Neurotransmitter-Induced Novel Modulation of a Nonselective Cation Channel by a cAMP-Dependent Mechanism in Rat Pineal Cells

Nissim Darvish and James T. Russell

Laboratory of Cellular and Molecular Neurophysiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Darvish, Nissim and James T. Russell. Neurotransmitter-induced novel modulation of a nonselective cation channel by a cAMP-dependent mechanism in rat pineal cells. J. Neurophysiol. 79: 2546-2556, 1998. In the rat, circadian rhythm in melatonin is regulated by noradrenergic and neuropeptide inputs to the pineal via adenosine 3',5'-cyclic monophosphate (cAMP)- and Ca2+-dependent mechanisms. We have identified a large conductance (170 pS), voltage-dependent, nonselective cation channel on rat pineal cells in culture that shows a novel mode of modulation by cAMP. Pituitary adenylate cyclase activating peptide (PACAP), norepinephrine, or 8-Br-cAMP increase channel open probability (Po) with a hyperpolarizing shift in voltage dependence such that the channel becomes active at resting membrane potentials. The increase in Po was accompanied by a change in current rectification properties such that the channel was transformed from being inactive at rest to an inwardly rectifying cation conductance in the presence of agonist, which depolarizes the cell. This channel is calcium insensitive, is blocked by Cs+, and shows a permeability sequence: K+ > Na+ >=  NH+4 > Li+. The data suggest thatPACAP and norepinephrine acting through a cAMP-dependent mechanism modulate this nonselective cation channel, resulting in a slow onset depolarization that may be important in regulation of pineal cell excitability.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

The pineal gland maintains a circadian rhythm in the biosynthesis and secretion of melatonin. In mammals, this is controlled by the circadian clock in the suprachiasmatic nucleus through the superior cervical ganglion (Klein 1985). Noradrenergic outflow from the superior cervical ganglion serves as the major regulator of pineal melatonin (see Klein 1985, 1986), in addition to peptidergic innervation by neuropeptide-containing fibers, e.g., pituitary adenylate cyclase activating peptide (PACAP) and vasoactive intestinal peptide (VIP). Norepinephrine (NE), PACAP, and VIP, all act through dual second-messenger pathways, increasing both adenosine 3',5'-cyclic monophosphate (cAMP) and Ca2+ (Klein et al. 1992; Saez et al. 1994; Schaad et al. 1993; Yuwiler et al. 1995). The two second messengers act in concert to increase expression of N-acetyl transferase, the rate-limiting step in melatonin biosynthesis (see Sugden 1989). In the case of NE, this is achieved by stimulation of both alpha  and beta  adrenergic receptors, where the former is coupled to phospholipase-C activation, whereas the latter to adenylate cyclase (Klein et al. 1992; Sugden et al. 1986, 1987; Vanacek et al. 1985). PACAP belongs to the family of neuropeptides that includes VIP, glucagon, and secretin (Arimura 1992). Two types of PACAP receptors have been identified. Type 1 receptors are coupled both to adenylate cyclase and phospholipase C and prefer the PACAP-38 form of the peptide (Spengler et al. 1993). Type 2 receptors do not distinguish between PACAP or VIP and are coupled to adenylate cyclase alone. Pineal cells express type 1 PACAP receptors in high density, and their stimulation in vitro causes increases in both cAMP and Ca2+ and results in melatonin secretion (Arimura 1992; Darvish et al. 1995a; Masuo et al. 1992; Olcese et al. 1996; Simonneaux et al. 1993).

In rats, melatonin synthesis and secretion increases in the evening and is maintained at an elevated level over several hours during the night (Klein 1985; Klein et al. 1992). The cellular mechanisms by which long-lasting increases in both [Ca2+]i and cAMP levels occur upon receptor activation and the mechanisms by which N-acetyl transferase expression is regulated are not completely understood. In many sensory systems, cyclic nucleotides act through nonselective cation channels (NSCC) in the transduction of sensory stimuli (see Kaupp and Koch 1992). In addition, different types of NSCC, expressed in different cell types (Distler et al. 1994), are central to the control of membrane potential and thus excitability (Kaupp and Koch 1992). In avian pineal cells, a NSCC controlled by the intrinsic circadian clock recently has been identified (D'Souza and Dryer 1996). In this study, we investigated membrane currents modulated by the two major regulatory neurotransmitters that control melatonin secretion by the rat pineal, namely, NE and PACAP, and have identified a nonslective cation channel expressed in high density on these cells the activity of which shows a novel form of cAMP-dependent modulation.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Cell culture

Pineal glands were isolated from adult male Sprague-Dawley rats (150-200 g) kept under a 12-h light/dark cycle as described previously (Schaad et al. 1993) with the following modifications. Animals were routinely killed within 3-4 h after lights on, and pineal glands (5-8 per preparation) were removed quickly and placed in a cold dissecting solution containing (in mM) 130 NaCl, 5.4 KCl, 1.5 CaCl2, 0.8 MgCl2, 1 Na2PHO4, 2.5 NaHCO3, 20 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 25 dextrose, and 1 sodium ascorbate. Under a binocular dissecting microscope, glands were disrupted gently, and tissue pieces were incubated in the same solution with a mixture of papain (20 U/ml; Worthington Biochemicals, Freehold, NJ) and DNAase (0.1 mg/ml) for 60 min at 37°C in a shaking water bath. Cells were centrifuged at 300 g, resuspended in fresh solution, and 200 µl of the suspension was plated on glass cover slips coated with collagen (0.3 mg/ml) and poly-L-lysine (10 µg/ml). Cells on cover slips were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS), penicillin (100 U/ml), and streptomycin (100 µg/ml) under 5% CO2 at 37°C. Immunocytochemistry using antibodies against S-antigen revealed that >96% of the cells stained positively for this protein, identifying them as pineal cells.

