Correspondence to: Juan I. Korenbrot, Dept. of Physiology, School of Medicine, Box 0444, University of California at San Francisco, San Francisco, CA 94143. Fax:415-476-4929 E-mail:juan{at}itsa.ucsf.edu.
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Abstract |
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We determined the Ca2+ dependence and time course of the modulation of ligand sensitivity in cGMP-gated currents of intact cone photoreceptors. In electro-permeabilized single cones isolated from striped bass, we measured outer segment current amplitude as a function of cGMP or 8Br-cGMP concentrations in the presence of various Ca2+ levels. The dependence of current amplitude on nucleotide concentration is well described by the Hill function with values of K1/2, the ligand concentration that half-saturates current, that, in turn, depend on Ca2+. K1/2 increases as Ca2+ rises, and this dependence is well described by a modified Michaelis-Menten function, indicating that modulation arises from the interaction of Ca2+ with a single site without apparent cooperativity. CaKm, the Michaelis-Menten constant for Ca2+ concentration is 857 ± 68 nM for cGMP and 863 ± 51 for 8Br-cGMP. In single cones under whole-cell voltage clamp, we simultaneously measured changes in membrane current and outer segment free Ca2+ caused by sudden Ca2+ sequestration attained by uncaging diazo-2. In the presence of constant 8Br-cGMP, 15 µM, Ca2+ concentration decrease was complete within 50 ms and membrane conductance was enhanced 2.33 ± 0.95-fold with a mean time to peak of 1.25 ± 0.23 s. We developed a model that assumes channel modulation is a pseudofirst-order process kinetically limited by free Ca2+. Based on the experimentally measured changes in Ca2+ concentration, model simulations match experimental data well by assigning the pseudo-first-order time constant a mean value of 0.40 ± 0.14 s. Thus, Ca2+-dependent ligand modulation occurs over the concentration range of the normal, dark-adapted cone. Its time course suggests that its functional effects are important in the recovery of the cone photoresponse to a flash of light and during the response to steps of light, when cones adapt.
Key Words: retina, ligand-gated ion channel, phototransduction, adaptation, teleost fish
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INTRODUCTION |
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Rods and cone photoreceptors in the vertebrate retina continuously adjust the gain and kinetics of their photoresponse as a function of background light intensity. These adjustments, known as adaptation, allow photoreceptors to respond to signals that differ little in intensity from the background light, regardless of the absolute intensity of that background. The extent of adaptation reflects the relative contribution of three distinct cellular mechanisms. (a) Response compression, which arises from the nonlinear relation between response amplitude and light intensity. (b) Neural adaptation, which arises from modulation of the biochemical cascade that underlies phototransduction. (c) Pigment adaptation, which arises from biochemical events associated with photopigment bleaching (reviewed in
In isolated, dark-adapted photoreceptors, adaptation begins to be discerned by the time the peak of the photoresponse is reached, and it then smoothly reaches a stationary state within a second or two (reviewed in
In rods and cones, light-dependent changes in outer segment cytoplasmic Ca2+ must occur for neural adaptation to proceed. If these changes are prevented, dark-adapted photoreceptors respond to light, but the photoresponses are altered in their light sensitivity, time course, and adaptation features (reviewed in
In rods, every one of the events described above has been shown to depend on Ca2+ at concentrations that overlap those expected in dark- and light-adapted cells, somewhere between 10 and 1,000 nM. The time course of each of the various possible Ca2+ biochemical actions is poorly understood and the relationship between these dynamics and the time course of adaptation is largely undefined (1 s (
Ca2+ modulates the ligand sensitivity of CNG channels. The value of K1/2, the concentration necessary to activate half-maximal current, increases as Ca2+ rises. In membrane patches detached from outer segments of either rods or cones, K1/2 changes 1.5-fold between its extreme values (
1.5 (
50 nM, but the midpoint is
290 nM in cones. Because in rods the extent of change in K1/2 is small and occurs at Ca2+ concentrations expected only under bright illumination, Ca2+-dependent channel modulation is expected to play a limited role in the control of gain and kinetics of the transduction signal. The converse arguments suggest that Ca2+-dependent channel modulation may be of significant physiological consequence in cones.
