Department of Neuroscience, Ohio State University, College of Medicine, Columbus, Ohio 43210-1239
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ABSTRACT |
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Bovine adrenocortical zona fasciculata (AZF) cells express a novel ATP-dependent K+-permeable channel (IAC). Whole cell and single-channel recordings were used to characterize IAC channels with respect to ionic selectivity, conductance, and modulation by nucleotides, inorganic phosphates, and angiotensin II (ANG II). In outside-out patch recordings, the activity of unitary IAC channels is enhanced by ATP in the patch pipette. These channels were K+ selective with no measurable Na+ or Ca2+ conductance. In symmetrical K+ solutions with physiological concentrations of divalent cations (M2+), IAC channels were outwardly rectifying with outward and inward chord conductances of 94.5 and 27.0 pS, respectively. In the absence of M2+, conductance was nearly ohmic. Hydrolysis-resistant nucleotides including AMP-PNP and NaUTP were more potent than MgATP as activators of whole cell IAC currents. Inorganic polytriphosphate (PPPi) dramatically enhanced IAC activity. In current-clamp recordings, nucleotides and PPPi produced resting potentials in AZF cells that correlated with their effectiveness in activating IAC. ANG II (10 nM) inhibited whole cell IAC currents when patch pipettes contained 5 mM MgATP but was ineffective in the presence of 5 mM NaUTP and 1 mM MgATP. Inhibition by ANG II was not reduced by selective kinase antagonists. These results demonstrate that IAC is a distinctive K+-selective channel whose activity is increased by nucleotide triphosphates and PPPi. Furthermore, they suggest a model for IAC gating that is controlled through a cycle of ATP binding and hydrolysis.
potassium channel; adenosine 5'-triphosphate; nucleotide; angiotensin II
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INTRODUCTION |
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BOVINE ADRENAL ZONA FASCICULATA (AZF) cells express a novel K+-permeable channel (IAC) that functions pivotally in the regulation of cortisol secretion. IAC channels may set the resting potential of AZF cells, while inhibition of these channels by ACTH and angiotensin II (ANG II) is coupled to membrane depolarization, Ca2+ entry, and cortisol secretion (15, 39, 40).
Whole cell patch-clamp studies have shown that IAC combines properties unique among ionic currents described thus far. Specifically, IAC is activated when the patch electrode contains ATP at millimolar concentrations (14), while it is inhibited by cAMP through an A-kinase-independent mechanism (16). IAC is also inhibited by antagonists of both cyclic nucleotide-gated (CNG) cation channels and K+ channel blockers (21).
Inhibition of IAC through multiple G protein-coupled receptors, including those activated by ACTH, ANG II, and multiple P1 and P2 nucleotide receptors, requires ATP hydrolysis (16, 39, 40, 47, 48). For at least one of these receptors and its associated second messenger (ACTH and cAMP), inhibition of IAC is independent of any known protein kinases (16).
Together, these results identify IAC as a novel channel combining properties of K+-selective and CNG cation channels. The overall sequence similarity between voltage-gated K+ channels and CNG cation channels suggests a common origin (26). Several such intermediate forms have been identified, including the ether-à-go-go (eag) family of K+ channels, which are modulated by cAMP. Some eag channels may also display Ca2+ permeability (11, 24).
A large family of K+ channels directly gated by ATP exists.
However, these inwardly rectifying K+ channels are
uniformly inhibited by ATP (5). IAC
channels are the first K+-permeable channel whose activity
depends on the presence of ATP in either hydrolyzable or
nonhydrolyzable forms. In this regard, our results are consistent with
a model in which IAC channel gating is coupled
to a cycle of ATP binding and hydrolysis with similarities to that
proposed for the cystic fibrosis transmembrane conductance regulator
(CFTR) Cl channel (7). In the proposed
scheme, IAC channel open probability is enhanced
upon ATP binding, while activation of G protein-coupled receptors
promotes channel closing subsequent to ATP hydrolysis.