Electrophysiology

Membrane potential and current recordings were made with a axopatch 200 A patch-clamp amplifier (Axon Instruments, Foster City, CA) with patch pipettes made from borosilicate glass capillaries with 1.5-mm ID (World Precision Instruments). Pipettes were pulled with a double pull electrode puller (Narishige, PP83, tip resistance between 2 and 10 MOmega ). Recordings were made in the on cell, single channel mode and in perforated patch whole cell configurations. Amphotercin B (240 µg/ml) was used to obtain perforated patches that were employed to prevent channel rundown and loss of cellular components (Horn and Marty 1988). In this configuration, after formation of a high-resistance membrane seal (typically >5 GOmega ), increase in membrane capacitance was monitored until steady state was reached. The series resistance was determined and in every case >= 75% of it was compensated. The zero current potential was determined and later was used as the holding potential. Diffusion offset potential was corrected at the beginning of each experiment. Voltage-clamp protocols and data acquisition were performed using "Synapse" (Synergistic Research Systems, Silver Spring, MD) running on apple Quadra-950 computer with an ITC-16 A/D board (Instrutech). Perforated patch clamp data were filtered at 2 kHz and digitized at 5 kHz. Single-channel data were stored on a videocassette recorder using a Vetter PCM recorder (model-200). During analysis, recorded data were low-pass filtered through an 8-pole Bessel at cutoff frequencies between 2 and 3 kHz. All experiments were conducted on 2- to 4-day-old cells at room temperature. During the experiments, cells were maintained under continuous perfusion using the bath solution (see following section) unless otherwise stated, and drug treatments were accomplished in the perfusion solution.

Solutions

SINGLE-CHANNEL EXPERIMENTS. The bath solution contained (in mM) 150 NaCl, 5 KCl, 10 HEPES, and 1.5 CaCl2; pH adjusted to 7.4 with NaOH. Inside the pipette, we used the following solutions: 1) (in mM) 130 KCl, 10 NaCl, 1 CaCl2, 11 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 10 HEPES, and 1 MgCl2, (pH 7.4). 2) (in mM) 70 KCL, 10 NaCl, 5 EGTA, 125 D-glucose, and 10 HEPES. This composition was chosen so that, in the inside-out configuration, the EK+ would be +66 mV and ENa+ would be -66 mV and thus enable us to determine the cation selectivity of the channel. 3) (in mM) 150 K-glutamate, 10, HEPES, and 1.0 EGTA. This solution was used when bi-ionic conditions were required in ion selectivity studies. Bath solution was the same in both single channel and perforated-patch whole cell experiments. In the latter, the pipette contained pipette solution 4 of the following composition (in mM): 75 K2SO4, 55 KCl, 10 NaCL 10 HEPES, and 8 MgCl2, (pH 7.4 with KOH). All solutions were adjusted to 280-300 mOsm/kg and pH to 7.4.

Data analysis

AxoGraph 3 (Axon instruments) for Apple Macintosh was used for data analysis. During analysis of single-channel data, open probability of the channel was expressed as Po, when there was only one channel in the patch and Np, if there were more than one; where N denotes number of channels and p, open probability. Po, Np, and current amplitude were calculated from the all amplitude histograms created for each voltage or time period using Axograph 3. The voltage values described throughout the manuscript refer to membrane potentials calculated from the pipette voltage commands based on an average resting membrane potential of -50 mV (see RESULTS). In single-channel experiments, the permeability ratio K+:Na+ was calculated from data obtained with pipette solutions 1 or 2 using the Goldman equation. Biionic media conditions was employed in some experiments to investigate ion selectivity between K+, Na+, and other cations, e.g., NH+4, Li+. We used pipette solution 3 in these experiments. The K+, Na+, NH+4, or Li+ permeability ratios were calculated from the reversal potential (Erev) as PA/PB = [B]i/[A]o* exp(Erev*zF/RT), where [B]i/[A]o is the ratio of intracellular concentration of ion B versus extracellular concentration of ion A, z is the charge, F is the Faraday constant, R is the gas constant, and T is absolute temperature. Reversal potentials were points on the linear regression of the I-V relationship with zero current and were obtained by extrapolation of data. Data are presented as means ± SE.

