger linking NMDA receptor activation to modulation of voltage-gated ion channels because of the NMDA receptor's permeability to calcium. We found that calcium influx through both NMDA and non-NMDA receptors triggered CICR from intracellular stores and that CICR is necessary for sodium channel modulation to occur. Besides catfish, glutamate receptor activation in other systems has also been linked to CICR (13, 27). At rest, the intracellular concentration of calcium is near 100 nM. Release of calcium from intracellular stores has been shown to increase this concentration to >1 µM for transient periods of time (32). This type of increase in intracellular calcium concentration can result in the activation of a myriad of calcium signaling pathways. Calcium ions can directly modulate voltage-gated ion channels, resulting in changes in neuronal activity (8, 57), or calcium may combine with the ubiquitous intracellular calcium protein CaM to initiate intracellular signaling events. Activation of CaM-dependent signaling pathways by NMDA has been demonstrated in a variety of systems (16, 25, 73). Ca2+/CaM exerts its effects by binding with various cellular proteins. One of the most common is the CaM kinase II. CaM kinase II phosphorylates other cellular proteins, including ion channels, thereby regulating their activity (61, 72). CaM kinase II is associated with the NMDA receptor near the plasma membrane (28) and has been shown to regulate NMDA receptor activity in the retina (25). Once activated by Ca2+/CaM, CaM kinase II can remain active long after the initial signaling event is over because of autophosphorylation of the enzyme until the autophosphorylation is reversed by phosphatases in the cytoplasm. This ability of CaM kinase II to remain active long after the initial signaling event is over has been proposed to be an important mechanism in some types of learning and memory (3, 16, 54). In catfish cone horizontal cells, this mechanism may be an important mechanism during light adaptation (5). Therefore, we tested the hypothesis that NMDA modulation of voltage-gated sodium and calcium conductances was dependent on CaM activation and found that two CaM antagonists blocked NMDA's modulatory effect on voltage-gated sodium channels. Thus it is likely that Ca2+/CaM activates CaM kinase II to directly or indirectly phosphorylate the voltage-gated ion channels, resulting in their modulation.
Previous biochemical and functional studies have examined sodium
channel phosphorylation (29). Sodium channel proteins in the mammalian brain consist of - and
-subunits (6,
7). Expression of
-subunits alone is sufficient for the
formation of functional sodium channels (17, 45, 56), but
-subunits are required to give the characteristic kinetic properties
and voltage dependence of sodium channel activation and inactivation (22, 40). Biochemical studies of purified brain sodium
channels show that
-subunits are exceptionally good substrates for
phosphorylation by cAMP-dependent protein kinase (PKA; Ref.
9) and by protein kinase C (PKC; Ref. 10),
whereas no phosphorylation of the
-subunits occurs. Similar findings
have been identified in synaptosomes (9) and in intact
brain neurons in cell culture (46, 55, 69). Although
activation of PKA and PKC may be a common mechanism associated with
sodium channel phosphorylation in some systems, other signaling
pathways may also be directly or indirectly important in neuronal
development and function. For example, phosphorylation and
dephosphorylation of sodium channels on tyrosine residues has been
found to modulate sodium channel inactivation (21, 52) and
direct interaction of sodium channels with G proteins may regulate
voltage-dependence gating of the sodium channel (34, 35).
Few neuronal studies, however, have linked the calcium-dependent CaM
pathway to regulation of sodium channel activity, although the
calcium-dependent CaM pathway was found to modulate sodium channel in
an isoform-specific manner via direct interaction with skeletal muscle
sodium channels (12).
In the present study, the effect of the CaM pathway on sodium channel activity in catfish cone horizontal cells may be direct or indirect. Because a PKII site has not been identified on voltage-gated sodium channels in biochemical studies of other systems, it is likely that the CaM-PKII action is indirect, working on a cAMP- or diacylglycerol-dependent pathway, although we cannot rule out the possibility that the voltage-gated sodium channel in catfish is unique and contains a PKII phosphorylation site. However, the interaction between two second messenger systems exists in rat olfactory neurons, in which odorant-evoked elevations in cAMP activate cyclic nucleotide-gated channels, leading to external calcium influx, while at the same time, the excitatory action of calcium activates the calcium-dependent CaM to activate PKII and inhibit adenylate cyclase (66, 67). In this olfactory system, the inhibition of adenylate cyclase may contribute to termination of olfactory signaling (69).