In the present study, single-channel recording from outside-out patches was used to characterize these novel K+-permeable channels with respect to ionic selectivity, conductance, and rectification. In whole cell recordings, the modulation of IAC by nucleotides, inorganic phosphates, and ANG II was studied to determine whether IAC gating might be controlled through an ATP hydrolysis cycle.
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MATERIALS AND METHODS |
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Tissue culture media, antibiotics, fibronectin, and fetal bovine
serum were obtained from GIBCO (Grand Island, NY). Coverslips were from
Bellco Glass (Vineland, NJ). Enzymes, ANG II, MgATP, NaATP, NaUTP,
NaCTP, KATP, 5-adenylylimidodiphosphate (AMP-PNP, lithium salt),
guanosine 5'-O-(2-thiodiphosphate) (GDPS),
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA), the inorganic phosphates polytriphosphate (PPPi), pyrophosphate (PPi), Pi, and
phosphatidylinositol 4,5-bisphosphate (PIP2), and
staurosporine were obtained from Sigma Chemical (St. Louis, MO).
Penfluridol was purchased from Jansen Pharmaceuticals (Beerse,
Belgium). Calphostin C, AG-490, genistein, herbimycin, and PD-98059
were purchased from Calbiochem (San Diego, CA).
Isolation and culture of AZF cells. Bovine adrenal glands were obtained from steers (age range: 1-3 yr) within 60 min of slaughter at a local slaughterhouse. Fatty tissue was removed immediately, and the glands were transported to the laboratory in ice-cold PBS containing 0.2% dextrose. Isolated AZF cells were prepared as previously described (16). Cells were plated in 35-mm dishes containing 9-mm2 glass coverslips that had been treated with fibronectin (10 µg/ml) at 37°C for 30 min and then rinsed with warm, sterile PBS immediately before cells were added. DMEM/F-12 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and the antioxidants 1 µM tocopherol, 20 nM selenite, and 100 µM ascorbic acid was used. Dishes were maintained at 37°C in a humidified atmosphere of 95% air-5% CO2.
Patch-clamp experiments.
Patch-clamp recordings of K+ channel currents were made in
the whole cell and outside-out patch configuration. For whole cell recordings, the standard pipette solution consisted of 120 mM KCl, 2 mM
MgCl2, 1 mM CaCl2, 20 mM HEPES, 11 mM BAPTA,
200 µM GTP, and 5 mM MgATP, with pH buffered to 7.2 using KOH.
Titration of pH to 7.2 with KOH raised the total K+
concentration to 160 mM. Pipette solution of this composition yielded a
free Ca2+ concentration of 2.2 × 108 M,
as determined by the Bound and Determined software program (10). In many experiments, MgATP was replaced with other
nucleotides or an inorganic phosphate, as described in the text. The
external solution consisted of 140 mM NaCl, 5 mM KCl, 2 mM
CaCl2, 2 mM MgCl2, 10 mM HEPES, and 5 mM
glucose, with pH adjusted to 7.4 using NaOH.
Calculation of IAC channel activity. Because of uncertainty about the number of channels in any given patch (N) and the nonstationary character of IAC activity in outside-out patches, channel activity was expressed in terms of NPo rather than Po (open probability). NPo was calculated from the expression I = NPoî, where I is the measured mean current, N is the number of active channels in the patch, î is the single-channel current, and Po is the open probability for samples of 90-100 consecutive traces, each 400 ms in duration.
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RESULTS |
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ATP-dependent activity of unitary IAC current in outside-out patches. Previously, in whole cell recordings from AZF cells, we found that IAC K+ current is present initially but grows 10-fold or more to a stable amplitude over a period of minutes, provided that ATP is present in the recording pipette at millimolar concentrations (14, 39). In excised outside-out patch recordings, single IAC channels showed a similar time- and ATP-dependent increase in channel open probability.
