Calcium-Sensitive Calcium Influx in Photoreceptor Inner Segments
William H. Baldridge,
Dmitri E. Kurennyi, and
Steven Barnes
Neuroscience Research Group, University of Calgary, Faculty of Medicine, Calgary, Alberta T2N 4N1, Canada
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ABSTRACT |
Baldridge, William H., Dmitri E. Kurennyi, and Steven Barnes. Calcium-sensitive calcium influx in photoreceptor inner segments. J. Neurophysiol. 79: 3012-3018, 1998. The effect of external calcium concentration ([Ca2+]o) on membrane potential-dependent calcium signals in isolated tiger salamander rod and cone photoreceptor inner segments was investigated with patch-clamp and calcium imaging techniques. Mild depolarizations led to increases in intracellular Ca2+ levels ([Ca2+]i) that were smaller when [Ca2+]o was elevated to 10 mM than when it was 3 mM, even though maximum Ca2+ conductance increased 30% with the increase in [Ca2+]o. When external calcium was lowered to 1 mM [Ca2+]o, maximum Ca2+ conductance was reduced, as expected, but the mild depolarization-induced increase in [Ca2+]i was larger than in 3 mM [Ca2+]o. In contrast, when photoreceptors were strongly depolarized, the increase in [Ca2+]i was less when [Ca2+]o was reduced. An explanation for these observations comes from an assessment of Ca2+ channel gating in voltage-clamped photoreceptors under changing conditions of [Ca2+]o. Although Ca2+ conductance increased with increasing [Ca2+]o, surface charge effects dictated large shifts in the voltage dependence of Ca2+ channel gating. Relative to the control condition (3 mM [Ca2+]o), 10 mM [Ca2+]o shifted Ca2+ channel activation 8 mV positive, reducing channel open probability over a broad range of potentials. Reducing [Ca2+]o to 1 mM reduced Ca2+ conductance but shifted Ca2+ channel activation negative by 6 mV. Thus the intracellular calcium signals reflect a balance between competing changes in gating and permeation of Ca2+ channels mediated by [Ca2+]o. In mildly depolarized cells, the [Ca2+]o-induced changes in Ca2+ channel activation proved stronger than the [Ca2+]o-induced changes in conductance. In response to the larger depolarizations caused by 80 mM [K+]o, the opposite is true, with conductance changes dominating the effects on channel activation.
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INTRODUCTION |
If the driving force acting on an ion to produce an ionic current is considered, it is straight forward to show that changes in the external concentration of a permeant ion should affect the intracellular concentration of that ion. This relation follows from both the Goldman-Hodgkin-Katz current equation or from calculation of the ion's equilibrium potential using the Nernst equation and an estimate of the conductance. In the present work, dealing specifically with Ca2+ ions, an increase in the external concentration of this ion would be expected to lead to increases in the intracellular concentration when Ca2+-permeable channels are opened.
However, divalent cations also alter membrane surface charge, affecting the gating of voltage-dependent ion channels, and divalent cations can interact with the permeation pathway of many channels, affecting conductance. Thus when making changes in external [Ca2+], several biophysical actions must be considered to work simultaneously in a prediction of how voltage-gated Ca2+ influx could change.
These various considerations take on relevance as the physiological levels of extracellular Ca2+ change in some neuronal preparations, including photoreceptors (Gallemore et al. 1994
). At the photoreceptor output synapse, for example, graded presynaptic potential changes control the release of glutamate primarily via L-type calcium channels (Wilkinson and Barnes 1996
). Because the presynaptic voltage excursions range over the lowermost region of the calcium channel activation curve, it has been shown that small changes in calcium channel activation properties translate into significant effects on synaptic transmission (Barnes et al. 1993
; Piccolino et al. 1996
). Thus in the present work we examine the influence of changes in driving force, channel activation, and channel permeation in assessing changes in Ca2+ signaling in response to external [Ca] changes.
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METHODS |
Preparation of isolated photoreceptors and loading with calcium indicator dye
Isolated photoreceptors were obtained by trituration of retinas from tiger salamander (Ambystoma tigrinum) in a retinal saline solution (RSS) of the following composition (in mM): 90 NaCl, 2.5 KCl, 3 CaCl2, 8 D-glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) at pH 7.6. Isolated photoreceptors were plated on 35-mm2 polystyrene tissue culture dishes (Corning 25000, Corning, NY) and then incubated in 10 µM Fluo-3 AM (Molecular Probes, Eugene, OR) in RSS for 2 h in the dark at 4°C. Fluo-3 AM was first dissolved in anhydrous dimethyl sulfoxide (DMSO; 0.1% final concentration in RSS) and then solubilized in RSS containing 0.1% Pluronic F-127 (Molecular Probes). After incubation in Fluo-3 solution, cells were rinsed in RSS.