To better understand the properties of CNG channel modulation in cones, we report here on an analysis of the Ca2+ dependence of K1/2 in electropermeabilized cones (ep-cones), a preparation in which the cytoplasmic content of a single, intact outer segment can be rapidly controlled while measuring currents through the cGMP-gated channels. We also analyze the kinetics of this modulation in intact cones in which sudden changes in Ca2+ concentration are achieved with the use of a "caged" Ca2+ buffer, diazo-2 (
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MATERIALS AND METHODS |
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Materials
Striped bass (Morone saxitilis) were obtained from Professional Aquaculture Services and maintained in the laboratory for up to 6 wk under 10:14-h dark:light cycles. The UCSF Committee on Animal Research approved protocols for the upkeep and killing of the animals. Diazo-2 and bis-fura-2 were purchased from Molecular Probes. Enzymes for tissue dissociation were obtained from Worthington Biochemicals. Zaprinast was from CalBiochem, all other chemicals were from Sigma-Aldrich.
Photoreceptor Isolation
Under infrared illumination and with the aid of a TV camera and monitor, retinas were isolated from the eyes of fish dark adapted for 3045 min. Single cones were isolated by mechanical trituration of retinas briefly treated with collagenase and hyaluronidase as described in detail elsewhere (
In experiments with tight-seal electrodes, cones were first isolated in a modified Ringers' in which pyruvate isosmotically replaced glucose. Cells were transferred in this solution onto the electrophysiological chamber, the bottom of which was a glass coverslip derivatized with Concanavalin A (3 mg/ml) (
Suction Electrodes and Electropermeabilization
We measured the outer segment currents from single cones using suction electrodes, as described previously (
Because the seal between suction electrode and single cones is low in impedance (few molarohms), there is an unavoidable drift in the recorded current. To minimize uncertainties due to this drift, we used a track-and-hold circuit before data digitization. The data acquisition software controlled this circuit and zeroed the current immediately before the delivery of test solutions. Data were accepted for analysis only if the holding current before and after the application of test solution (an interval of 4570 s) differed by less than approximately ±20 pA (typically approximately ±5% of the maximum cGMP-dependent current). In any given data set, the error in the value of K1/2 due to drift can be calculated to be no worse than 1%. This error is negligible when compared with the variance from cell to cell, which is the limiting source of error in our data.
The cytoplasmic content of the cone outer segment was controlled by electropermeabilizing the inner segment membrane and continuously superfusing with the "ep-solution," composed of (mM): 140 cholineCl, 5 glucose, 0.4 Zaprinast, and 10 HEPES. pH was 7.5 and osmotic pressure 310 mOsM. The ep-solution contained 1 mM free Mg2+ and various amounts of free Ca2+, cGMP, or 8Br-cGMP, according to the design of the experiment. The solutions of defined Ca2+ and Mg2+ concentration were prepared by mixing appropriate amounts of CaCl2, MgCl2, and the buffering agents EGTA (2 mM) or HEDTA (2 mM), calculated according to the equilibrium constant listed in
The cone electropermeabilization method is described in detail elsewhere (
Membrane Currents in Intact, Single Cones
We measured membrane currents under voltage clamp using tight-seal electrodes in the whole-cell mode. Electrodes were produced from aluminosilicate glass (1.5 x 1.0 mm o.d. x i.d., No. 1724; Corning Glassworks). Single cones were firmly attached to the glass bottom of a recording chamber and maintained in darkness on the stage of an inverted microscope equipped with DIC. Under IR illumination, and observing with the aid of TV camera and monitors, electrodes were sealed onto the side of the inner segment. Currents were recorded with a patch-clamp amplifier (Axopatch 1D; Axon Instruments, Inc.). Analogue signals were low-pass filtered below 200 Hz with an eight-pole Bessel filter (Kronh-Hite) and digitized online at 1 kHz (FastLab).