In Fig. 1, unitary currents were recorded with a pipette containing 2 mM MgATP in response to voltage steps applied at 4-s intervals from a holding potential of
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K+ selectivity and conductance of IAC channels. In whole cell recordings, IAC appears as a noninactivating, weakly voltage-dependent, ATP-activated K+ current (14, 16, 39). Properties of unitary IAC current, including ionic selectivity, conductance, and rectification, in symmetrical K+ solutions have not been described.
To obtain a measure of the K+ selectivity and conductance of IAC channels, unitary IAC currents were recorded from outside-out patches at voltages ranging from
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Effect of M2+ on unitary conductance and rectification of IAC channels. The rectifying properties of some K+-selective channels are caused by the unidirectional block of K+ flux by M2+. In particular, the rectification of many inward rectifier K+ channels occurs because intracellular Mg2+ blocks the outward flow of K+ (37).
In outside-out patch recordings made in symmetrical K+ solutions, it was discovered that the presence of M2+ on either side of the membrane dramatically altered unidirectional K+ flow through these channels. Specifically, with standard external and pipette solutions containing Ca2+ and Mg2+ at approximately physiological concentrations (external: 2 mM Ca2+, 2 mM Mg2+; pipette: 22 nM Ca2+, 2 mM Mg2+), IAC channels were outwardly rectifying in symmetrical K+ with chord conductance of 94.5 pS measured between 0 and +80 mV, compared with 27 pS between 0 and
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Activation of IAC by poorly hydrolyzable
nucleotides.
Two types of K+ channels expressed by bovine AZF cells are
easily distinguished in whole cell recordings. In addition to the rapidly inactivating A-type K+ current
(IA), the noninactivating
IAC current grows continuously over a period of
minutes in whole cell recordings (14, 16, 39, 41). The
absence of time-dependent inactivation of IAC allows it to be isolated for measurement with the use of either of two
voltage-clamp protocols. When voltage steps of 300-ms duration are
applied from a holding potential of 80 mV to a test potential of +20
mV, IAC can be selectively measured near the end
of a step, at a point where IA has completely
inactivated (Fig. 5A,
left). With the second protocol, IAC
can be selectively activated by an identical voltage step after a 10-s
prepulse to
20 mV has fully inactivated IA
(Fig. 5A, middle).
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Activation of IAC by inorganic phosphates.
Inorganic phosphates inhibit ATPases by occupying sites on the enzyme
from which phosphate is released after ATP is cleaved, interrupting
further cycles of ATP hydrolysis (7, 22). Inorganic phosphates such as PPi and PPPi may promote the
opening of CFTR Cl channels via this mechanism (7,
22, 23).
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Activation of unitary IAC currents by NaUTP and
PPPi.
NaUTP and PPPi both activated IAC
channels in outside-out patches at the same concentrations that were
effective in whole cell recordings. Figure
9 shows representative recordings made with pipettes containing 1 mM NaUTP (Fig. 9A) or 2 mM
PPPi (Fig. 9B). As in outside-out patch
recordings made with MgATP, NPo typically increased with time in the presence of UTP and PPPi.
However, with UTP and PPPi, channel activity was unstable
and sometimes disappeared abruptly.
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Effect of nucleotides and PPPi on AZF cell membrane potential. IAC channels may set the resting membrane potential (Vm) of AZF cells. If so, then Vm should depend on the presence of nucleotides and polyphosphates that have been shown to activate these channels. We measured Vm of AZF cells in whole cell current-clamp recordings with patch electrodes containing standard saline supplemented with MgATP, PPPi, or NaUTP.