Imaging
Photoreceptors loaded with Fluo-3 were imaged using a Photometrics CH250 cooled charge-coupled device (CCD) camera (Photometrics, Tucson, AZ) fitted to a Nikon UM-2 fluorescent microscope employing a Zeiss ×40 water-immersion objective (N.A. 0.75). Fluo-3 fluorescence was produced by excitation from a 100-W mercury-vapor lamp and an appropriate filter set (Nikon B-2A, excitation 450-490 nm; emission 520-560 nm; dichroic 510 nm). To reduce photobleaching, excitation illumination was filtered by a
1.6 log neutral density filter and the period of illumination limited by an electronic shutter (Uniblitz, Rochester, NY). Images (100 × 100 pixels2, 16 bit) were captured using IPLab Spectrum (Signal Analytics, Vienna, VI) and saved to the hard disk of a Quadra 800 Macintosh computer. Images were typically captured every 5.5 s with an exposure time of 200 ms. At the conclusion of each experiment, bright-field images of the cells were also captured to record their identity.
Images were analyzed using IPLab Spectrum. The mean intensity over a large area of the inner segment, 25-50 µm2, well separated from the edge of the cell, was measured for each captured image. Changes in fluorescence were quantified as the difference in intensity from baseline (
F) divided by the baseline intensity (F). Changing values of
F/F are not linearly related to changes of [Ca2+]i and are intended only to provide a qualitative indication of changing [Ca2+]i.
Treatments
During treatment, dishes containing Fluo-3-labeled photoreceptors were constantly superfused with RSS-based solution at a rate of 2 ml/min. Solutions with different [K+]o and [Ca2+]o were made equiosmotic by adjusting [Na+]o as required. Cells were exposed to different solutions using an eight-way valve. Initially cells were superfused with RSS containing 2.5 mM [K+]o, but during experiments the most stable baselines were obtained with 1 mM [K+]o RSS. To activate voltage-dependent calcium channels the cells were depolarized by raising [K+]o from 1 to 10 or 80 mM. Nisoldipine (Miles, Kankakee, IL) and Bay K 8644 (Sigma, St. Louis, MO) were applied via the superfusate. Apparent differences between mean
F/F, due to treatments, were tested for statistical significance by normalizing the data to the fluorescence increase produced by the initial treatment with elevated [K+]o and comparing the mean normalized data to the normalized control value of 1.0 using population t-test.
Patch-clamp recording of isolated cells
Calcium current (ICa) was recorded from mechanically isolated cone photoreceptors using perforated patch-clamp techniques. Nystatin (150 µM, Sigma, St. Louis, MO) was added to the pipettes containing (in mM) 95 CsCl, 3 MgCl2, 1 ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 10 HEPES, pH 7.2. The recording chamber was constantly perfused with a control solution containing (in mM) 70 NaCl, 2.5 KCl, 5 CsCl, 3 CaCl2, 10 tetraethylammonium (TEA), 10 D-glucose, and 20 HEPES, pH 7.6. In test solutions, CaCl2 concentration was reduced or increased as described. The magnitude of ICa was assessed at the end of voltage steps from the holding potential of
70 mV. After leak subtraction, activation curves were constructed and three parameters estimated from a Boltzmann fit: the maximum conductance (Gmax), the half activation potential (V1/2), and the slope factor. Changes in these parameters induced by a test solution application were estimated using linear interpolation between control and wash measurements. Data from different cells were averaged and expressed as means ± SE. Statistical significance was evaluated using paired two-tail Student's t-test. The Gouy-Chapman-Stern model for surface charge screening was employed to fit the data. We applied both binding and nonbinding models for Ca2+ interactions with surface charges [as described by Kwan and Kass (1993)
, for cardiac L-type Ca channels]. The equations were modified to include only changes in [Ca2+]o because all experiments were done at constant pH. A simple iterative process was used to fit the data by adjusting the binding constant, KCa, and surface charge density.