[Ca2+]i measurements

[Ca2+]i was measured using previously published methods (Fatatis and Russell 1992; Yagodin et al. 1994). Briefly, cells on cover slips were loaded with Fura-2 AM (1 µM) for 10 min at 37°C in a solution of the composition same as that of dissection solution given above and containing, in addition, sodium pyruvate (1 mM) and bovine serum albumin (1.5 mg/ml; osmolarity 320 mOsm/Kg; pH 7.4). Cells were perfused with the same solution in a Leiden cover slip chamber mounted on the microscope stage. Drug treatments were accomplished by applying the agents in the perfusion system. Fura-2 fluorescence was measured with an inverted microscope on a vertical optical bench using a Nikon ×40/1.3 NA objective lens. The cell were illuminated with a mercury arc lamp with two outputs and two collector lenses (Oriel Optics). A single quartz crystal fiber-optic cable bussed to form a "Y" (Laser Sciences, Newton, MA) was used to bring light from both sides of the lamp housing to the microscope. The input sides of the fiber were 0.6 µm in diameter, and the single output was 0.8 µm in diameter. Shutters (Uniblitz) and band-pass and neutral density filters were mounted in the light path, and a computer controlled the shutters such that the cells were illuminated alternatively with 340 or 380 nm excitation beams. Fluorescence images were acquired through an intensifier with a CCD camera (Videoscope International, Washington, DC) and images were digitized using a Quadra 950 computer (Apple Computers) and a LG-3 digitizer board (Scion, Frederick, MD). The time interval between image pairs was varied depending on the required resolution. After all the image pairs were collected and stored, ratio images were generated on a pixel by pixel basis after subtraction of appropriate blanks. Ratio values were converted into Ca2+ concentration with calibration values obtained as described earlier (Fatatis and Russell 1992; Yagodin et al. 1994) (Kd = 150 nM). The average Ca2+ concentration in each cell in the field was measured from the average pixel values within the cell boundary as described earlier (Fatatis and Russell 1992; Yagodin et al. 1994)

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Nonselective cation channel in rat pineal cells

Membrane currents were recorded in cell-attached patches of rat pineal cells in culture. In 40% of the patches (of >200), a nonselective cation channel activity was elicited by depolarizing the patch by >60 mV (Fig. 1A). Current voltage curves, obtained by changing the pipette command voltage in steps (Fig. 1C), revealed a cation selective channel with a slope conductance of 175 ± 6 pS (n = 17) with 130 mM KCl in the pipette and 146 ± 5 pS (n = 18) when the pipette contained 70 mM KCl. Extrapolation of the current voltage data showed that the channel is cation selective (ERev = +2 mV with 130 mM K+ and 10 mM NaCl in the pipette, pipette solution 1). Patches held at hyperpolarizing voltage commands for as long as 20 min did not show any channel activity. The possibility that either low open probability (Po) or asymmetrical K+ concentration was responsible for this lack of channel activity was excluded by single-channel tail current recordings with 130 mM KCl in the pipette. In these experiments, the voltage jump from depolarized membrane potentials should have allowed us to record channel activity as instantaneous current before inactivation occurs. No instantaneous inward currents were recorded in 10 such trials (Fig. 1B). Figure 1D depicts the voltage dependence of the channel in the cell-attached configuration with an e-fold change in Po for every 14-mV depolarization.


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FIG. 1. Biophysical characterization of the nonselective cation channel in rat pineal cells. A: cell-attached patch recordings showing single-channel current traces obtained at different membrane potentials. Cell was perfused with the bath solution and the pipette contained KCl (130 mM) and NaCl (10 mM) (pipette solution 1, see METHODS). Note increase in channel open probability with depolarization of the patch. Note also that no inward currents are observed under hyperpolarizing voltage commands. Membrane potentials shown were calculated from pipette potential using the average resting membrane potential of -50 mV. B: measurement of single channel tail currents. A pineal cell membrane patch was held under depolarizing voltage commands (+70 mV) and instantaneously jumped to the resting membrane potential (-50 mV), which failed to evoke inward currents (n = 10. Pipette contained 130 mM KCl and 10, NaCl) and the cell was perfused with bath solution. C: I-V relationship measured from records shown in A. Channel with a slope conductance of 160 pS on extrapolation showed a reversal potential of +2 mV, as expected for a nonslective cation channel. D: voltage dependence. Graph shows Boltzman fit of pooled data from 15 different experiments (mean ± SE). Cell-attached patches were held at each voltage command for >= 30 s, and probability of opening was calculated from recorded data. Experimental conditions were the same as in A. Note, Po increases on membrane depolarization with an e-fold change for every 14 mV.

On patch excision to the inside-out configuration, channel Po increased significantly (Fig. 2A), and the voltage dependence was shifted to the left (compare with Fig. 1D). Data in Fig. 2A was recorded in the same patch shown in Fig. 1A after excision. In this mode, with 150 mM NaCl and 5 mM KCl facing the inner mouth of the channel and 10 mM NaCl and 70 mM KCl in the pipette, only inward currents could be observed, and the calculated current reversal potential (Erev) was +32 ± 5 mV (n = 10). Under these ionic conditions, the permeability ratio, P+K:P+Na was found tobe 2. In another set of experiments, permeability sequence of cations was examined with 150 mM K-glutamate in the pipette (solution 3, see METHODS) and 150 mM of either Na+, NH+4, or Li+ supplemented with HEPES (10 mM) in the bath. In this biionic condition, excised patches, in the inside-out configuration, showed both inward and outward currents and current voltage relationships were constructed (Fig. 2B). From this data the cation permeability sequence was found to be K+ > Na+ >=  NH+4 > Li+ 1.0:0.55:0.5:0.1. Cs+ (10 mM) applied to the bath (the inner mouth of the channel) blocked channel activity (n = 5; Fig. 2C). The channel, however, was insensitive to changes in Ca2+ because lowering Ca2+ concentration to 10 nM (verified using an ion selective electrode), did not alter Po (n = 7; Fig. 2D), suggesting that the channel is not Ca2+ dependent. In addition, changes in bath concentrations of Ca2+ did not alter current rectification (n = 7).


