Desensitization of the mGluR6 transduction current in tiger salamander On bipolar cells

Abstract

Light depolarizes retinal On bipolar cells, opening the cation-selective channels that are responsible for producing the synaptic current. In this study, the basic features of light-induced signals were mimicked by bathing slices of salamander retina with an agonist for the mGluR6 receptor that is expressed on the dendrites of On cells, and then displacing the agonist with the mGluR6 antagonist (RS)-a-cyclopropyl-4-phosphonophenylglycine (CPPG). The transduction current that is activated by this protocol rapidly shuts off, or desensitizes. Desensitization was highly correlated with the concentration and the type of Ca²⁺ buffer that was dialysed into the cell: When Ca²⁺ buffering was minimized by dialysing cells with 0.5 mm EGTA, the steady-state response was reduced to approximately 40% of the peak response. Buffering with 10 mm EGTA reduced desensitization, while BAPTA completely eliminated it. Removing external Ca²⁺ also prevented desensitization, suggesting that entry of Ca²⁺ through the transduction channel provides the trigger. The time course of desensitization was measured by using a voltage jump protocol to rapidly increase Ca²⁺ influx, and could be fitted with a single time constant on the order of 1 s, in good agreement with previously published rates of desensitization to steps of light in this species. It is proposed that Ca²⁺-dependent shut-off of the On bipolar cell transduction current may contribute to the conversion of sustained to transient light responses that predominate in the inner retina.

Photoreceptors synapse with two kinds of bipolar cells, distinguished by the polarity of their responses to light and glutamate agonists. Off bipolar cells hyperpolarize in response to light because light closes AMPA or kainate channels (DeVries & Schwartz, 1999; DeVries, 2000) by decreasing the concentration of glutamate in the synaptic cleft. On the other hand, light depolarizes On bipolar cells by activating a cation-selective transduction current (Shiells et al. 1981; Nawy & Copenhagen, 1987). The current is negatively coupled to a metabotropic receptor, mGluR6 (Nakajima et al. 1993) that is localized to the dendrites of On bipolar cells (Nomura et al. 1994; Vardi & Morigiwa, 1997; Vardi et al. 2000), and mediates synaptic transmission between On bipolar cells and photoreceptors.

A number of studies have characterized the actions of glutamate and l-2-amino-4-phosphonobutyrate (AP-4), a group III metabotropic receptor agonist (reviewed in Pin & Duvoisin, 1995), and have conclusively demonstrated that they hyperpolarize On bipolar cells by closing the cation-selective transduction channels that are opened by light (Shiells et al. 1981; Slaughter & Miller, 1981; Nawy & Copenhagen, 1987; Nawy & Jahr, 1991; Thoreson & Miller, 1993; de la Villa et al. 1995). Most of these studies utilized an experimental protocol in which glutamate or AP-4 was applied intermittently under conditions where there was minimal endogenous release of transmitter (i.e. light-adapted preparations or dissociated cells). This protocol probably does not mimic physiological stimuli to On bipolar cells, which are designed to detect light-induced decrements from a background of essentially continuous transmitter release. Responses to exogenous application of glutamate or AP-4 are essentially flat for the duration of the application period, showing no obvious desensitization (Nawy & Jahr, 1990; de la Villa et al. 1995). This makes sense for a cell which is designed to be continuously bombarded with glutamate. Fewer studies have examined the effect of agonist withdrawal on the On bipolar cell, as occurs during light stimulation.

This issue was addressed in two recent studies of On bipolar cell responses to steps of light (Shiells & Falk, 1999; Awatramani & Slaughter, 2000). Both studies, one of the all-rod dogfish retina, and the other of the mixed rod—cone salamander retina, found that the transduction current, revealed by maintained illumination, strongly desensitized. One of these studies (Shiells & Falk, 1999) also showed that buffering Ca²⁺ with BAPTA eliminated the transient nature of the response, suggesting that Ca²⁺ might play a role in shaping the synaptic response. These studies provide evidence for postsynaptic regulation of synaptic transmission which allows the membrane potential to recover toward the dark potential during continuous illumination.

