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Introduction |
Postsynaptic
neuronal nicotinic acetylcholine receptors (nAChRs) mediate fast
excitatory synaptic transmission at synapses in the central (primarily
on interneurons) and peripheral nervous system (sympathetic and
parasympathetic ganglia). A second population of neuronal nAChRs is
localized to presynaptic terminals and can modulate release of a
variety of transmitters. Despite the widespread distribution and varied
synaptic localization of nAChRs in the nervous system, a degree of
functional and pharmacological specificity is achieved as a result of
the diversity of nAChR subtypes. Each subtype is composed of five
subunits arranged around a central ion channel. Two families of nAChRs
are likely present in the nervous system: 1) heteromeric receptors
consisting of 
2, 3, 4, and/or 6 subunits in combination
with 
2 and/or 4 subunits (other modulatory subunits such as 3
and 5 may also be present although not strictly required for
function), and 2) receptors capable of functioning as homomers
including 7-9. The 10 subunit has also been cloned recently
and appears to function only in combination with 9 (Elgoyhen et al.,
2000
).
Correlative evidence for the involvement of brain nAChRs in cognitive
dysfunction associated with neurodegenerative disorders such as
Alzheimer's disease, Parkinson's disease, and schizophrenia has
focused attention on brain nAChR subtypes as therapeutic targets (Kem,
2000; Leonard et al., 1996; Quik and Jeyarasasingam, 2000). The
homomeric 7 nAChR is widely expressed in the nervous system and has
received particular scrutiny because of its high calcium permeability
and consequent potential for activation of second-messenger systems.
The possible therapeutic application and experimental utility of drugs
directed to this receptor subtype has driven the development and
characterization of 7-selective agonists and antagonists (Mullen et
al., 2000; de Fiebre et al., 1995). We have previously shown that
cocaine inhibits heteromeric nAChR subtypes that contain the 4
and/or 4 subunits with high affinity (Francis et al., 2000). In the
present study, we report the selective activation of 7 nAChR by a
quaternary cocaine derivative, cocaine methiodide. Whereas cocaine
inhibits 7 receptors, addition of a methyl group to the amine moiety
of cocaine forms the quaternary compound cocaine methiodide, which
permits high-affinity interaction with the agonist binding site and
potent and specific activation of 7 receptors expressed in either
Xenopus laevis oocytes or transiently transfected PC12 cells.
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Materials and Methods |
Chemicals.
Cocaine and cocaine methiodide were provided by
the National Institute on Drug Abuse (Bethesda, MD). Reagents for cell
culture were purchased from Life Technologies (Rockville, MD). All
other chemicals were purchased from Sigma (St. Louis, MO).
Preparation of RNA and Oocyte Injection.
Rat nicotinic
receptor cDNA clones were provided by Drs. Steve Heinemann (Salk
Institute, La Jolla, CA) and Jim Boulter (UCLA, Los Angeles,
CA). The chick 7 nAChR subunit clone was provided by Dr. Marc
Ballivet (University of Geneva, Geneva, Switzerland) and the human 7
nAChR subunit clone (in the PMXT oocyte expression vector) was provided
Dr. Jon Lindstrom (University of Pennsylvania, Philadelphia, PA). After
linearization and purification of cloned cDNAs, RNA transcripts were
generated using the mMessage mMachine in vitro RNA transcription kit
(Ambion, Austin, TX). Resultant RNA transcripts were evaluated by UV
spectroscopy and agarose gel electrophoresis under denaturing
conditions (visualized with ethidium bromide). RNAs were diluted to a
concentration of 600 ng/µl and stored frozen in ribonuclease-free
water at 
80°C.