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FIG. 2. Current measurements in the inside-out configuration A: plot of the open probability (Po) vs. the patch membrane potentials for the nonselective cation channel in an excised patch. Data were obtained from the experiment shown in Fig. 1A. After measurement of I-V relationship in the cell-attached mode, the patch was excised into bath solution (see METHODS) and measurements were made. Channel activity increased on patch excision and in all the experiments, mostly inward currents were measured under these conditions. Channel Po increased with patch depolarization. B: permeability ratio measurements. Cation permeabilities through the channel was measured under biionic condition in excised patches with 150 mM K-glutamate, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 1 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) in the pipette. Bath solution contained 150 mM X-Cl and 10 mM HEPES, where X refers to either Na+ (bullet ), NH4+ (square ), or Li+ (black-triangle). Figure shows I-V relationship under all 3 conditions and thus the permeability sequence K+ > Na+ >=  NH+4 > Li+. C: gating of >= 2 channels in this patch could be blocked by Cs+ (10 mM) applied to the inner mouth of the channel. Recording was made in the inside-out configuration with bath solution in the bath. Pipette contained pipette solution 1. Cs+ was added to the bath during the test period. Washing out Cs+ reverses the block (data not shown). Patch was held at 0 mV and channel openings are in the downward direction (inward currents). D: effect of changes in Ca2+ concentration on channel activity. Excised patch in the inside-out configuration was in bath solution with solution 1 in the pipette. Patch was held at 0 mV, and reducing Ca2+ concentration to 10 nM by adding HEPES-buffered EGTA in the bath (on the cytoplasmic side) had no effect on channel Po or current rectification. Inset: data in higher resolution.

The experiments in Fig. 1 show that in the resting cell, in the on cell configuration (under all 3 pipette solution conditions), only outward currents could be measured. On patch excision, however, channel gating in the inward direction was measured (Fig. 2B). Taken together, the above data suggest that interaction of the channel with some intracellular component prevents inward currents being carried by the channel in the cell-attached configuration and that the channel block is "washed out" when the patch is excised from the cell.

Neurotransmitter mediated channel modulation

We next investigated the modulation of the NSCC by norepinephrine and PACAP, the two major neurotransmitters involved in the control pineal function (Klein 1986; Klein et al. 1992; Sugden 1989). Cell-attached patches were held at depolarizing voltage commands for >= 4 min before the agonist was applied, ensuring that there was no spontaneous increase in channel activity with time after seal. Addition of PACAP (1 µM) resulted in a dramatic increase in channel activity in 12 of 15 experiments. Np increased from 0.002 ± 0.0005 to 0.507 ± 0.1 (n = 12; Fig. 3), and this activation occurred 3-10 min after application of agonist (Fig. 3, B and C). Removal of agonist did not reverse the increase in channel activity (n = 4), suggesting a long-lasting modification of the channel complex. Concurrent with activation of this channel by PACAP, outward unitary current amplitude decreased in 9 of 12 experiments (Fig. 3C). In four of these experiments, current amplitude reduced to 10% of the original level such that we could not resolve channel openings. In control experiments, patches held at depolarizing voltage commands for 20 min without agonist application did not show either spontaneous increase in channel activity or decrease in the amplitude of the outward current (n = 3).


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FIG. 3. Effect PACAP on channel activity and rectification properties. A: recordings were made in the cell-attached mode with the membrane potential held at +80 mV, and pituitary adenylate cyclase activating peptide (PACAP; 1 µM) was applied to the bath, diluted in bath solution. Pipette contained pipette solution 1. Single-channel activity before (top) and 7 min after (bottom) agonist application showing channel activation. Channel opening is shown as upward deflection. Current amplitude histograms created from 30 s of recording shows increase in channel Po and a 20% decrease in current amplitude under the same voltage command. Prolonged exposure of agonist results in a 60% decrease in current amplitude (not shown). B: modulation of rectification. Cell-attached patch recordings with two channels in the patch clamped at +30 mV. Cell was perfused with bath solution and the pipette contained pipette solution 1. Every 1-2 min, a voltage ramp from +90 to -130 mV was applied. Activation of the outward current is observed 6 min after PACAP application and in 8 min unitary conductance is reduced. Inward currents were measured in the same patch 15 min after agonist application. Inset: trace obtained 16 min after application is shown in high resolution. C: changes in open probability (NP) and current amplitude after PACAP application. Data are from the experiment shown in B. Patch containing channel activity was held at +30 mV, and the Np values were extracted from the all points amplitude histogram as shown in A. Np increased with time, whereas current amplitude decreased ~10 min after drug application. Measurement of current amplitude (I) was normalized by dividing the values with the highest current amplitude measured in that patch.