Besides calcium-dependent protein kinases, it is well known that activated CaM also binds to neuronal nitric oxide (NO) synthase and can stimulate guanylyl cyclase to form cGMP (65). NO by itself can serve as an intra- or intercellular messenger (14), or cGMP can effect a diversity of biological responses including modulation of agonist-gated currents in retinal neurons (39, 70). In addition, there are several other calcium-dependent processes described in the literature that can ultimately lead to modulation of voltage-gated channel activity in neurons (15, 49, 64). Therefore, although the results of this study provide evidence that CaM is involved in the pathway linking NMDA receptor activation to modulation of voltage-gated sodium channels in catfish cone horizontal cells, the influence of other calcium-dependent pathways contributing to this modulation is certainly a possibility.
Together these results provide insight into a potential mechanism for
the NMDA receptor-mediated modulation of voltage-gated sodium channels
in catfish cone horizontal cells (Fig.
9). We conclude that activation of both
NMDA and non-NMDA receptors present on catfish cone horizontal cells
results in calcium permeation through agonist-gated channels and
induces CICR from calcium-sensitive intracellular stores. Modulation of
the voltage-sensitive sodium conductance is dependent on this and the
activation of the calcium-binding protein CaM. NMDA receptor activation
has been linked to CICR and CaM activation in other systems, most
notably in hippocampal models of long-term potentiation (3, 16,
54). Our studies showing that NMDA receptor activation results
in modulation of voltage-gated sodium conductance via CICR and CaM
provide new insight into the role of the NMDA receptor in synaptic
plasticity.
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Catfish cone horizontal cells as well as cells in other systems coexpress both NMDA- and non-NMDA glutamate receptors. The question still remains as to how the role of the NMDA receptor differs from the role of the non-NMDA glutamate receptors in synaptic plasticity. At rest, it is likely that only non-NMDA glutamate receptors are activated, because of NMDA receptor channel blockage by physiological concentrations of magnesium. Activation of the non-NMDA glutamate receptors, however, would depolarize the cell and release the voltage-dependent magnesium block of the NMDA channels. Therefore, the presence of non-NMDA glutamate receptors on catfish cone horizontal cells may mediate fast excitatory responses that depolarize the cell and allow activation of NMDA channels. AMPA receptors rapidly desensitize, but we have demonstrated that NMDA activation of the voltage-gated sodium channel leads to relatively long-term reduction of the voltage-gated sodium channel. A previous study demonstrated that modulation of the voltage-gated sodium channel in salamander retina plays a role in contrast adaptation at the level of the ganglion cells (24). Approximately one-half of the contrast adaptation to variable light stimuli in the salamander retina can be attributed to a change in the input-output relationship of the retinal ganglion cells correlated with a change in sodium channel function (24). At the horizontal cell level, it is likely that modulation of voltage-gated ion channels would directly affect the input-output relationship of the cell and directly contribute to physiological processes associated with horizontal cell function, such as light and dark adaptation and surround inhibition.
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ACKNOWLEDGEMENTS |
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We thank Dr. John Cork for advice and imaging assistance and Dr. David Linn for helpful discussions throughout the course of this study.
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FOOTNOTES |
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This study was supported by National Eye Institute Grant EY-11133 awarded to C. L. Linn and a Louisiana State University Board of Regents Grant to S. F. Davis.
Address for reprint requests and other correspondence: C. L. Linn, Dept. of Biological Sciences, Western Michigan Univ., 1903 W. Michigan Ave., Kalamazoo, MI 49008 (E-mail: clinn{at}unix.cc.wmich.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 29, 2003;10.1152/ajpcell.00256.2002
Received 3 June 2002; accepted in final form 19 December 2002.
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