Ovarian lobes were surgically removed from anesthetized adult female
X. laevis and cut open to expose the oocytes. The
ovarian tissue was then treated with collagenase for about 2 h at
room temperature [1 mg/ml in oocyte saline solution (82.5 mM NaCl, 2.5 mM KCl, 1 mM NaH2PO4, 15 mM
HEPES, and 1 mM MgCl2, pH 7.4)]. Following
harvest, healthy stage 5 oocytes were isolated and injected with 50 nl
each of a mixture of the appropriate subunit RNAs. Sterile oocyte
storage medium (oocyte saline solution supplemented with 1.8 mM
CaCl2, 5 U/ml penicillin, 5 µg/ml streptomycin,
and 5% horse serum) was changed daily. Recordings were made 2 to 7 days after injection depending on the RNAs being tested.
Two-Electrode Voltage-Clamp Recording.
Two-electrode
voltage-clamp recordings were made at room temperature in oocyte saline
solution supplemented with 1.8 mM CaCl2 and 1 µM atropine as described previously (Francis et al., 2000). All
recordings were made using a Turbo Tec 01C amplifier (NPI Electronics,
Tamm, Germany) at a holding potential of 50 mV unless otherwise
noted. Recording electrodes were filled with 3 M of KCl and typically
had resistances in the range of 0.5 to 3 M
.
Oocyte recording solution was perfused at a rate of 5 ml/min through a
Lucite recording chamber via a large-bore pipette (1.5 mm diameter)
placed about 0.5 mm above the oocyte. Agonist and antagonist solutions
were applied by loading a loop near the terminus of one arm of the
perfusion line. Constant perfusion was maintained by switching to the
other arm of the perfusion line during loading of the drug loop.
Perfusion of oocyte saline solution from an independent reservoir at a
rate of 2 ml /min maintained bulk flow through the recording chamber at
all times. Based on the rise time of current responses of slowly
desensitizing receptor subtypes (e.g., skeletal muscle nAChR), solution
exchange time is estimated to be in the range of 500 to 800 ms under
these conditions (Vazquez and Oswald, 1999). Data were collected at a
sampling rate of 100 Hz on a Compaq personal computer using pClamp
5.5.1 (Axon Instruments, Foster City, CA) and filtered at 30 Hz using
the lowpass filter in the amplifier. From the time of each drug
application, 2 min of data were acquired.
Each experimental response was normalized to an initial control
response to agonist alone. A second control application of agonist
alone subsequent to the experimental application permitted assessment
of inhibition time course and receptor rundown. Each drug application
was separated by a wash period of approximately 3 min. Values for
EC50, Hill coefficient and
IC50 were estimated from curve fits to normalized
data using Kaleidagraph 3.08 (Abelbeck/Synergy Software, Reading, PA).
Data for receptor activation were plotted using a nonlinear,
least-squares fit of the Hill equation: Response = Imax [agonist]nH /
([agonist]nH + (EC50)nH).
IC50 was calculated with a nonlinear,
least-squares fit of the equation: Response = [IC50]nH /
([cocaine]nH + (IC50)nH).
Cell Culture and Binding Assays in Stably Transfected
GH4C1 Cells.
Both nontransfected
GH4C1 cells and
GH4C1 cells that had been
stably transfected with the rat 7 cDNA in the pcep4 vector were
generously provided by Dr. Maryka Quik (The Parkinson's Institute, Sunnyvale, CA). Cells were grown in F10 medium supplemented with 8%
fetal bovine serum (FBS), 50 U/ml penicillin, and 50 µg/ml streptomycin. For purposes of maintaining stable expression, hygromycin B (0.2 mg/ml) was periodically added to the culture medium. Treatment with this concentration of antibiotic was sufficient to kill
nontransfected cells within 3 to 4 days. Cells were incubated in a 95%
oxygen/5% carbon dioxide environment at 37°C.
Binding assays were performed on intact cells as previously described
by Quik et al. (1996). Briefly, cells were plated on 24-well plates and
grown to confluence. Immediately before the binding assay, cells were
washed with Dulbecco's modified Eagle's medium (DMEM) containing 3.7 mM NaHCO3 and 0.1% bovine serum albumin. Cells
were incubated with the drug of interest at the appropriate concentration at 37°C for 1 h.