In 7 of 12 experiments, 7-15 min after stimulation with PACAP inward currents were detected in the same patches after the initial increase in channel activity (Fig. 3B). Analysis of these traces from the seven experiments revealed that the reversal potential did not change >10-12 mV at any time after stimulation (compare trace at 8 vs. 16 min, Fig. 3B). In addition, at certain time intervals after agonist application, both outward and inward currents could be observed (see inset, Fig. 3B), and the unitary current amplitude of the inward currents did not change with time. In several experiments, although Po increased >20-fold, inward currents were not detected, whereas in the other patches, with a sixfold increase in Po, inward currents were measured. Thus no correlation was found between the increase in channel Po and the ability to record inward currents. Both the outward currents and the inward currents appearing after agonist treatment showed similar voltage dependence, such that Np decreased with hyperpolarization (Fig. 4A). Furthermore, the I-V relationship of the outward currents before application of PACAP, when plotted together with the inward currents measured in the presence of the agonist, could be fitted with a simple linear regression (r = >0.9; Fig. 4B). It remains possible that agonist treatment completely shuts down the outward current and activates a new inwardly directed cation channel in the patch. Because both the inward and the outward currents, however, showed identical biophysical properties (voltage dependence, I-V relationship and unitary conductance), the simplest interpretation of this data are that agonist application modifies the outwardly directed cation channel complex originally present in the patch.


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FIG. 4. Characterization of inward currents after agonist application. A: plot of the open probability (Np) vs. membrane potential for the nonselective cation channel in the outwardly (open circle ) and inwardly (bullet ) rectifying conditions. Inward current measurements were made 16 min after PACAP application. Data are from the experiment shown in Fig. 3B in the cell-attached mode. Although the voltage dependence shows a marked shift to the left, the voltage sensitivity remains unchanged as shown by the similar slopes on an exponential fit of the data. B: plot of the current voltage relationship of the data from experiment shown in Fig. 3B. Data points obtained before agonist (outward currents, open circle ) and after (inward currents, bullet ) were well fitted with a simple linear regression (r = 0.995), suggesting that both ion selectivity and unitary conductance are identical for both outward current before agonist application and inward currents 16 min after agonist addition. C: norepinephrine (NE) induced changes in the channel properties. Cell-attached patch recordings were made in a pineal cell, and the cell was stimulated with NE (2 µM) while under perfusion with bath solution. Pipette contained pipette solution 1. Patch was held at +30 mV, and a voltage ramp (+80 to -130 mV) was applied every 1-2 min and current measurements were made. Three minutes after application of NE, NP increased followed by significant reduction in unitary conductance of the channels. Inward currents were also measured through the same channels 19 min after agonist application.

Norepinephrine, the major neural control of pineal function, was tested next for its effect on the NSCC. Similar to PACAP, NE (2 µM) application resulted in increased channel activity (n = 4; Fig. 4C). Po increased from 0.0024 ± 0.0016 to 0.374 ± 0.13 within 4 min of NE application, and in all the experiments, unitary current amplitude decreased. In two of three experiments, inward currents were elicited after NE application (see Fig. 4C). In this way, the effects of NE were indistinguishable from that of PACAP, in that NE increased channel Po, decreased unitary conductance, and profoundly modified the rectification properties.

Modulation of whole cell currents

We then investigated the effect of PACAP on whole cell currents using the perforated-patch technique. Before drug application, pinealocytes were voltage clamped from the zero current potential to hyperpolarizing and depolarizing steps, to obtain the current voltage relationship (I-V, see Fig. 5) The zero current potential in the perforated patch configuration was -52 ± 11 mV (n = 82), and this measurement was not significantly different from values obtained in the whole cell configuration (-46 ± 7; n = 6). PACAP (1 µM) application caused activation of outward currents within 4 min followed by depression after 5-10 min in eight of nine experiments (Fig. 5, A and B). In four of these experiments, prolonged PACAP treatment reduced the outward current amplitude to levels less than before agonist treatment. In addition, using a tail current protocol, activation of inward current was observed in two of three experiments, and in contrast with PACAP-induced activation of outward currents, this inward current persisted as long as PACAP was present (Fig. 5C). Tail current analysis also suggested that the channel underlying the agonist-induced current is a NSCC with a permeability ratio, P+K:P+Na of 2:1 (Erev = -56 ± 5 mV, n = 3) similar to values obtained in single channel measurements. Input resistance and capacitance were 0.95 ± 0.4 GOmega and 8.0 ± 0.5 pF (n = 15), respectively, and no spontaneous membrane potential oscillations or firing activity was observed in any of the cells.