One problem with the use of light to control postsynaptic responses is that if the photoreceptors themselves are not monitored, it is difficult to be certain that the recovery in the postsynaptic response is not due to a parallel recovery of presynaptic release, as would occur during photoreceptor adaptation (Fain et al. 2001). To avoid this problem in the present study, the presynaptic cell was bypassed and responses were elicited by applying glutamate or AP-4 to On bipolar cells (as in darkness), and the mGluR6 antagonist (RS)-a-cyclopropyl-4-phosphonophenylglycine (CPPG) to mimic light. The findings obtained using this approach strongly support a Ca²⁺-feedback model of channel desensitization which decreases the output of the On pathway in the face of steady illumination.

Methods

Slices of retina from larval tiger salamanders (Charles Sullivan) were prepared as previously described (Walters et al. 1998; Nawy, 1999). Briefly, salamanders were anaesthetized with 3-aminobenzoic acid ethyl ester, decapitated, and the eyes enucleated. The experimental protocol was approved by the Albert Einstein College of Medicine Institute for Animal Studies committee. Whole retinas were isolated and placed on a 0.65 μm cellulose acetate/nitrate membrane filter (Millipore) which was secured with vacuum grease to a glass slide adjacent to the recording chamber. Slices were then cut to a thickness of 150–200 µm with a tissue slicer (Stoelting), transferred to the recording chamber while remaining submerged, and viewed with a Zeiss fixed-stage upright microscope equipped with a water-immersion 40× objective modified for Hoffman modulation. Slices were bathed in solution containing (mm): 108 NaCl, 2 CaCl₂, 2.5 KCl, 1.2 MgCl₂, 10 Hepes, 10 glucose, 0.1 picrotoxin (pH 7.6 with NaOH). AP-4 (4 µm) was added to the bath immediately before the start of each experiment. This concentration is sufficient to shut off the transduction current. Nominally Ca²⁺-free solution was prepared by omitting Ca²⁺ without substitution. Solution was perfused continuously through the recording chamber at a rate of approximately 1 ml min⁻¹.

Glutamate analogues were applied via flowpipes, which consisted of two polymer-coated fused silica tubes (o.d. 350 µm, i.d. 250 µm) positioned close to the cell. One tube contained 1 mm glutamate dissolved in the bath solution, and the other contained 300 µm CPPG. The tubes were mounted onto a computer-controlled piezo-bimorph (Morgan-Matroc). In some experiments, a Picospritzer was used to apply 600 µm CPPG to On bipolar cells by pressure ejection (typically 1–2 p.s.i.) from an unpolished pipette.

Patch pipettes were fabricated from borosilicate glass (WPI) using a two-stage vertical puller (Narishige). Whole-cell currents were recorded with the Axopatch 1D amplifier. Recordings had input and series resistances of 300–600 MΩ and 5–15 MΩ. Three internal solutions were used. For experiments with 10 mm EGTA, the pipette solution contained (mm): 85 potassium gluconate, 10 KCl, 10 Hepes, 10 EGTA, 4 MgATP, and 1 LiGTP (pH 7.4 with KOH). For experiments with 0.5 mm EGTA, potassium gluconate was raised to 95 mm. For faster buffering of Ca²⁺ the pipette solution contained 20 mm BAPTA and 65 mm potassium gluconate but was otherwise unchanged. All chemicals were obtained from Sigma except for CPPG and AP-4, which were obtained from Tocris. Holding potentials were corrected for the liquid junction potential, which was measured to be −10 mV with the 10 mm EGTA—gluconate-based pipette solution. Data were acquired with Axograph software and the Digidata 1322A interface and analysed with Kaleidagraph and Axograph software.

Results

On bipolar cell transduction current desensitizes upon removal of agonist

Slices were bathed in solution containing the mGluR6 agonist AP-4 throughout the experiment. This constant exposure to agonist mimicked the continuous bombardment of transmitter from photoreceptors that is a basic feature of darkness. Although glutamate is the native transmitter at this synapse, its application for extended periods of time proved to be toxic to the retina, and AP-4 was therefore used, with no obvious detrimental effects. To pharmacologically imitate the conditions of low synaptic release during light, a stream of solution containing CPPG was used to blow agonist away from the cell. Early in this study, CPPG was paired with AP-4 as the agonist in the flowpipes. However glutamate was used in later experiments, not only because it allowed for unambiguous identification of On and Off cells, but also because CPPG turned on the transduction current more rapidly when paired with glutamate (data not shown), presumably because glutamate has a faster unbinding rate than AP-4.