125I--bungarotoxin was added to dishes to the
desired concentration and the cells were incubated at 37°C for 90 min. Binding was terminated by removal of the
125I--bungarotoxin and repeated washes with
the DMEM solution. After termination of binding, cells were solubilized
in 0.5 M NaOH and transferred to vials for gamma counting. Binding in
the presence of methyllycaconitine (MLA, 10 µM) was defined as
nonspecific binding. Experiments with nontransfected cells showed
125I--bungarotoxin binding that was not
significantly different from nonspecific binding. Nonspecific binding
typically represented 10 to 20% of total radioligand binding for 1 nM
125I--bungarotoxin (the concentration used for
displacement studies). In each experiment, data representing specific
binding for the displacement curves were normalized to maximal specific
binding. Values for Ki were calculated from
IC50 values for inhibition of
125I--bungarotoxin binding by the equation of
Cheng and Prusoff (1973): Ki = IC50 / [1 + ([bungarotoxin] /
Kd)].
Cell Culture and Whole-Cell Recording of Transiently Transfected
PC12 Cells.
Rat pheochromocytoma cells (PC12; obtained from the
laboratory of either Dr. Rick Cerione or Dr. George Hess, both at
Cornell University, Ithaca, NY) were maintained in DMEM supplemented
with 10% (v/v) horse serum and 5% (v/v) FBS,
100 U/ml sodium penicillin G, and 100 µg/ml streptomycin sulfate in a
95% oxygen/5% carbon dioxide environment at 37°C. For experiments
using HEK 293 cells, cells were cultured as above with the exception
that DMEM was supplemented with 10% FBS. Cells were passaged 24 to
48 h before transfection and plated onto 35-mm dishes. Upon
reaching 40 to 60% confluence, cells were cotransfected with the human
7 cDNA (provided by Dr. Roger Papke, University of Florida,
Gainesville, FL) in the PCI-neo (Promega, Madison, WI) mammalian
expression vector and a cDNA coding for green fluorescent protein (GFP)
in the pEGFP-N1 plasmid (CLONTECH Laboratories, Palo Alto, CA) using the Superfect (QIAGEN, Valencia, CA) transfection reagent according to
the manufacturer's recommendations. A total of 7 µg DNA was added to
each dish in a 4:3 ratio of 7 to GFP. Transfection efficiency was
evaluated by visual inspection under blue light (395 nm) 24 to 36 h after transfection using a TMD-EF epi-fluorescence attachment on a
Nikon Diaphot-TMD inverted microscope (Nikon, Melville, NY).
Current responses from transfected and nontransfected PC12 cells were
recorded using conventional whole-cell patch clamp recording methods.
Pipettes were pulled from borosilicate glass capillary tubes (World
Precision Instruments; Sarasota, FL) to a tip diameter of 2 to 3 µm
with resistances in the range of 2 to 3 M using a PP-83 pipette
puller (Narishige, Greenvale, NY). Recording pipettes were filled with
a solution containing 120 mM CsF, 10 mM CsCl, 10 mM EGTA, and 10 mM
HEPES, pH 7.4 with CsOH. Recordings were obtained using an Axopatch
200B amplifier (Axon Instruments) and filtered at a cutoff frequency of
5 kHz using the lowpass filter in the amplifier before being digitized
at a sampling frequency of 20 kHz. Data were acquired on a Gateway 2000 PC using pClamp 6 software (Axon Instruments). All recordings were
obtained at a holding potential of 80 mV 36 to 60 h after
transfection. Two hours before recording, cells were replated at a
lower density. Immediately before study, the culture medium was removed
and the cells were washed gently with extracellular recording buffer
(145 mM NaCl, 3 mM KCl, 2 mM CaCl2, 1 mM
MgCl2, 10 mM HEPES, and 5 mM glucose, pH 7.4).