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FIG. 5. Perforated patch recordings of outwardly rectifying currents in rat pineal cells. A: cell was perfused with bath solution, and the pipette contained pipette solution 4 (see METHODS). Cell was held at its resting potential (-54 mV) and a voltage-clamp protocol was executed from -130 to +110 mV. Top: control recording; middle and bottom: recordings 4 and 13 min after PACAP (1 µM) application, respectively. Initially, PACAP application resulted in increased outward currents followed by a significant reduction in current amplitude while PACAP was still present. Series resistance was compensated by 85%, and traces shown are not leak subtracted. B: I-V curves obtained from data in A, after leak subtraction using P/N protocol (N = -4), during the control period and 4 min and 12 min after PACAP (1 µM) application. C: tail current experiments, where the cell membrane was 1st depolarized to +80 mV for 100 ms and then stepped to -120 and back to the resting membrane potential (-54 mV). Cells were perfused with bath solution, and the pipette contained pipette solution 1. In this protocol, PACAP application initially resulted in an increase of both inward and outward currents. Over time, however, the outward currents reduced below control levels, whereas inward currents increased or remained the same without detectable inactivation. Note that the scale bars are different for the traces. D: I-V curves obtained from data in C, after off-line subtraction of control currents at 4, 13, and 20 min after PACAP (1 µM) application.

Modulation by cAMP analogues

NE acting through alpha  and beta  receptors (Cena et al. 1991; Yu et al. 1993) and PACAP acting through type 1 receptors (Masuo et al. 1992) increase cAMP levels as well as [Ca2+]i in rat pineal cells (Darvish et al. 1995a; Olcese et al. 1996; Simonneaux et al. 1993). Because the gating properties of the NSCC were Ca2+ insensitive, we asked if cAMP analogues would mimic the effects of PACAP and NE. When pineal cells held under cell-attached patch configuration were perfused with 8-Br-cAMP (2 mM), a marked increase of channel activity resulted. In 4-10 min, channel Po increased from 0.0049 ± 0.0025 to 0.43 ± 0.06 in five of six experiments (Fig. 6A) and similar to agonist treatment, concomitantly, the current amplitude decreased (Fig. 6, A and B). In some experiments, current amplitude reduced to 20% of control levels (Fig. 6B) with an average reduction of 40% (n = 5). In addition, similar to PACAP and NE, inward currents were elicited by 8-Br-cAMP in three of five experiments that were voltage dependent, and the channel Po decreased with patch hyperpolarization. The current voltage relationship of the inward currents measured after application of 8-Br-cAMP were well fitted with a simple linear regression to the I-V data for the outward currents before agonist treatment (Fig. 6C).


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FIG. 6. Adenosine 3',5'-cyclic monophosphate (cAMP) analogues modulate activity of channels in cell-attached patches. A: traces showing single channel activity before and after perfusion with 2 mM 8-Br-cAMP. Cell was perfused with bath solution, and the pipette contained pipette solution 1. Patch was held at +50 mV and exposed to 8-BR-cAMP, and in this experiment, activation of the channel was observed 5 min after application. Note also marked reduction in current amplitude with time after 8-Br-cAMP. B: plot of the open probability against time highlights the channel activation and reduction in current amplitude. Data are from experiment shown in A. C: I-V curve of data from experiment in A and B. Outward currents (open circle ) before 8-Br-cAMP application were plotted together with mostly inward currents measured 16 min after (bullet ). Both sets of points are well fitted with a simple linear regression (r = 0.996, Erev = +10 mV). D: plot of open probability and current amplitude measured in on cell patches containing the nonselective cation channel. Cell was perfused with bath solution, and the pipette contained pipette solution 1. Rp-cAMPs was perfused for 18 min, and data show that neither open probability nor current amplitude were altered by this analogue of cAMP.

Cyclic nucleotides are known to modulate channel activity either by direct binding to the channel subunit (Darvish et al. 1995b; Matthews 1991) or through initiation of phosphorylation-dephosphorylation cascade (Darvish et al. 1994; White et al. 1993). To test whether the activation of the channel by cAMP is direct or via the phosphorylation cascade, we perfused pineal cells with Rp-cAMPS in the cell-attached patch configuration. Rp-cAMPS is known to competitively inhibit the cAMP-induced activation of protein kinase A but can mimic the direct actions of cAMP on effector proteins (Kelley et al. 1995; Reale et al. 1995; Rothermel and Botelho 1988). Unlike 8-Br-cAMP, application of Rp-cAMPS (2 mM) had no effect on NSCC gating activity or on current amplitude during a 20-min period (n = 5; Fig. 6D). This result is consistent with the suggestion that a cAMP-mediated phosphorylation step is essential for channel modulation.

Consequences of NSCC gating

In a resting pineal cell, with the membrane potential between -35 and -56 mV (Freschi and Parfitt 1986; Poling et al. 1995), the NSCC will remain closed. Activation of the NSCC by NE or PACAP would be expected to cause inward currents through the channel, Na+ being the major charge carrier, causing depolarization of the cell. This prediction was tested in seven experiments, and PACAP effects on pineal cell membrane potential was monitored in the current-clamp condition with the whole cell perforated-patch configuration. In six of the seven experiments, PACAP application resulted in a slow depolarization of 12 ± 2 mV with a latency of 65 ± 22 s which lasted as long as the agonist was present (Fig. 7A). Because this depolarization is expected to open voltage-gated Ca2+ channels, in four experiments we measured [Ca2+]i levels in pineal cells during treatment with 8-Br-cAMP, [Ca2+]i increased slowly after a 2-min latency in all cells and remained elevated as long as 8-Br-cAMP was present (Fig. 7B). Similarly, exposure to forskolin, which increases cAMP levels in pineal cells (Vanacek et al. 1985), also resulted in a slow increase in [Ca2+]i levels (Fig. 7B; 3 separate trials, 36 cells). In two experiments, we showed that the forskolin-induced increase in [Ca2+]i was abolished when extracellular Ca2+ ions were removed (Fig. 7C). The figure also shows that the cell was responsive to metabotropic agonists after exposure to low Ca2+ (50 nM) solutions because a subsequent challenge with NE (1 µM) elicited a large [Ca2+]i signal.