The tight-seal electrode-filling solution was composed of (mM): 115 K+ gluconate, 20 K+ aspartate, 33 KCl, 1 diazo2, 0.1 bis-fura2, 1.04 MgCl2 (to yield 1 mM free Mg2+), 0.258 CaCl2 (to yield 600 nM free Ca2+), and 10 MOPS. pH was 7.25 and osmotic pressure was 305 mOsM. Free Ca2+ and Mg2+ were calculated using the equilibrium constants reported for dark diazo-2 (
Uncaging Diazo-2 and Measuring the Rate of Ca2+ Sequestration In Vivo
The instrument we developed to uncage and simultaneously measure membrane current and fluorescence in isolated, dark-adapted bass single cones is described in detail elsewhere (Ohyama, T., D.H. Hackos, S. Frings, V. Hagen, U.B. Kaupp, and J.I. Korenbrot, manuscript submitted for publication). Ca2+ concentration was monitored using bis-fura-2, a structural analogue of fura-2 with slightly lower affinity for Ca2+ (Kd 520 nM in the presence of 1 mM Mg2+) and higher molar extinction coefficient than fura-2. Fluorescence was excited by 380-nm light and emission intensity measured in the range between 410 and 600 nm using a photomultiplier operated in photon counting mode (50-ms counting bins). Our instrument allowed us to use relatively low intensity 380-nm light to excite bis-fura-2 fluorescence (2.55 x 108 photons · µm-2 s-1), an important feature necessary to avoid uncaging diazo-2 with this light. In each cell, fluorescence was corrected for cross talk between excitation and emission paths by subtracting from the cell signal the mean of the signal measured under identical conditions, but in the cell's absence. To assure that fluorescence intensity signaled only Ca2+ concentration in the outer segment, fluorescence excitation light was restricted to the outer segment alone with the use of an aperture that created an 18-µm diameter circle of light centered on the outer segment (Ohyama, T., D.H. Hackos, S. Frings, V. Hagen, U.B. Kaupp, and J.I. Korenbrot, manuscript submitted for publication).
To uncage diazo-2, we delivered the light of a Xe flash (200 J, 0.3-ms duration at half-peak) (Chadwick-Helmuth) by focusing the lamp's arc onto a single cone using the same microscope objective used to visualize the cell. Uncaging light was spectrally selected below 400 nm using optical filters (Hoya Filters). Uncaging and measurement of the consequent changes in membrane current and fluorescence began 3 min after achieving whole cell mode, a sufficient time for equilibration of small molecules, as evidenced by attainment of steady state cell bis-fura2 fluorescence intensity. Experiments were completed within the following 35 min. After that time, loss of the modulator was apparent by drift in current amplitude and weakening of the effects of uncaging light.
Data Analysis
Selected functions were fit to experimental data using nonlinear, least square minimization algorithms (Origin; Microcal Software). Experimental uncertainty throughout is presented as standard deviation. Simulations were executed with dynamic simulation software (Tutsim; Actuality Corp.).
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RESULTS |
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We measured the Ca2+ dependence of ligand sensitivity in cGMP-gated currents of electropermeabilized, single cones (ep-cones). In this preparation, outer segment membrane currents are measured with suction electrodes at 0-mV holding potential. To generate an electromotive force that drives the cGMP-gated currents, measurements are conducted under an ion concentration gradient with Na+, a permeable cation, in the extracellular medium and choline+, an impermeant cation, in the intracellular medium. Also, there is only 1 µM Ca2+ in the extracellular medium to reduce the ion's influx through open CNG, and thus to minimize intracellular Ca2+ loading. Under these conditions, activation of cyclic nucleotide-gated channels generates a sustained, inward current (Fig 1).