Atropine (1 µM) was included in the recording buffer to inhibit
potential muscarinic receptor responses. After identification of
GFP-positive cells, the whole-cell configuration was obtained and cells
were lifted off the culture dish and placed <50 µm from the 150 µm
outflow of a u-tube application device (Udgaonkar and Hess,
1987). Solution exchange time was measured experimentally via two
independent methods (Fig. 6A). Open-tip measurements of voltage changes
in response to 500 mM CsCl indicated that a solution exchange time
(10-90%) of ~1 ms can be obtained. However, because open-tip
measurements reflect solution exchange at the tip of the pipette rather
than across the surface of a cell, this technique may underestimate
whole-cell solution exchange time. Therefore, we also measured solution
exchange by u-tube application of a saturating concentration of
noncompetitive inhibitor (1 mM cocaine) during the plateau phase of the
whole-cell response (to 100 µM ACh) of a slowly desensitizing nAChR
subtype expressed in HEK 293 cells (34, kindly provided by Dr.
Ken Kellar). Using this technique, solution exchange time
(10-90%) was estimated to be ~3 ms. We take these values to
represent lower and upper limits for solution exchange time because the
open-tip response does not reflect exchange across the entire cell and
solution exchange measured by the application of inhibitor may reflect a small binding component in addition to exchange. Apparent
desensitization time constants were calculated from exponential fits to
the rising and falling phase of the data traces employing
desensitization correction as described by Udgaonkar and Hess (1987).
Comparable values were obtained by fitting the falling phase of the
responses with a single exponential using Clampfit 8.0 (Axon Instruments).
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Results |
Effects of Cocaine and Cocaine Methiodide on 7 nAChRs Expressed
in X. laevis Oocytes.
In contrast to the relatively
high-affinity binding of cocaine to inhibitory sites on the 4 and
4 subunits described in previous work
(KD ~2 µM for 44 nAChRs; Francis
et al., 2000), cocaine exhibits weaker binding to an inhibitory site
associated with the 7 receptor. Whereas application of 1 mM cocaine
alone has no effect (n = 3, data not shown),
coapplication of 100 µM cocaine with 300 µM ACh produces
qualitatively similar levels of inhibition for the rat, human, and
chick isoforms of the receptor (Fig. 1A).
Moreover, analysis of the concentration dependence of inhibition for
rat and human 7 nAChR yields comparable IC50 values (55 ± 10 and 98 ± 19 µM, respectively); the rat
isoform exhibits slightly higher affinity (Fig. 1B). We have previously shown for other nAChR subtypes that a brief equilibration with cocaine
before agonist application increases inhibitory effects (Francis et
al., 2000). However, for 7 nAChRs, no significant effects of
pre-equilibration with inhibitor were observed. Cocaine inhibition of
7 receptors also exhibits voltage dependence (not shown), most
consistent with binding to a channel site (as was observed for 34
nAChRs).
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Fig. 1.
Cocaine inhibits the rat, human, and chick forms of
the 7 nAChR. A, responses of oocytes expressing 7 nAChRs to 300 µM ACh in the absence and presence (middle trace) of 100 µM
cocaine. ACh was applied for 15 s in each case and the bar above
the upper left trace shows the timing of application (in this figure
and all subsequent figures). Each response is separated by a wash
period of 3 min. B, concentration dependence of cocaine inhibition of
rat and human 7 nAChR. Data are normalized to an initial response to
300 µM ACh. Each data point represents the mean (± S.E.M.) of three
to six responses.