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FIG. 7. Consequences of nonselective cation channels gating. A: PACAP-induced membrane depolarization was measured under current-clamp condition. Pineal cell was held in the perofrated-patch configuration and was perfused with PACAP (0.1 µM) in bath solution. Pipette contained pipette solution 1. PACAP caused a slow depolarization of ~10 mV that persisted as long as the agonist was present. B: intracellular Ca2+ increase caused by 8-Br-cAMP and forskolin in pineal cells. Rat pineal cells on cover slips were loaded with Fura-2 and under perfusion were challenged with 8-Br-cAMP (1 mM) or forskolin (100 µM). Cells were perfused with bath solution, and drugs were applied in the perfusion medium. Both agents caused a significant, slow increase in [Ca2+]i after a brief latency. C: cells were challenged with forskolin at low extracellular Ca2+ concentration (50 nM) (square ). Cells were perfused with bath solution containing no added Ca2+ and 1 mM EGTA. When [Ca2+]o was 50 nM, the forskolin-induced [Ca2+]i increase was abolished and subsequent challenge with NE (1 µM, black-square) in normal medium shows that the cell and the indicator are responsive.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

In this paper, we describe a novel nonselective cation channel found in rat pineal cells the gating of which is regulated by the two major neurotransmitters, NE and PACAP, that mediate circadian changes in melatonin secretion. This NSCC is voltage dependent, is of large conductance (170 pS), and is quiescent under resting membrane potentials. We show that NE, PACAP, and 8-Br-cAMP, all activate the channel to increase Po, cause reduction of unitary conductance of the outward current on prolonged stimulation, and change the rectification properties such that the channel now carries inward currents. Our results suggest that this channel is expressed at high density in rat pineal cells because we could record activity in 40% of the patches. From whole cell current measurements and single channel data, we calculate that between 10 and 20 channels are present per pineal cell.

Recently, a large conductance (211 pS) nonselective cation channel was described in cerebrovascular smooth muscle cells (Mathers and Zhang 1995), and another current activated by protein kinase C was reported in rabbit portal vein cells (Oike et al. 1993). The NSCC described here is different from these and other nonselective cation conductances previously identified in vertebrate pineal cells, which are cyclic nucleotide-gated cation channels of the type found in the retina (Dryer and Henderson 1991, 1993). Indirect evidence for the existence of a similar nucleotide gated NSCC in mammalian pineal cells was published recently from this laboratory (Schaad et al. 1995). The NSCC described here has a unitary conductance >20 pS, unlike the nucleotide-gated channels found in chicken pineal cells (Dryer and Henderson 1991, 1993) and is more permeable to K+ than Na+. Our results clearly indicate that the conductance measured here is a nonselective cation channel because both single channel measurements and whole cell (perforated patch) recordings showed that the permeability ratio, PK+:PNa+ was 2:1 based on measurement of reversal potentials. The permeability to Na+ and the fact that the channel is insensitive to Ca2+ show that the NSCC is distinct from the large conductance Ca2+-dependent potassium channel (BK channels) present in these cells (Cena et al. 1991). Calcium-activated potassium channels with unitary conductance of >100 pS were observed in ~10% of the patches (total 200 patches). During the preparation of this manuscript, a novel nonselective cation conductance activated by neurotensin, with an unitary conductance of 31 pS, was described in dopaminergic neurons (Chien et al. 1996), but the paucity of data in that study made direct comparison with the channel described here difficult.

Our data show that NE, PACAP, and 8-Br-cAMP increase channel Po by changing the voltage dependence such that channel activity could be measured even under hyperpolarized membrane potentials (Fig. 4A). The slope of the exponential fit to the voltage dependence curves were not significantly different before and after treatment with agonist, suggesting that there was no change in the intrinsic voltage sensitivity of the channel. In addition to the increase in channel Po, prolonged treatment results in a large decrease of unitary conductance of the outward current measured in single-channel and whole cell recordings. The molecular mechanisms that underlie this phenomenon remain largely unknown, and several possibilities exist. The fact that thereduction in current amplitude was measured in perforated-patch experiments under voltage clamp suggests that changes in the resting membrane potential, and thus the driving force for ion flow, are not responsible for this decrease. In addition, we measured PACAP-induced membrane depolarizations to be only 12 ± 2 mV in six separate trials, and this small change in membrane potential cannot account for the >50% change in the outward single-channel current amplitude nor the change in voltage dependence. Furthermore, the reduction in current amplitude after agonist application was observed even under voltage clamp. Receptor desensitization alone could not explain the change in conductance because single-channel experiments show that channel Po did not decrease with time and, in some instances, even after removal of the agonist. Another possibility is intracellular accumulation of a blocker with time after agonist exposure which causes a fast channel block (Hille 1992) and our experiments cannot rule out this possibility.