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Ca2+-dependent CNG current modulation in cones is mediated by the interaction of the CNG channel protein with a diffusible, unidentified factor (
Ca2+ Dependence of Cyclic Nucleotide Sensitivity in Electropermeabilized Cones
The dependence of membrane current on cytoplasmic cGMP in dark-adapted ep-cones in the presence of varying concentrations of cytoplasmic Ca2+ is illustrated in Fig 1. All solutions contained 1 mM free Mg2+ and 0.4 mM Zaprinast, an effective inhibitor of cone cGMP-specific phosphodiesterase (PDE) (
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(1) |
where I is the current amplitude measured at the cGMP concentration, cGMP. Imax is the maximum current measured, K1/2 is the cGMP concentration at which I has the value Imax/2, and n is an adjustable parameter that denotes cooperative interactions. The same function applied to data measured in the presence of all Ca2+ concentrations tested, but the values of K1/2 (and not n) changed with Ca2+.
To analyze Ca2+ dependence in detail, we determined the value of K1/2 in the presence of Ca2+ at concentrations between 0 and 20 µM. Each Ca2+ concentration was tested in a different cone and K1/2 values were averaged (Fig 2). Ca2+ dependence is well described by a modified Michaelis-Menten equation:
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(2) |
where K1/2 (Ca2+) is the value of K1/2 at a given Ca2+ concentration. Khiand K
low are the extreme values of K1/2, at 20 and 0 µM Ca2+, respectively. CaKm is the Ca2+ concentration at the midpoint between K
hiand K
low.The values of K1/2, their errors, and details of the statistical universe sampled are presented in Table 1. Optimum fit of the Michaelis-Menten equation (Equation 2) to the mean of the data was obtained with CaKm = 857 ± 68 nM.
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To insure that the effects reported for cGMP are not confounded by the possible hydrolysis of cGMP by PDE in the outer segment (in spite of the presence of Zaprinast), we carried out the same class of measurements using 8Br-cGMP also in the presence of Zaprinast. 8Br-cGMP is an effective activator of cone CNG channels (
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Cytoplasmic Loading of Intact Single Cones
To investigate the time course of the CNG channel modulation, we studied isolated single cones with tight-seal electrodes in the whole-cell mode. The electrode filling solution lacked nucleotide triphosphates and, therefore, cones could not sustain endogenous synthesis of cGMP (
To detect CNG channel modulation in the absence of ATP and GTP, we activated the channels with exogenous ligand. We added 8Br-cGMP and 0.4 mM Zaprinast to the electrode-filling solution, which also contained 1 mM diazo-2 and 0.1 mM bis-fura-2 with 1 mM free Mg2+ and 600 nM free Ca2+, a value close to that of CaKm (see above). In Fig 4, we illustrate typical membrane currents measured in different cones at -35 mV with electrode filled with solution containing either 15 or 30 µM 8Br-cGMP. After attaining whole-cell mode, an inward current developed slowly that reached a peak and then declined to a stationary value. This time course reflects the activation of the outer segment inward current as the nucleotide loads the cell and cGMP-gated channels open, followed by channel block due to increasing cytoplasmic Ca2+ concentration. Because of this associated cytoplasmic Ca2+ load, we elected to carry out these experiments in a Ringers' solution containing 0.1 mM Ca2+, rather than the normal 1 mM. This allowed us to maintain cytoplasmic Ca2+ within reasonable bounds.
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With 15 µM 8Br-cGMP in the electrode, the time for current to reach peak had an average value of 15.8 ± 2.4 s (n = 8, range 13.320 s). The decay from peak to steady state values was well described by a single exponential with average time constant of 32.7 ± 7.6 s (n = 8, range 21.441.0). The stationary current was, on average, -46 ± 17 pA for 15 µM 8Br-cGMP (n = 21) and -415 ± 107 pA for 30 µM 8Br-cGMP (n = 8). Since the photocurrent in the bass single cone outer segment at -40 mV is, on average, 23 ± 8 pA in amplitude (
Ca2+-dependent CNG Channel Modulation in the Intact Cone
We caused a rapid (<< 50 ms, see below) decrease in cytoplasmic Ca2+ by uncaging diazo-2 in intact cone outer segment. In the presence of 15 µM cytoplasmic 8Br-cGMP, the uncaging flash caused a slow increase in current that reached a peak and then slowly returned towards its starting value (Fig 5). Within the first 68 min after establishing whole-cell mode, responses of similar features could be generated repeatedly by simply waiting 2 min between flashes. After longer intervals, the responses became smaller and even disappeared. The change and eventual loss of the response is almost certainly due to the slow and irreversible loss of modulator, just as occurs in the ep-cones. Data presented here were collected in the interval between 3 and 6 min after achieving whole-cell mode.