After breaking into an On bipolar cell, a stream of solution containing glutamate was directed onto the cell. Figure 1A contrasts the response of a cell to the application and removal of agonist. On the left is a response to glutamate, which binds to the mGluR6 receptor and shuts off an inward current by closing a cation-selective channel. The channels remained closed for the duration of the application. On the other hand, rapid removal of agonist by exposing the cell to a stream of CPPG (right) produced an inward current that decayed to a plateau whose magnitude was approximately 40% of the peak response. On bipolar cells therefore respond to glutamate application in a sustained fashion, but to glutamate removal in a transient manner.

Figure 1. Transient response to removal of agonist originates in the dendrites of On bipolar cells.

A, two responses from the same cell. Left panel illustrates the sustained outward response to 1 mm glutamate. Record is the average of 4 trials. Right panel illustrates the transient nature of the mGluR6 transduction current, revealed by the rapid application of the mGluR6 antagonist CPPG (300 µm). Record is the average of 3 trials. Cell was recorded with a pipette solution containing 0.5 mm EGTA at a holding potential of −40 mV. B, response of another cell to pressure ejection (2 p.s.i.) of 600 µm CPPG aimed at the dendrites or axon terminal as indicated in the figure. Right and left panels differ only in the duration of the application.

The stream of CPPG that was delivered to On bipolar cells in these experiments was wide enough to cover the entire cell. As CPPG is an inhibitor of all group III metabotropic receptors, the inward current produced by CPPG might have resulted from inhibition of group III receptors that are expressed on the axon terminals of On bipolar cells (Awatramani & Slaughter, 2001), rather than by inhibition of mGluR6. To test this possibility, CPPG was focally applied to the inner or outer retina by pressure ejection from a fine-tipped pipette. When aimed in the direction of the bipolar cell dendrites, CPPG produced an inward current with short latency, while CPPG directed to the inner retina produced a smaller, long latency response that was probably due to diffusion of CPPG to the dendrites (Fig. 1B). Thus, the predominant effect of CPPG under these conditions was to turn on the mGluR6 transduction current by binding to mGluR6 receptors that are expressed on the dendrites.

Ca²⁺ is required for desensitization

Manipulation of Ca²⁺ buffering had a dramatic effect on desensitization of the mGluR6 current. To quantify this, CPPG was applied for 30 s and the amplitude of the peak and plateau response were measured as in Fig. 2A. The desensitization ratio, defined as one minus the ratio of the amplitude of the plateau and peak responses, was 0.60 ± 0.03 (mean ± s.e.m., n = 13) when Ca²⁺ was buffered with an internal solution containing 0.5 mm EGTA. On the other hand, buffering Ca²⁺ with 20 mm BAPTA essentially eliminated desensitization (Fig. 2A, right panel). Overall, in cells buffered with BAPTA, the desensitization ratio was 0.01 ± 0.03 (n = 12). These data, summarized in Fig. 2B, suggest that desensitization of the mGluR6 current is mediated by a Ca²⁺-dependent process.

Figure 2. Buffering with BAPTA converts the CPPG response from transient to sustained.

A, left: response to CPPG in a cell buffered with 0.5 mm EGTA. Also shown is the method for calculating the desensitization ratio. Dashed line indicates the amplitude of the sustained, or plateau component, and the thick line indicates the peak of the mGluR6 current. The desensitization ratio was defined as 1 − (I_sustained/I_peak). Holding potential was −40 mV. A, right: response of another cell that was dialysed with 20 mm BAPTA. Holding potential was −70 mV. B, summary of the desensitization ratio in 12 cells recorded with BAPTA (either at −70 or −40 mV) and 13 cells recorded with 0.5 mm EGTA (all at −40 mV).