The membrane potential of AZF cells was strongly dependent on the presence of nucleotides or polyphosphates in the recording pipette and was well correlated with their potency as activators of IAC. With 1 mM MgATP in the recording pipette, average Vm reached
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Anionic phospholipid PIP2 does not activate IAC channels. The plasmalemmal phospholipid PIP2 activates a number of ATP-sensitive channels by altering their sensitivity to ATP or by direct interaction with the channels (8, 17, 29, 44). PIP2 failed to enhance the expression of IAC current in whole cell recordings from AZF cells. In these experiments, PIP2 was added to pipette solutions at several different concentrations along with 1 mM MgATP and 200 µM GTP. At PIP2 concentrations of 5, 10, and 50 µM, IAC reached maximum current densities of 9.2 ± 1.4 (n = 4), 13.6 ± 2.6 (n = 8), and 9.4 ± 2.0 pA/pF (n = 2), respectively, values not significantly different from the control value of 13.0 ± 1.0 pA/pF (n = 24) obtained in the absence of PIP2. The addition of 10 µM PIP2 to the patch electrode in addition to 5 mM MgATP also failed to alter the inhibition of IAC current by ANG II (10 nM) (data not shown).
ANG II-mediated inhibition of IAC and ATP hydrolysis. The activity of IAC K+ channels is promoted by the binding of hydrolyzable and poorly hydrolyzable nucleotide triphosphates. However, inhibition of IAC through activation of a number of G protein-coupled receptors, including ANG II receptors, requires the presence of hydrolyzable ATP (16, 39, 40, 48). These results are consistent with a model in which IAC opening and closing are controlled through an ATP hydrolysis cycle involving ATP binding and metabolism by an ATPase. However, the requirement for hydrolyzable ATP might implicate a protein kinase rather than an ATPase in IAC inhibition.
Experiments were done to determine whether ATP-dependent inhibition of IAC by ANG II was mediated through a mechanism requiring an ATPase or, alternatively, a protein kinase. The inhibition of IAC by ANG II was studied by using pipette solution containing 5 mM NaUTP and various concentrations of MgATP. Although UTP is more potent than ATP as an activator of IAC channels, it is a poor substrate for phosphate transfer enzymes, including protein kinases and ATPases (6, 33). ATP, on the other hand, is a substrate for both protein kinases and ATPases, although typically at different concentrations. Protein kinases are fully activated by 50 µM ATP, whereas cellular ATPases frequently display significantly higher Michaelis-Menten constant (Km) values for ATP (18, 34). As previously reported, ANG II effectively inhibits IAC when the pipette solution contains MgATP (5 mM) and GTP (200 µM) as the only nucleotides. Under these conditions, ANG II reduced IAC by 82 ± 5% (n = 6) (Fig. 10, A and B). When NaUTP replaced ATP in the pipette, ANG II was much less effective, inhibiting IAC by only 10 ± 5% (n = 8). The addition of 50 µM or even 1 mM MgATP to pipette solution containing 5 mM NaUTP failed to restore IAC inhibition by ANG II. Under these conditions, ANG II inhibited IAC by only 14 ± 4% (n = 3) and 14 ± 7% (n = 3), respectively (Fig. 10, A and B). Raising intracellular MgATP to 2 mM in the presence of 5 mM NaUTP partially restored IAC inhibition by ANG II to 57.6% (n = 5).
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DISCUSSION |
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We have described the properties and regulation of a unique ATP-activated ion channel in bovine AZF cells. Unitary IAC channels were shown to be K+ selective with no measurable Na+ or Ca2+ conductance. IAC channels were outwardly rectifying in the presence of physiological concentrations of M2+, but conductance became almost ohmic in the absence of M2+ on either side of the membrane.
The potent activation of IAC channels by poorly hydrolyzable nucleotides and PPPi and the failure of ANG II to inhibit these channels when these agents replace ATP in the pipette solution are consistent with a model for IAC gating coupled to an ATP hydrolysis cycle. Overall, these results identify IAC as a distinctive new type of K+ channel with properties that would allow it to couple hormonal and metabolic signals to membrane potential and cortisol production.
Nucleotide-dependent activation of IAC channels in membrane patches. Nucleotide-dependent activation of unitary IAC channels in outside-out patch recordings resembled, in time course and concentration dependence, previous results obtained in whole cell recordings (14). Specifically, IAC channels were active in outside-out patches only when ATP or UTP were present in the patch pipette at millimolar concentrations. These results are consistent with the idea that nucleotide-dependent activation of these channels is independent of cytoplasmic proteins that are absent in outside-out patches and of protein kinases for which UTP is a poor substrate.