Prolonged receptor activation results also in appearance of inward currents through what appears to be the same channel. Such a transmitter-induced change in current rectification properties suggests modulation of the permeation properties of the channel which has not been described before. The following observations argue that the outward and inward currents are measured through the same channel complex. 1) The I-V relationships of the outward and inward currents measured through the NSCC could be fitted with a simple linear regression (r = >0.9), suggesting that the unitary conductance and the ion selectivity are identical under both circumstances (Fig. 4B). 2) The voltage sensitivity was not different between the outwardly directed openings and the inward currents (Fig. 4A). And 3) inward currents appeared in 13 of 21 trials. It could be argued that cAMP accumulation increases channel Po by shifting the voltage dependence such that channel gating could now be observed under hyperpolarized voltage commands. If the appearance of inward currents is due to a shift in Erev, the resting membrane potential has to change as much as 70-90 mV; this was never seen in our current-clamp experiments. There, however, was no correlation between the increase in channel Po and the appearance of inward current. In several experiments, even though channel Po increased dramatically, we did not observe any inward currents, whereas in others, small increases in channel Po were associated with appearance of inward currents. If a different dormant channel is present in the same patch carrying inward current, this second channel should have the same conductance, ion selectivity and voltage dependence and most of the time be expressed together with the outward channel on the membrane.

We interpret the data presented here to mean that NE and PACAP acting through their common second messenger, cAMP, cause a change in the rectification characteristics of the current through this NSCC. The fact that inward currents were never observed through this channel, in cell-attached patches, in the absence of stimulation and that single channel tail currents under symmetrical K+ and Na+ concentrations failed to demonstrate inward currents suggests that mechanisms other than voltage dependence or asymmetric ion concentration underlie this behavior. This result strongly argues for channel modulation by cAMP accumulation, which results in a change of the channel protein or related proteins enabling inward currents through the channel pore. The molecular mechanism responsible for this change is unclear. We found that in inside out patches both outward as well as inward currents could be observed. It is likely, that in these experiments an intracellular component that modulates channel gating and rectification was washed out on patch excision, resulting in appearance of inward currents. One hypothesis is that a cAMP-mediated change in the channel complex reduces the affinity of the channel to the intracellular component which then dissociates. Because Rp-cAMPs did not mimic the effects of cAMP, it is likely that a cAMP-dependent phosphorylation step is important for this change to occur. This novel mode of second-messenger-mediated channel modulation of an ion channel in the vertebrate to our knowledge has not been observed before.

Under resting membrane potentials (-35 to -50 mV) (Freschi and Parfitt 1986; Poling et al. 1995), the NSCC of rat pineal cells will remain closed most of the time. On noradrenergic or PACAP activation, however, the channel will open at these membrane potentials contributing to depolarization of the cell because under physiological conditions, Na+ is expected to be the major current carrier. Indeed, under current-clamp conditions, we found that PACAP induces a slow onset, long-lasting depolarization of ~12 mV, which increases Ca2+ influx across the plasma membrane (Fig. 7). Because pineal cells maintain a circadian rhythm in plasma levels of melatonin in rats, their activation is unique and lasts many hours each evening (4:00 P.M. to midnight) (Klein 1985). NE outflow from the superior cervical ganglion and PACAP act via cAMP and Ca2+ (Cena et al. 1991; Darvish et al. 1995a; Olcese et al. 1996; Simonneaux et al. 1993). Both second messengers are required for maximal activation of N-acetyl transferase activity to increase melatonin synthesis (Klein et al. 1992). Because Ca2+ release from the endoplasmic reticulum stores is finite (Berridge 1993), a long-lasting depolarization could provide the mechanism for refilling the Ca2+ stores by gating voltage-gated Ca2+ channels found in these cells (Chick et al. 1995; Harrison and Zatz 1989) and activation of NSCC by cAMP could support such a mechanism. Indeed, 8-Br-cAMP and stimulation of cAMP accumulation by forskolin treatment resulted in calcium influx in these cells (Fig. 7). In summary, we describe here a nonselective cation channel in rat pineal cells that is modulated by neurotransmitter receptors through a cAMP-dependent mechanism. This modulation results in a marked increase in channel gating activity, a shift in the voltage dependence followed by a change in its rectification properties.

    ACKNOWLEDGEMENTS

  We are indebted to Drs. Chris McBain and David Klein for discussions throughout the course of this work. We thank Dr. Daniel Dagan, Technion, Haifa, Israel; Dr. Mark L. Mayer and Dr. Chris McBain, National Institutes of Health, Bethesda, for painstakingly reading and commenting on the draft manuscript; and L. Hotzclaw for technical help during the development of the pineal cell culture.

    FOOTNOTES

  Address for reprint requests: J. T. Russell, Laboratory of Cellular and Molecular Neurophysiology, NICHD, NIH, Bldg. 49, Room 5A-78, Bethesda, MD 20892.

  Received 5 August 1997; accepted in final form 8 January 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society