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RESULTS |
Increasing [Ca2+] reduces the mild depolarization-evoked increase of [Ca2+]i
Figure 1 illustrates the effect [Ca2+]o had on [Ca2+]i, as revealed by Fluo-3 fluorescence, in two salamander cone photoreceptors subject to mild depolarization by increasing [K+]o from 1 to 10 mM for 2 min. The shift from 1 to 10 mM [K+]o was the smallest concentration increase that provided consistent increases in Fluo-3 fluorescence in control (3 mM [Ca2+]o) conditions. As illustrated by the cone shown in Fig. 1A, 10 mM [K+]o increased Fluo-3 fluorescence (
F/F) by 0.067 when [Ca2+]o was 3 mM (Fig. 1B). When [Ca2+]o was increased to 10 mM, however, the same cell showed only a small increase in fluorescence (
F/F = 0.006) in response to 10 mM [K+]o (Fig. 1C) but showed full recovery when [Ca2+]o was returned to 3 mM (
F/F = 0.066; Fig. 1D). When [Ca2+]o was lowered, as illustrated by the cone shown in Fig. 1E, the effect of 10 mM [K+]o was enhanced from
F/F = 0.057, in the presence of 3 mM [Ca2+]o (Fig. 1F), to
F/F = 0.108, in the presence of 1 mM [Ca2+]o (Fig. 1G). After return to 3 mM [Ca2+]o, the response to 10 mM [K+]o was reduced somewhat (
F/F = 0.051; Fig. 1H) compared with control.

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| FIG. 1.
Effect of [Ca2+]o on Fluo-3 fluorescence increases in isolated cones produced by 10 mM [K+]o. A: bright-field image of a salamander cone used to illustrate the effect of elevated [Ca2+]o on the increase of Fluo-3 fluorescence produced by 10 mM [K+]o. B: representation of the Fluo-3 fluorescence increase ( F/F) produced by 10 mM [K+]o in the presence of 3 mM [Ca2+]o, 10 mM [Ca2+]o (C), and after return to 3 mM [Ca2+]o (D). In another cone (E) the effect of decreasing [Ca2+]o on the fluorescence increase due to 10 mM [K+]o was tested by shifting [Ca2+]o from 3 mM (F) to 1 mM (G) and then back to 3 mM (H). All the fluorescence images are an average of 5 images, acquired every 5.5 s, at the peak of the response to a 2-min application of 10 mM [K+]o.
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Similar results are shown from two other cones (Fig. 2, A and B) with fluorescence intensity illustrated graphically and throughout the entire time course of the experiment. In the first cell (Fig. 2A), raising [Ca2+]o from 3 to 10 mM produced a small decrease in baseline fluorescence and abolished the response to 10 mM [K+]o. When 3 mM [Ca2+]o was restored, there was an increase in fluorescence that, for a time, exceeded the initial baseline fluorescence. Subsequently, fluorescence returned to baseline, and the response to 10 mM [K+]o showed a full recovery. In the second cell (Fig. 2B) reducing [Ca2+]o from 3 to 1 mM increased both baseline fluorescence and the response to 10 mM [K+]o. This cell showed good recovery of the response to 10 mM [K+]o when [Ca2+]o was returned to 3 mM.

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| FIG. 2.
Graphic representations of the effect of [Ca2+]o on Fluo-3 fluorescence increases in photoreceptor inner segments produced by 10 mM [K+]o. A: effect of increasing [Ca2+]o to 10 mM on the Fluo-3 fluorescence increase produced by 10 mM [K+]o in an isolated cone inner segment. B: optical recordings from another cone inner segment showing the effect of decreasing [Ca2+]o to 1 mM [Ca2+]o on the increase in fluorescence produced by 10 mM [K+]o. Mean results, after normalization to the initial response to 10 mM [K+]o in 3 mM [Ca2+]o, showing the effect of raising [Ca2+]o to 10 mM (n = 7; C) and reducing [Ca2+]o to 1 mM (n = 6; D). In all cases (A-D) 10 mM [K+]o was applied for 2 min. E: graphic representation of the change in Fluo-3 fluorescence in a cone produced by shifting [Ca2+]o from 10 to 1 mM during constantly elevated (10 mM) [K+]o. Error bars in C and D are means ± SE. ***P < 0.001. **P < 0.01.