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In the presence of constant 8Br-cGMP, the increase in current caused by uncaging diazo-2 can reflect Ca2+-dependent modulation of channel sensitivity as proposed here, but other mechanisms could also explain the finding. For example: (a) Ca2+-dependent activation of guanylyl cyclase (GC), which in turn synthesizes cGMP; (b) activation of Ca2+-dependent currents that are not cyclic nucleotide gated; and (c) a direct effect of light. The GC hypothesis is most unlikely because the cones were intentionally made free of nucleotide triphosphates and, without GTP, GC cannot synthesize cGMP. The absence of endogenous cGMP is made evident by the fact that in darkness and in the absence of GTP and ATP, cGMP-gated channels close and the holding current at -35 mV drifts from a starting value near zero to a steady state value of 21.1 ± 12.3 pA (n = 35) (due to the activity of voltage-gated K+ channels, as discussed above). Moreover, under these conditions, dark-adapted cones do not respond to light, however bright it may be.
To test that the flash-generated current is indeed caused by CNG channel modulation, we tested the response to uncaging flashes in cones loaded with solutions modified as follows: (a) free of 8Br-cGMP (to test whether a cyclic nucleotideindependent current is activated by lowering Ca2+) and (b) diazo-2 replaced by 1 mM BAPTA (to test whether light alone, in the absence of Ca2+ changes can cause channel activation). Typical results of these tests are shown in Fig 5. In the absence of 8Br-cGMP, the holding current at -35 mV was outward, 21.1 ± 12.3 pA (n = 35), and reflects the activity of voltage-dependent K+ channels in the inner segment. Uncaging flashes caused a decrease in Ca2+, but failed to generate changes in current. In the absence of diazo-2, 15 µM 8Br-cGMP sustained a stationary inward current and light flashes also failed to change the current. The same occurred in every cell we tested (8Br-cGMP free, n = 4; diazo-2 free, n = 5). Also, adding 10 mM BAPTA to the electrode-filling solution blocked the flash-generated current change (data not shown). Examination of the data, however, shows a large and very fast spike at the moment of flash (Fig 5 Fig 6 Fig 7). The spikes are inevitable artifacts caused by the high-energy discharge of the capacitors in the Xe flash instrument. Thus, control experiments indicate that the slow change in current generated by uncaging diazo-2 arise specifically from activation of cyclic nucleotidegated current caused by lowering cytoplasmic Ca2+.
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Time Course of Flash-generated Sequestration of Cytoplasmic Ca2+
The electrode-filling solution was buffered with dark diazo-2 and bis-fura-2 to attain 1 mM free Mg2+ and 600 nM free Ca2+; however, the effective Ca2+ concentration in the cone outer segment may differ from this value because of the action of endogenous mechanisms that control cytoplasmic Ca2+ (
To determine the speed of Ca2+ sequestration in a single cone outer segment caused by uncaging diazo-2, we measured the fluorescent signal of cytoplasmic bis-fura-2 excited at 380 nm and emitted in the range between 410 and 600 nm. Under these conditions, a decrease in Ca2+ causes an increase in fluorescence intensity. Fluorescence intensity signaled only free Ca2+ in the outer segment because an optical aperture restricted fluorescence excitation light to the outer segment alone. Fig 6 illustrates a typical result in a cone loaded with 15 µM 8Br-cGMP (with Zaprinast). In the absence of an uncaging flash, cell fluorescence intensity was constant, indicating a steady Ca2+ concentration unaffected by the 380-nm fluorescence excitation light. This is important because it demonstrates that the fluorescence monitoring light alone did not uncage diazo-2. Presentation of an uncaging flash caused an instantaneous increase in fluorescence, which then slowly recovered towards its starting value (Fig 6). The increase in fluorescence indicates that Ca2+ concentration decreased within 50 ms of the flash. This value, however, is an upper limit on the actual speed of sequestration imposed by the bandwidth of the photon counting instrumentation. Ca2+ reached a minimum, and then slowly recovered, presumably because of additional Ca2+ influx through the newly activated CNG channels.