The transduction channel has been shown previously to be Ca²⁺ permeable (Nawy, 2000). If desensitization follows from the entry of Ca²⁺ through open transduction channels, then it should be reduced when Ca²⁺ is removed from the extracellular solution. To test this, responses to CPPG were first recorded in normal solution (2 mm Ca²⁺). The cell was then exposed to streams of nominally Ca²⁺-free solution, and the response to CPPG was measured again. Now desensitization was largely abolished (Fig. 3A). In five cells in which CPPG responses were recorded in both 2 mm Ca²⁺ and in nominally Ca²⁺-free solutions, the mean desensitization ratio decreased from 0.64 ± 0.03 to 0.02 ± 0.06, respectively (Fig. 3A, right panel). As a second test, cells were held at −40 mV and at +40 mV during application of CPPG. If Ca²⁺ entry is required for desensitization, then holding the cell at potentials where Ca²⁺ influx is reduced should prevent desensitization. At +40 mV, desensitization was dramatically reduced compared with −40 mV (Fig. 3B). These data imply that Ca²⁺ entry through the transduction channel is the trigger for desensitization of the current. Alternatively,Ca²⁺ might enter through voltage-gated Ca²⁺ channels. However this seems unlikely, since desensitization persisted in cells that were held at −70 mV, and L-type Ca²⁺ channels, the predominant Ca²⁺ channel in On bipolar cells (Heidelberger & Matthews, 1992; Tachibana et al. 1993), are closed at that voltage.

Figure 3. Elimination of Ca ²⁺ influx prevents desensitization of the transduction current.

A, left: responses to CPPG in the presence of 2 mm Ca²⁺ (dark trace) and nominally Ca²⁺-free solution (light trace). A, right: summary of the desensitization ratio in 5 cells that were exposed to 0 and 2 mm Ca²⁺. Elimination of Ca²⁺ consistently reduced desensitization. B, left: a pair of responses to CPPG from a cell that was held at +40 mV and at −40 mV. Cell was buffered with 0.5 mm EGTA. B, right: summary of desensitization for 8 cells held at +40 mV and −40 mV. Reducing the driving force for Ca²⁺ through the transduction channel similarly reduced desensitization. *P < 0.001 (unpaired Student t test).

Measurement of transduction current following voltage jumps

Measurement of both the degree and kinetics of desensitization using rapid switching of solutions requires the near simultaneous arrival of antagonist at all of the mGluR6 receptors in the dendritic arbor. Otherwise, desensitization of some channels might begin before others have opened, thus blunting the peak amplitude of the response. This is analogous to the manner in which the speed of AMPA delivery determines the amplitude of the peak of the AMPA response in horizontal cells (Eliasof & Jahr, 1997). When tested on the same cell, responses to CPPG were usually larger in Ca²⁺-free than Ca²⁺-containing solution, probably because the application of CPPG was too slow to reach all of the mGluR6 receptors before desensitization set in. To avoid this problem, an alternative strategy was devised to measure the kinetics and Ca²⁺ dependence of desensitization using a method that did not require fast application of CPPG (Fig. 4). This approach took advantage of the finding that desensitization was minimal at positive potentials, thus affording the opportunity for CPPG to reach all of the receptors before desensitization was initiated. After a 5 s application of CPPG, the holding potential was jumped to a new test voltage. Stepping from +40 to −40 mV elicited an inward current which was likely to be the transduction current, as the same voltage step applied when the transduction channels was closed (by glutamate) yielded only ohmic currents (data not shown).

Figure 4. Kinetics of desensitization revealed by voltage jumps.

A, left panel is an On bipolar cell trace illustrating the voltage jump protocol. The cell was held at +40 mV during CPPG application and then stepped to a negative voltage, −40 mV in this example. To isolate the mGluR6 current, the voltage protocol was repeated during continuous exposure to glutamate, and that result was subtracted from the raw record to obtain the trace that is shown here. Right panel is an expansion of the decay of the transduction current indicated by the box on the left. Dark line is the fit with a single time constant of 0.95 s. Inset: filled bars represent the mean ± s.e.m. of the time constant at each voltage in cells that were buffered with 0.5 mm EGTA. Open bar is the time constant measured in cells buffered with 10 mm EGTA at −40 mV. For 10 mm EGTA, time constants measured by the voltage jump method or the fast switching method were virtually identical, and results from both methods have therefore been pooled. B, left: desensitization ratio plotted as a function of voltage in cells buffered with 0.5 mm EGTA, The desensitization ratio was calculated as 1 − (I_plateau/I_{instantaneous}). B, right: desensitization ratios obtained in cells dialysed with 10 mm EGTA. There was less desensitization overall, compared with 0.5 mm EGTA, and desensitization was strongly voltage dependent. **P < 0.001, *P < 0.01 (unpaired Student t test). n = 7, 17 and 8 for −20, −40 and −70 mV, respectively. Data using the rapid application of CPPG and the voltage jump protocol were pooled. C, I–V relation obtained by measuring the instantaneous and plateau currents of 0.5 EGTA cells from B. Data point at +40 mV were obtained from the initial and final size of the response to CPPG application prior to the voltage jump. Desensitization confers a pronounced outward rectification to the transduction current measured 5–10 s after the voltage jump. Continuous line is the linear fit to the instantaneous values.