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The mean response to 10 mM [K+]o for all cells studied, normalized to the magnitude of the primary response to 10 mM [K+]o, is depicted in Fig. 2, C and D. Of the seven cells where the effect of 10 mM [Ca2+]o was studied, five were cones and two were rods. No difference between rod and cone data were noticed, and therefore mean results represent data pooled from both photoreceptor types. In the presence of 10 mM [Ca2+]o, the mean response to 10 mM [K+]o was only 13 ± 3% (SE; P < 0.001) of the mean response in the presence of 3 mM [Ca2+]o (Fig. 2C). After return to 3 mM [Ca2+]o, the response to 10 mM [K+]o was completely restored (105 ± 4%; Fig. 2C). Increasing [Ca2+]o from 3 to 10 mM did not, on average, alter baseline fluorescence (mean
F/F = 0.003 ± 0.005), although fluorescence in two cells decreased slightly (e.g., Fig. 2A) and in one cell increased slightly.
All six cells used to study the effect of 1 mM [Ca2+]o were cones. Decreasing [Ca2+]o to 1 mM from 3 mM produced, on average, a small increase in baseline fluorescence (mean
F/F = 0.009 ± 0.004), and the response to 10 mM [K+]o was increased (P < 0.01) more than twofold in the presence of 1 mM [Ca2+]o (231 ± 34%; Fig. 2D). After return to 3 mM [Ca2+]o, the response to 10 mM [K+]o was, on average, less (59 ± 10%, P < 0.01) than the control (1st) treatment with 10 mM [K+]o (Fig. 2D).
To directly compare the effects of 1 and 10 mM [Ca2+]o on the response to 10 mM [K+]o in the same cell, [K+]o was maintained at 10 mM and [Ca2+]o then shifted from 10 to 1 mM (Fig. 2E). Even after a long (4 min) exposure to 10 mM [K+]o, in the presence of 10 mM [Ca2+]o there was only a slight increase in fluorescence, apparent mostly during the latter 2 min of the treatment. When [Ca2+]o was then reduced to 1 mM, there was a dramatic increase in fluorescence. When [Ca2+]o was returned to 10 mM, fluorescence dropped to baseline, and no difference was noted when [K+]o and [Ca2+]o were subsequently returned to resting levels (1 and 3 mM, respectively). Similar results were obtained in all of six cones studied. In the presence of 10 mM [Ca2+]o, the 4-min treatment with 10 mM [K+]o increased fluorescence, on average, by
F/F = 0.020 ± 0.006. When [Ca2+]o was shifted to 1 mM, fluorescence increased (P < 0.01; paired t-test), on average, to
F/F = 0.293 ± 0.059.
Decreasing [Ca2+]o reduces the strong depolarization-evoked increase of [Ca2+]i
To compare the effect of [Ca2+]o on the increase of [Ca2+]i produced by larger depolarizations, higher concentrations of [K+]o were employed. Changing [Ca2+]o had no effect on the response elicted by 2-min applications of 40 mM [K+]o (not shown). We were concerned that very large increases of [K+]o, over a 2-min exposure time, might produce increases in [Ca2+]i so great that they could 1) be toxic, 2) lead to more complex Ca2+-handling responses by the cell, or 3) saturate the Fluo-3 signal [Kd = 390 nM (Haughland 1996); although Rieke and Schwartz (1996)
estimated the Kd in salamander rod cytoplasm to be 800 nM] to the point where differences due to [Ca2+]o could not be detected. Therefore we chose to use 20-s applications of 80 mM [K+]o. Such treatment should result in much greater depolarization of the cell than 10 mM [K+]o but for a shorter time period and did in fact translate into greater, but briefer, Ca2+ influx. Therefore the maximum amplitude of the Fluo-3 signal (
F/F) with this treatment was comparable with the response to 10 mM [K+]o, even though the exposure time to 10 mM [K+]o was five times longer. We found that the effects of the brief applications of 80 mM [K+]o on Fluo-3 fluorescence were altered when [Ca2+]o was decreased from 3 to 1 mM (Fig. 3). Figure 3A illustrates the decrease in the response of a cone (from
F/F = 0.093 to 0.058) to a brief application of 80 mM [K+]o when [Ca2+]o was shifted from 3 to 1 mM. After the return to 3 mM [Ca2+]o, there was partial recovery of the response to [K+]o (
F/F = 0.074). Similar results were found in the four cones and two rods tested, as illustrated in Fig. 3B. The response to the short application of 80 mM [K+]o was 53 ± 14% (P < 0.01) in 1 mM [Ca2+]o compared with the first treatment with 80 mM [K+]o in 3 mM [Ca2+]o. On average, there was complete recovery (104 ± 9%) of the response to 80 mM [K+]o when [Ca2+]o was returned to 3 mM.