We found that in all cells tested the Ca2+ concentration change caused by uncaging diazo-2 was well described by an exponential process (Fig 6):
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(3) |
where Cadark2+ is the concentration preceding the flash, Capeak2+ is the maximum decrease in concentration, and Cass2+ is the concentration at the end of the sampling period (23 s after the uncaging flash). t = 0 is the moment the flash is presented and is the time constant of decline between the peak and ss values. We note that ss is not truly a stationary value; 23 min after a flash, Ca2+ concentration returned to its original "dark" value. This very slow recovery is expected from the eventual equilibrium between cell cytoplasm and electrode lumen.
We did not calibrate in situ the 380-nm bis-fura-2 fluorescence in absolute units of Ca2+ concentration, a difficult task that is even less reliable when only a single excitation wavelength is used ( = 0.52 ± 0.07,
= 0.70 ± 0.08, and
Ca = 5.38 ± 0.35 s.
A Kinetic Model of CNG Channel Modulation
Examination of the data in Fig 5 and Fig 6, each a different cone, shows that, after an uncaging flash, cytoplasmic Ca2+ changed more rapidly than did the membrane current. The difference in the early time course of the fluorescent and current signals is illustrated in detail in Fig 7, data measured in yet a different cone. After an uncaging flash, Ca2+ concentration reached its minimum within 50 ms, yet current increased with an exponential time course and reached a peak only after 0.9 s. At the peak, the nucleotide-gated conductance was enhanced 3.85-fold. In seven cells, Ca2+ was always minimal within 50 ms, and current reached peak in a mean time of 1.25 ± 0.23 s. Conductance enhancement at the peak was, on average, 2.33 ± 0.95-fold.
We developed a kinetic model that successfully matched experimental data. Let us simply assume that CNG channels exist at concentration g1 in conductance state S. Conductance state is the product of single channel conductance and probability of opening. Thus, membrane conductance is the product of g1 and S. Upon Ca2+ binding, and in the presence of the modulator, the channels change to a new concentration g2 of conductance state S. Membrane conductance changes as channel concentration changes from g1 to g2. We do not specify the molecular mechanisms underlying the change in channel concentration (and, therefore, membrane conductance), we simply assume that concentration change is pseudo first order, characterized by a time constant g (
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(4) |
then, when g1(0) >> Ca(t) (Equation 5):
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(5) |
where (Equation 6):
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(6) |
and Ca(t) is the Ca2+ concentration given by Equation 3.
To test the adequacy of the model, we fit data simulated by the model to experimental results. To this end, and for each set of experimental data: (a) we fit Equation 3 to the measured fluorescence change and used the resulting analytical expression to describe Ca(t) for that data set. (b) We simulated changes in membrane conductance using the model above; the simulation predicts the change in conductance caused by a defined change Ca2+ provided as an input. (c) We systematically varied the values of g to best match simulated and experimental data. The kinetic model could be made to simulate experimental data well (Fig 8). For seven cells, with fits of similar quality to that shown in Fig 8, mean value was
g = 0.40 ± 0.14 s.
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DISCUSSION |
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Activation of cGMP-gated ion channels in intact cone photoreceptors depends on agonist concentration, but their sensitivity, how much agonist is necessary for activation, depends on Ca2+. Ligand sensitivity decreases as Ca2+ increases, and this relationship is well described by a Michaelis-Menten function (Equation 2), suggesting that modulation arises from the noncooperative binding of Ca2+ to a single site. This apparent simplicity belies the fact that the molecular events underlying the Ca2+-dependent modulation involve interaction among at least three distinct molecules: Ca2+ ions, a modulator protein, and the ion channels (themselves constituted by alpha and beta subunits;
In the experiments with ep-cones, we minimized the extent of modulator loss by conducting all measurements in the presence of 1 mM Mg2+ and completing them within 180 s of inner segment permeabilization. In intact cones under whole-cell voltage clamp, modulator was still lost, but at a much slower rate. We minimized modulator loss in intact cones by completing all measurements in a window between 3 and 6 min after attaining whole-cell mode. This period was long enough to allow equilibration between the cell and electrode lumen, and yet not so long as to loose the modulator.