To isolate the transduction current, the ohmic current measured in glutamate was subtracted from the response to CPPG application to yield the trace in Fig. 4A. For this cell, the rate of decay of the transduction current at early times was well fitted by a single exponential with a time constant of 0.95 s (right panel). For twelve cells, the decay rate was measured following jumps from +40 mV to either two or three test voltages and the results are summarized in the inset of the right-hand panel of Fig. 4A (filled bars). When buffered with 0.5 mm EGTA, the time constant was on the order of 1 s. Although there was a trend toward an acceleration of the rate of decay with hyperpolarization, the difference was not statistically significant. When measured in cells buffered with 10 mm EGTA, the decay rate was an order of magnitude slower (open bar).

At each voltage the instantaneous mGluR6 current was measured within 50 ms after the jump, as was the plateau current 15 s later. The desensitization ratio was expressed as one minus the ratio of the amplitudes of the plateau and the instantaneous current, in keeping with the convention established for the fast application of CPPG. As with the decay rate, the desensitization ratios of the transduction current were weakly dependent on voltage between −20 and −70 mV when Ca²⁺ was buffered with 0.5 mm EGTA. However, when buffering of Ca²⁺ was increased with 10 mm EGTA, the overall amount of desensitization decreased, and became more steeply dependent on voltage (Fig. 4B). It remains to be determined if the endogenous Ca²⁺ buffering capacity of On bipolar cell dendrites is more similar to 10 mm or 0.5 mm EGTA.

Data from the voltage jump experiments in 0.5 mm EGTA are plotted as a current—voltage (I–V) relation in Fig. 4C. The peak and plateau points at +40 mV were obtained by measuring at the beginning of the CPPG application and then again immediately before jumping to the test voltage. The instantaneous I–V relation was found to be very linear, implying that there is no fast block of the channel by divalent cations, and that the channel itself is inherently ohmic over the entire range of physiologically relevant voltages. On the other hand, the I–V relation of the mGluR6 current approaching steady state was highly rectifying. The plot shows that at −20 mV, well within the range of membrane potentials during On bipolar cell light responses, the transduction current generated by a light stimulus will be reduced by approximately 50%. Coupled with the time constant measurements, the results imply that the response to a saturating light stimulus will be reduced by one-half, and 90% of this reduction will occur within 2.5 s of light onset.

Direct evidence that the relaxation of transduction current induced by voltage jumps is due primarily to Ca²⁺ influx was obtained by performing voltage jumps in nominally Ca²⁺-free bathing solution (Fig. 5). The panel on the upper left of Fig. 5A compares two responses to CPPG in a cell held at +40 mV. At this membrane potential, removing Ca²⁺ had a negligible effect on the response, as the driving force for Ca²⁺ was minimal. The cell's holding potential was then jumped from +40 to −120, −40 and −70 mV, as shown in the shaded area at the top of the figure. Removing Ca²⁺ from the bath prevented the relaxation of the transduction current at every voltage. The effects of Ca²⁺ removal on relaxation of the transduction current are summarized in Fig. 5B, which compares the desensitization ratio of the transduction current in normal Ca²⁺, and in Ca²⁺-free media in the same cell population. In Ca²⁺-free solution, the plateau of the transduction current no longer rectified, as shown in Fig. 5C. Thus, in the absence of external Ca²⁺, there was no significant relaxation of the transduction current following a voltage step, and therefore no rectification of the I–V relation of the steady-state current. The voltage-dependent reduction of the steady-state transduction current is therefore conferred by Ca²⁺.