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| FIG. 3.
Effect of [Ca2+]o on Fluo-3 fluorescence increases produced by 80 mM [K+]o. A: example from a cone showing the effect of reducing [Ca2+]o from 3 to 1 mM on the Fluo-3 fluorescence increase produced by a brief (20 s) application of 80 mM [K+]o. B: mean normalized results of the effects of 1 mM [Ca2+]o on the fluorescence increased produced by 80 mM [K+]o (n = 6). Error bars are mean ± SE. **P < 0.01.
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Increase of [Ca2+]i depends on voltage-gated calcium channels
To determine whether the fluorescence signal produced by elevated [K+]o was related to voltage-gated calcium channel activity, the effect of two agents known to affect L-type voltage-gated calcium channels was tested. Nisoldipine, a blocker of L-type Ca channels (Rieke and Schwartz 1994
; Wilkinson and Barnes 1996
), at 0.5 µM reversibly reduced the increase of fluorescence produced by 10 mM [K+]o (Fig. 4A). On average (results from 3 rods and 3 cones), the response to 10 mM [K+]o in the presence of 0.5 µM nisoldipine was only 11 ± 4% (P < 0.001) of the control response to 10 mM [K+]o (Fig. 4B). Bay K 8644, an L-type Ca channel agonist (Nowycky et al. 1985
; Wilkinson and Barnes 1996
), at 2 µM enhanced the response to 10 mM [K+]o (Fig. 4A), an effect that was reversed by washing. On average, Bay K 8644 increased (P < 0.05) the response to elevated [K+]o nearly fourfold (390 ± 99%) in five cones where the effect of Bay K 8644 alone was studied. Dihydropyridine actions were slow to wash out.

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| FIG. 4.
Increase of Fluo-3 fluorescence produced by 10 mM [K+]o was reduced by nisoldipine and enhanced by Bay K 8644. A: effect of 0.5 µM nisoldipine and 2 µM Bay K 8644 on the Fluo-3 fluorescence elicited by 10 mM [K+]o. B: mean normalized effect of nisoldipine on the fluorescence increase produced by 10 mM [K+]o (n = 6). C: mean normalized effect of Bay K 8644 on the response to 10 mM [K+]o (n = 5) in cells where this drug was studied alone (not after prior nisoldipine treatment, as shown in A). In all cases 10 mM [K+]o was applied for 2 min. Error bars are mean ± SE. ***P < 0.001. *P < 0.05.
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Electrophysiological correlate of calcium imaging observations
To check further the possibility that the observed effects of different [Ca2+]o on calcium signals in photoreceptors were due to changes in calcium influx, we studied ICa in cones and modeled the results using surface charge screening theory (Kwan and Kass 1993
). Figure 5A shows typical current-voltage (I-V) relationships obtained in a cone photoreceptor at the three different [Ca2+]o used in the imaging studies. When [Ca2+]o was increased from 3 to 10 mM, the maximum amplitude of ICa increased, but activation of the channels shifted markedly to more positive potentials. Conversely, when [Ca2+]o was reduced to 1 mM, a much smaller peak current was observed, and the I-V relation shifted to more negative values. Thus in the voltage range between
30 and
10 mV, the amplitude of ICa varies according to the combined gating and permeation properties of the channel and does not simply follow what might be expected from varying [Ca2+]o.

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| FIG. 5.
ICa activation shifts induced by extracellular calcium. A: current-voltage plots obtained from the same cone in control ( ), 1 mM ( ), and 10 mM ( ) [Ca2+]o. B: pooled data for normalized Gmax. C: shifts in V1/2 as function of [Ca2+]o (n = 5 for 0.3 and 10 mM, n = 8 for 1 mM). Solid line in B represents equation g = [1 + (K/3)m]/[1 + (K/[Ca2+]o)m], where K = 11.96 mM and m = 0.396. Solid line in C represents the best fit of the data to the surface charge screening model with an apparent negative charge density of 1 e /(13.25 Å2) and a binding constant for calcium of 10.78 1/M. A model assuming no binding was also tested and gave similar fit, but with an extremely high charge density of 1 e /(0.19 Å2).