The Ca2+-dependent modulation of channel sensitivity, quantitatively expressed by the value of CaKm (Equation 2) is the same whether channels are activated with cGMP or 8Br-cGMP, 860 nM. This observation is important because it indicates that the interaction between Ca2+, modulator, and channel is independent of the ligand gating the channel. In a previous report (
50 nM Ca2+ (
In both rods and cones in the dark, 35% of all cGMP-gated channels are active. Because the ligand sensitivity of the channels in the two receptor types is so different (
10 µM in rod outer segments and 52 µM in cones.
Signal transduction in olfactory neurons arises from odorant-dependent changes in cytoplasmic cAMP, which, in turn, activates specific CNG-gated channels. The ligand sensitivity of these channels decreases with increasing Ca2+ (
In the intact cones, we found a relatively large variance in the values of K1/2 at any given Ca2+ concentration (Table 1). Also, the value of n, while independent of Ca2+, differs between cGMP and 8Br-cGMP, as reported before (
Modulation of K1/2 by phosphorylation has not been definitely demonstrated in cones, but probably occurs. Modulation by phosphorylation, however, is unlikely to play a role in the results presented here since nucleotide triphosphates, both ATP and GTP, were absent from the cells, both in ep-cones and in the whole-cell mode. The lack of nucleotide triphosphates in the intact cones was verified by the fact that holding currents recorded in the absence of 8Br-cGMP were positive, reflecting current flowing out of the cone through voltage-gated K+ channels in the inner segment unopposed by the activity of outer segment channels. Absence of triphosphate nucleotides substrates also assured us that Ca2+-dependent guanylyl-cyclase was not active in the cones studied in this report.
We measured the kinetics of sensitivity modulation at a fixed ligand concentration, 15 µM 8Br-cGMP. We elected this concentration because it activated channels to an extent comparable with that of a normal dark-adapted cone. In the presence of higher 8Br-cGMP concentrations, we observed, in addition to sensitivity modulation, small current changes that reflected flash-induced hydrolysis of the nucleotide. It must be recalled that 8Br-cGMP is a poor substrate of the photoreceptors' PDE when compared with cGMP, but the enzyme can nonetheless hydrolyze it. Indeed, photoresponses can be measured in rods loaded with 8Br-cGMP alone (
The time course of the Ca2+-dependent change in ligand sensitivity is slower than that of the Ca2+ change itself. The kinetics of the change in sensitivity is well described as a pseudofirst-order process rate limited by the Ca2+ concentration change with a time constant of 0.4 s. The kinetics of most of the Ca2+-dependent events in the phototransduction cascade of rods or cones is not known with precision (
In bass single cones, voltage-clamped current responses to dim light flashes reach peak in 80100 ms and recover fully within 300 ms (
In bass single cones, as in all cones, the response to a constant dim flash changes as a function of the intensity of background light against which the flash is presented. In general, the peak amplitude decreases as the background intensity rises, and the relationship between peak current and background intensity is described by the Weber law (1-s time constant (
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Footnotes |
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1 Abbreviations used in this paper: CNG channels, cyclic nucleotidegated ion channels; PDE, phosphodiesterase.
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Acknowledgements |
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We thank C. Chung, P. Faillace, A. Picones, A. Olson, T. Ohayama, and C. Paillart for their helpful criticism and continuing interest.
This study was supported in part by a student fellowship supported by the Fight for Sight research division of Prevent Blindness America (E. Kotelnikova) and a Grant-in-Aid of research from the National Academy of Sciences, through Sigma Xi, The Scientific Research Society. Research was also supported by the National Institutes of Health (EY-05498).
Submitted: 22 June 2000
Revised: 21 August 2000
Accepted: 21 August 2000
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