Figure 5. Voltage-dependent properties of the transduction current are conferred by Ca²⁺.

A, the shaded area at the top of the figure shows the voltage protocol for the four panels enclosed within the box. On the left is the response to application of CPPG at +40 mV in the presence and absence of Ca²⁺ in the bathing medium. The three panels on the right illustrate the effect of steps to the indicated voltage, also in the presence and absence of Ca²⁺. Removing Ca²⁺ essentially eliminates the voltage-dependent decay of the transduction current. B, summary of the effects of Ca²⁺ removal on desensitization. Included are only cells where desensitization was measured in both conditions, and thus this is a subset of the cells summarized in Fig. 4B. C, I–V relation of the plateau phase of the transduction current in 2 mm Ca²⁺ and Ca²⁺-free solution. Removing Ca²⁺ eliminates the rectification.

Desensitization is not regulated by calmodulin-dependent kinases or phosphatases

The involvement of Ca²⁺ suggests a potential role for the Ca²⁺-regulated kinase CaMKII, or the phosphatase calcineurin. In fact both calcineurin and CaMKII have previously been implicated in regulation of the mGluR6 pathway (Walters et al. 1998; Nawy, 2000; Shiells & Falk, 2000, 2001; Snellman & Nawy, 2002). To determine if either might be required for desensitization of the transduction current, we tested whether calcineurin and CaMKII inhibitors prevented desensitization induced by the rapid application of CPPG, or by voltage jumps. Dialysis of On bipolar cells with cyclosporin A, an inhibitor of calcineurin, or either KN-93 or (Ala9)-autocamtide-2 (AIP), inhibitors of CaMKII, did not reduce desensitization to fast application of CPPG (Fig. 6A). In separate experiments, inclusion of these inhibitors did not prevent desensitization of the mGluR6 current induced by voltage jumps (Fig. 6B). These results are summarized in Fig. 6C. Thus, although Ca²⁺ mediates desensitization of the transduction current, it does not appear to operate via activation of common Ca²⁺-dependent kinases and phosphatases.

Figure 6. Calcineurin and CaMKII inhibitors fail to prevent Ca²⁺-dependent desensitization of the transduction current.

A, responses to CPPG in two cells, one dialysed with 10 µm (Ala9)-autocamtide-2 (AIP) (left), and the other with 1 µm cyclosporin A (right). Records were obtained after at least 10 min of dialysis. Holding potential for both cells was −40 mV. Each trace is the average of three responses. B, voltage-dependent shut-off of the transduction current occurs normally in cells treated with AIP (left) or cyclosporin A (right). C, summary of experiments in A and B. Left panel, mean desensitization ratios of cells dialysed with cyclosporin A or AIP, measured within 30 s of breaking into the cell and after at least 10 min. There was no significant change in desensitization during the recording time, nor were values significantly different from controls. Right panel, I–V relation of 4 cells (2 with cyclosporin A and 2 with AIP). Rectification of the sustained component of the transduction current was readily observed even in the presence of inhibitors.

Discussion

Glutamate closes a cation-selective channel in the dendrites of On bipolar cells. Light generates a depolarizing response in On cells by decreasing release of glutamate from photoreceptor terminals. In this study, the essential features of light and dark signalling were mimicked by continuously bathing the retina in AP-4 and then, once a recording was obtained, by locally applying glutamate or the mGluR6 antagonist CPPG. Using this experimental approach, I show here that the transduction current revealed during the presentation of CPPG is not static, but instead begins to decay following application of CPPG with no observable latency. Both the rate of this decay, and the steady-state value to which it decayed depends upon Ca²⁺; the fast Ca²⁺ chelator BAPTA eliminated the decay, as did removing Ca²⁺ from the outside, while a high concentration of EGTA reduced it. The decay is indirectly regulated by voltage, because voltage drives Ca²⁺ into the cell through the transduction channel. Thus, On bipolar cells respond vigorously to incremental light, but desensitize in response to maintained illumination and therefore respond poorly to light decrements (Schiller et al. 1986; Schiller, 1992). In contrast, Off bipolar cells express glutamate receptors that desensitize quickly in the presence of glutamate (DeVries & Schwartz, 1999; DeVries, 2000). During maintained illumination, lower levels of synaptic glutamate allow these ionotropic receptors to recover from desensitization. Consequently, Off bipolar cells respond more robustly to decrements in illumination (Schiller et al. 1986; Schiller, 1992).