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On the other hand, closer to 0 mV, when Ca channel activation is for the most part complete, the [Ca2+]o-induced changes in calcium conductance dominate ICa, producing changes in current as a function of external concentration of permeant ion as predicted by the Goldman-Hodgkin-Katz equation (see Hille 1992
).
Figure 5, B and C, summarizes data on the effects of changing [Ca2+]o on maximum calcium conductance and shift in ICa activation, respectively, derived from Boltzmann fits. No significant changes were observed in the slope factor values in the range of [Ca2+]o tested. With increasing [Ca2+]o, the concurrent increase of maximum calcium conductance and positive shift in channel activation work against each other in the zone where calcium current activation is small, and this happens to be the range of voltages over which rod and cone photoreceptors operate. Over a narrow voltage range, despite the higher concentration of the current-carrying ion, current amplitude may be reduced.
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DISCUSSION |
The Goldman-Hodgkin-Katz current equation describes the dependence of ionic flux on concentration gradient and constant permeability. It is expected, therefore, that increasing the Ca2+ concentration gradient should increase the flux of Ca2+ into a cell, provided there is some permeability to the ions. However, as the imaging data from amphibian photoreceptors presented in this study clearly show, under certain conditions, increasing [Ca2+]o can reduce Ca2+ influx, and, conversely, decreasing [Ca2+]o can increase Ca2+ influx. The condition that produces this surprising result is depolarizing the cell only slightly, by using the smallest possible increase in [K+]o that produced an increase in Fluo-3 fluorescence under control (3 mM [Ca2+]o) conditions. Larger depolarizations, produced by greater increases in [K+]o, are less surprising and follow prediction, with reduced [Ca2+]o resulting in a decrease in the K+-induced rise of [Ca2+]i as revealed by Fluo-3 calcium imaging. The electrophysiological recordings support and extend these observations by showing that currents through photoreceptor inner segment calcium channels (ICa) are reduced when [Ca2+]o was elevated but only over the range of potentials close to the foot of the activation curve for these channels. Conversely, when photoreceptor inner segments were strongly depolarized, ICa increased when [Ca2+]o was elevated and decreased when [Ca2+]o was reduced.
These findings can be understood by realizing that divalent cations alter membrane surface charge, affecting the gating of voltage-dependent ion channels, in this case voltage-gated calcium channels. Such effects have been described by the Gouy-Chapman-Stern theory in a variety of preparations (see Hille 1992
), and the electrophysiological data reported here also follow the Gouy-Chapman-Stern model, making it likely that the results of the imaging experiments can be interpreted by reference to surface charge effects.
As [Ca2+]o increases, fixed negative surface charges are screened thereby increasing the electric field across the membrane, the field that affects the voltage sensors of the voltage-gated calcium channels. In the hypothetical case where all the negative charges are balanced, the electric field across the membrane would be due entirely to the resting membrane potential. At the other extreme, with no screening, the electric field across the membrane is reduced by the negative charges. Activation of voltage-gated calcium channels requires that the electric field across the membrane be reduced. Therefore when [Ca2+]o is increased and surface charges screened, greater depolarization is required to change the electric field across the membrane than in the case where [Ca2+]o is low and the charges are less screened. This has the effect of shifting the activation curve of ICa to the left, such that the range for voltage-gated calcium channel activation is more negative in low versus high [Ca2+]o. This shift applies only over the range for which activation of voltage-gated calcium channels is incomplete. At well depolarized potentials, when the electric field is substantially reduced by the change in membrane potential and voltage-gated ion channels are for the most part fully activated, the effect of surface charge on channel gating diminishes, and the Goldman-Hodgkin-Katz current equation, with its fixed permeability, dominates.