The failure of MgATP to activate IAC channels in excised inside-out patches suggests that key regulatory factors or associated membrane proteins are lost during this form of patch excision. This result is not surprising in view of the rich combination of metabolic factors that control the activity of this K+ channel (14, 16, 19, 39). In particular, because the molecular identity of IAC channels has not been determined, we do not know whether nucleotide binding sites are located on the pore-forming proteins or an associated subunit. The inability to directly activate IAC K+ channels in inside-out patches limits studies exploring the modulation of these channels. For example, we have been unable to describe the temporal pattern and reversibility of ATP-mediated IAC activation.K+ selectivity, conductance, and rectification of IAC channels. Although the molecular structure of IAC and its relationship to other channels is unknown, the results of the present study identify it as a true K+-selective channel with no measurable conductance to Na+ or Ca2+. In symmetrical K+ solutions, unitary IAC currents reversed at potentials slightly positive to 0 mV. This probably occurred through a reduction in pipette K+ activity through binding to ATP or BAPTA.
The identification of IAC as an authentic K+-selective channel is significant in view of the aforementioned similarities to CNG cation channels with respect to pharmacology and gating by cAMP (16, 20, 43). The lack of measurable Na+ and Ca2+ conductance clearly distinguishes IAC from CNG cation channels and Drosophila eag K+ channels (11). IAC K+ channels may represent a new intermediate form in a continuum linking true K+-selective and CNG cation channels.M2+ and rectification. Inwardly rectifying ATP-sensitive K+ (KATP) channels comprise a major class of K+-selective channels that include two rather than six membrane-spanning regions. Although KATP channels resemble IAC channels with respect to gating by ATP, KATP channels are uniformly inhibited, rather than activated, by the nonhydrolytic binding of ATP (5). In addition, KATP channels are sensitive to sulfonylurea agonists and antagonists, whereas IAC channels are not (1, 5, 20).
In the present study, a third fundamental difference between IAC K+ channels and KATP channels was identified. Specifically, in symmetrical solutions, KATP channels are inwardly rectifying due to the unidirectional block of K+ efflux by Mg2+ at physiological concentrations (5, 28). By comparison, in the presence of physiological concentrations of internal and external M2+ and symmetrical K+, IAC channels are outwardly rectifying. Removal of intracellular Mg2+ showed that outflow of K+ is only weakly blocked by this divalent cation, an effect that is more than matched by block of K+ influx by Ca2+ and Mg2+. Accordingly, when these two M2+ are deleted from external and pipette solutions, IAC channels conduct K+ almost equally well in either direction. The relative contributions of Ca2+ and Mg2+ to block of K+ influx were not determined in our experiments. K+ efflux through IAC channels is dramatically inhibited by intracellular Ca2+ at a concentration of only 2 µM (19). However, this effect appears to occur through an action on IAC gating rather than permeation. Overall, although they are both ATP-gated and K+-selective channels, IAC and KATP channels differ in several fundamental aspects. It is unlikely that they belong to the same family of K+ channels.Activation of IAC channels by nucleotides and inorganic
phosphates.
At concentrations 1 mM, nucleotides including AMP-PNP, NaUTP, NaCTP,
and NaTTP were much more effective than MgATP as activators of
IAC. In a model for IAC
gating in which nucleotide binding leads to channel opening and
nucleotide hydrolysis or dissociation allows the channel to close,
nonhydrolyzable nucleotides could be more potent through a reduction in
the effective "off rate," lowering the dissociation
constant. Accordingly, NaUTP was at least 10-fold more potent
than MgATP as an activator of IAC.
Nucleotides and membrane potential. Good correlation exists between the activation of IAC K+ channels by nucleotides and polyphosphates and the magnitude of the Vm installed by these same agents in AZF cells. This result provides evidence that IAC channels are primarily responsible for setting Vm. Furthermore, single-channel recordings showed that IAC channels remain active at very negative membrane potentials, as expected for a channel that sets Vm near the K+ equilibrium potential.