Desensitization is very evident during responses to maintained illumination. In a recent study of salamander On bipolar cells (Awatramani & Slaughter, 2000), the authors reported on two general types of On bipolar cells, both of which responded to maintained illumination in a transient manner. At −70 mV and under similar Ca²⁺ buffering conditions to those used in this study, the more transient of the two types decayed with a time constant of 800 ms, similar to the time constant of 870 ms obtained here by using voltage jumps to −70 mV. In that study, it was not determined if buffering with a high concentration of BAPTA or holding at positive membrane potentials could convert the light response from transient to sustained. A desensitizing response to illumination has also been observed in On bipolar cells of the dogfish retina (Shiells & Falk, 1999), in which study the authors demonstrated that buffering with BAPTA eliminated the desensitization. One difference between the present study and the study in dogfish retina was the substantial difference in the kinetics of desensitization. With little or no Ca²⁺ buffering present, the desensitization rate appeared to be about an order of magnitude slower than the rate reported here, although the authors did not explicitly measure this. A possible explanation for this discrepancy is that only moderately intense illumination was used in that study, sufficient to open only a fraction of the transduction channels. This might be expected to produce a smaller Ca²⁺ influx compared to the present study, resulting in a slower rate of desensitization.

How does Ca²⁺ produce desensitization? Evidence presented here suggests that calcineurin and CaMKII are not involved (but see Shiells & Falk, 2000). More likely, the effect is mediated by a direct interaction of Ca²⁺ with the channel or a Ca²⁺ binding protein such as calmodulin. The calmodulin inhibitors calmidazolium (n = 4) and W-7 (n = 2) were both ineffective at preventing desensitization (data not shown). However, in cases where Ca²⁺ acts via calmodulin to modulate channel activity, such as activation of the SK potassium channel (Xia et al. 1998) or inhibition of L-type Ca²⁺ channel (Peterson et al. 1999), inhibitors of calmodulin are often ineffective because of the constitutive binding of calmodulin to the channel (reviewed in Levitan, 1999). On the other hand, BAPTA is also generally ineffective in these systems, yet it was able to abolish desensitization in this study, suggesting a more distant relationship between the transduction channel and the Ca²⁺ effector in On bipolar cells. Thus, desensitization is likely to be mediated by a Ca²⁺ binding protein other than calmodulin.

Earlier studies from this laboratory (Nawy, 2000; Snellman & Nawy, 2002) reported on use-dependent depression of the transduction current. It was shown that the amplitude of the transduction current remained unchanged, provided that agonist was removed only briefly to measure the size of the response. Longer periods without agonist caused a cumulative depression of the current. The use-dependent depression was prevented by buffering with BAPTA, or by recording in low Ca²⁺, and thus shares a Ca²⁺ dependence with desensitization. However, many of the experiments in the previous two studies were performed without AP-4 in the bath, and so it is likely that the transduction current in all of the On bipolar cells was fully desensitized even before a recording was attempted. In support of this, in these previous studies the mean amplitude of the initial response in experiments where AP-4 was not included in the bath was 52.1 ± 1.7 pA at −40 mV, in reasonable agreement with the size of the desensitized (plateau) response obtained in this study (Figs 4 and 62.4 ± 7.1 pA). In contrast, the size of the full transduction current prior to desensitization was measured in this study to be 153.2 ± 18.9 pA (Fig. 4). Since use-dependent depression could still be observed in cells where the transduction current had been fully desensitized, it is likely to represent a different form of Ca²⁺-dependent regulation. It also proceeded at a significantly slower rate than desensitization, with an average time constant of about 11 min. Importantly, it was prevented by cyclosporin and exacerbated by AIP, and therefore is mechanistically distinct from desensitization. Thus there are at least two forms of Ca²⁺-dependent regulation, operating on different time scales, which conspire to limit the transduction current during prolonged illumination.