Although the imaging and electrophysiological data in this study agree, how closely coupled are the changes in Fluo-3 fluorescence to the influx of Ca2+ through voltage-gated calcium channels? Although it is possible that a substantial portion of the signal measured by Fluo-3 fluorescence is due to Ca2+ release from intracellular stores (for review see Simpson et al. 1995
), if such a release were triggered by Ca2+ influx, it seems reasonable to interpret the Fluo-3 signal as a reflection of Ca2+ influx. In fact, we demonstrated (Fig. 4) that the Fluo-3 signal is affected by pharmacological treatments directed at the type of voltage-gated calcium channels on cone inner segments (Wilkinson and Barnes 1996
). This suggests that if the Fluo-3 signal measured is due to "calcium-induced calcium release" it is related to the magnitude of Ca2+ influx through voltage-gated calcium channels. Changing [Ca2+]o might also alter the release of Ca2+ from intracellular stores directly or perhaps affect mechanism(s) that remove Ca2+ from the cytoplasm, either by pumping it back into stores (Friel and Tsien 1994
; Thayer and Miller 1990
; Werth and Thayer 1994
; White and Reynolds 1995
) or out of the cell via a Na+-Ca2+ exchanger (see Reeves 1990
; White and Reynolds 1995
) or an ATP-driven Ca2+ pump (see Carafoli 1992
; Garrahan and Regan 1990
). In fact, recent work has suggested that it is a calcium ATPase that removes most Ca2+ from the photoreceptor inner segment (Krizaj and Copenhagen 1997
) in contrast to the removal of Ca2+ from the outer segment, which is due to a Na+/K+/Ca2+ exchanger (Schnetkamp et al. 1989
). There is no evidence, however, that changing [Ca2+]o affects any of these mechanisms in a manner consistent with the data reported here. In fact, an explanation relating to Ca2+ clearance is unlikely given that the recovery rate following elevated K+ was unaffected by [Ca2+]o.
The results of this work provide additional evidence explaining the curious effect [Ca2+]o has on the responses of retinal horizontal cells, neurons that receive direct synaptic input from photoreceptors, in the intact retina. For example, Rowe (1987)
showed, in isolated superfused goldfish retina, that the light-evoked responses of cone-driven horizontal cells decreased and dark resting membrane potential hyperpolarized when [Ca2+]o was increased from 50 µM to 2 mM. Applying the results from the current work to the observations made by Rowe (1987)
suggests that synaptic transmission from photoreceptors, in this case cones, to horizontal cells was increased in low [Ca2+]o because of increased activation of voltage-gated calcium channels leading to increased Ca2+ influx and therefore increased [Ca2+]i. The elevated [Ca2+]i would promote transmitter release from the cone terminal, even in the dark, depolarizing horizontal cells and increasing the magnitude of the response during a flash of light. The dark resting membrane potential of photoreceptors is typically about
35 mV, but ICa is only detected at potentials positive to about
40 mV. Consequently, it is not expected that the decrease of Ca2+ influx during large artificial depolarizations of photoreceptor membrane potential, when [Ca2+]o is reduced, would be relevant to the light-evoked responses of horizontal cells. That is, over the entire voltage range that photoreceptors operate in vivo, the activity of voltage-gated calcium channels are susceptible to the effect of [Ca2+]o and surface charge screening.
These results are consistent with evidence that the block of photoreceptor synaptic transmission by specific divalent cations (Zn2+, Ni2+, Co2+), and recovery from block with reduced [Ca2+]o, is due to surface charge-mediated shifts in the activation range of voltage-gated calcium channels (Piccolino et al. 1996
). Our work supports the conclusion drawn recently by Piccolino and Pignatelli (1996)
that designation of a persistent synaptic event as "Ca2+-independent" can be made only after adequate measurements establish that [Ca2+]o at synaptic sites is indeed zero.
Finally, [Ca2+]o in the retina can change dramatically depending on physiological activity. At the level of photoreceptors, light evokes a sustained decrease in [Ca2+]o from 3 mM down to 2 mM (Gallemore et al. 1994
). Modulation of voltage-gated calcium channel activity by [Ca2+]o shown in the present work represents a mechanism by which Ca2+ influx and synaptic activity in vivo could therefore be enhanced during illumination.
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ACKNOWLEDGEMENTS |
This work was supported by the Medical Research Council of Canada, the Alberta Heritage Foundation for Medical Research (AHFMR), and the Johannsen Research Fund of Fight for Sight research division of Prevent Blindness America. W. Baldridge was an AHFMR Postdoctoral Fellow. S. Barnes is an AHFMR Senior Scholar and the Roy Allen Investigator at the University of Calgary.
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FOOTNOTES |
Present address of W. H. Baldridge: School of Optometry, Faculty of Science, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
Address for reprint requests: S. Barnes, Dept. of Physiology and Biophysics, University of Calgary, Faculty of Medicine, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada.
Received 12 September 1997; accepted in final form 6 March 1998.
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