A direct relationship exists among nucleotide triphosphate concentration, IAC activity, and membrane potential. This suggests a specific mechanism whereby membrane potential, Ca2+ entry, and cortisol secretion could be linked to the metabolic state of the cell and, therefore, to other variables such as blood glucose concentration. Cortisol is a glucose counterregulatory hormone that acts in opposition to insulin in maintaining blood glucose levels (9).Mechanism for ANG II-mediated inhibition of IAC. The failure of ANG II to inhibit IAC when NaUTP or PPPi replaced ATP in the pipette is consistent with our previous observation that ANG II was ineffective in the presence of the nonhydrolyzable ATP analog AMP-PNP (40). However, the requirement for ATP hydrolysis could signal the involvement of either an ATPase or a protein kinase. The failure of the addition of 0.05 or 1 mM MgATP to the pipette (in addition to 5 mM UTP) to restore inhibition by ANG II argues for the involvement of an ATPase rather than a kinase in this response. Nearly all kinases are fully activated by the substrate MgATP at concentrations of 50 µM, whereas cellular ATPases have higher Km values for ATP (18, 27, 34). Thus, if ANG II-mediated inhibition of IAC occurred through activation of a protein kinase, low concentrations of ATP should have been sufficient to restore this effect.
Results of experiments with multiple protein kinase antagonists support the conclusion that no protein kinase known to be activated by ANG II mediates inhibition of IAC. In various cells, including those of the adrenal cortex, ANG II acts through AT1 receptors to activate protein kinase C, tyrosine kinases, JAK/STAT (signal transducers and activators of transcription) kinases, and MAP kinases (4, 36, 38, 46, 49). The failure of specific antagonists of each of these kinases to attenuate IAC inhibition by ANG II indicates that none of these is involved in this response. The nonselective protein kinase antagonist staurosporine (1 µM) reduced the inhibition of IAC by ANG II from 82% to only 62%. At this concentration, staurosporine completely inhibits a wide range of protein kinases, including serine/threonine kinases, and tyrosine kinases (45). It is possible that an unidentified staurosporine-sensitive protein kinase contributes to IAC inhibition by ANG II.Identity and function of IAC K+ channels. Although the results of this study identify IAC as a true K+-selective ion channel, its molecular structure and relationship to other K+ channel gene families is unknown. Its weak voltage dependence, insensitivity to sulfonylureas and PIP2, and lack of inward rectification suggest that it does not belong to the six-membrane-spanning, voltage-gated channels or to the two-membrane-spanning inward rectifiers. In this regard, a new family of K+-selective channels with two pore domains in tandem has been identified in organisms ranging from yeast to humans (31, 32). A number of these noninactivating, outwardly rectifying channels display properties similar to those of IAC.
Regardless, the convergent inhibition of IAC by multiple G protein-coupled receptors through second messengers, including Ca2+ and cAMP, and the activation of these K+ channels by ATP at physiological concentrations indicate that IAC is a central control point where hormonal and metabolic signals are transduced to electrical events involved in cortisol secretion. In this scheme, the control of IAC K+ channel activity through a cycle of ATP binding and hydrolysis may be a fundamental mechanism linking biochemical signals to AZF cell membrane potential. In this regard, ATP-activated IAC K+ channels provide an interesting contrast to KATP channels of insulin-secreting cells, which are inhibited, rather than activated, by ATP. The opposing actions of ATP on the activity of these two metabolic sensors are consistent with their function in regulating insulin and cortisol secretion. In pancreatic ![]() |
ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-47875 (to J. J. Enyeart).
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. J. Enyeart, Dept. of Neuroscience, The Ohio State Univ., College of Medicine, 5190 Graves Hall, 333 W. 10th Ave., Columbus, OH 43210-1239 (E-mail: enyeart.1{at}osu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 13 April 2000; accepted in final form 28 August 2000.
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