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The effects of the quaternary cocaine derivative cocaine methiodide
(Fig. 2A) on nAChR function were also
evaluated. Surprisingly, current responses of rat 7 nAChRs to
coapplication of 300 µM acetylcholine with 30 µM cocaine methiodide
were on average 157 ± 5% of responses to 300 µM acetylcholine
alone (Fig. 2B, top), suggesting either agonist activity or allosteric
potentiation by cocaine methiodide. Application of cocaine methiodide
alone also activates rapidly desensitizing responses in oocytes
expressing either the rat, human, or chick isoforms of the receptor
(Fig. 2B), demonstrating this compound to be an agonist for the 7
nAChR subtype. Curve fits of the Hill equation to cocaine methiodide concentration-response data for both the rat and human forms of 7
yielded Hill slopes <1 and EC50 values of
56 ± 12 and 47 ± 14 µM for rat and human 7 nAChRs , respectively (Fig. 2C). Inhibition by cocaine methiodide at high
concentration is apparent (Fig. 2C, data point at 3 mM) and probably
influences the Hill slope. Notably, however, inhibitory effects of
cocaine methiodide do not appear to limit the efficacy of this compound
relative to ACh. In addition, cocaine methiodide is approximately
6-fold more potent than acetylcholine (EC50 = 370 ± 34 and 281 ± 32 µM rat and human 7 nAChRs , respectively) for activation of 7 receptors. Accurate measurement of
Hill slope and EC50 values for 7 agonists has
previously been shown to be limited by the slow solution exchange time
of the oocyte system relative to desensitization of 7 responses (Papke and Thinschmidt, 1998). Therefore, the
EC50 values determined from our data may
underestimate agonist potency. Comparison of the relative potency of
cocaine methiodide versus ACh should be unaffected by this limitation.
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Fig. 2.
Cocaine methiodide activation of the rat, human, and
chick isoforms of 7 nAChR. A, structures of cocaine and cocaine
methiodide. B, top, response of oocytes expressing rat 7 nAChR to
300 µM ACh in the absence and presence (middle trace) of 30 µM
cocaine methiodide. Bottom, response of oocytes expressing either rat,
human, or chick 7 nAChR to either 300 µM ACh (left and right
traces) or 100 µM cocaine methiodide (middle trace). C, concentration
dependence of cocaine methiodide and ACh activation of rat and human
7 nAChR. For both compounds, data are normalized to an initial
response to 300 µM ACh and plotted relative to the maximum response
to ACh. Each data point represents the mean (± S.E.M.) of 3 to 11 responses.
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Cocaine Methiodide Displaces 125I--Bungarotoxin
Binding.
To confirm binding of cocaine methiodide in the vicinity
of the agonist binding site, we also examined displacement of
125I--bungarotoxin from a pituitary cell line
(GH4C1) stably transfected with the rat 7 cDNA (Quik et al., 1996). Consistent with previous reports, our experiments indicate that this cell line shows
dose-dependent binding of 125I--bungarotoxin
with a KD value of 0.7 ± 0.2 nM,
whereas nontransfected cells do not exhibit appreciable specific
binding (not shown). 125I--Bungarotoxin
binding can be displaced by MLA, another specific inhibitor of 7
nAChRs, with a Ki value of 2.3 ± 0.6 nM, consistent with expression of 7 nAChRs. Moreover, cocaine
methiodide displaces 125I--bungarotoxin
binding with a Ki value of 0.19 ± 0.02 µM, indicating that cocaine methiodide is a high-affinity ligand
for the agonist binding site of 7 nAChRs (Fig.
3). Interestingly, the displacement curve
exhibits a high degree of apparent positive cooperativity (nH = 2.6), possibly reflecting the fact
that homomeric 7 receptors may include as many as five agonist
binding sites (Palma et al., 1996; Rangwala et al., 1997). Nicotine and
ACh have been reported previously to inhibit
125I--bungarotoxin binding in these cells with
Ki values of 0.9 ± 0.1 and 6.2 ± 0.2 µM, respectively (Quik et al., 1996). In contrast to cocaine
methiodide, cocaine does not effectively compete with 125I--bungarotoxin binding at cocaine
concentrations ranging from 0.1 to 10 µM, indicating that the
presence of an additional methyl group at the amine moiety of cocaine
confers affinity for the agonist binding site.
125I--Bungarotoxin binding in the presence of
100 µM cocaine (the highest concentration tested) was 83 ± 15%
of control binding (not shown), suggesting that cocaine may have low
affinity for the 7 agonist binding site. However, as noted above,
application of cocaine alone (up to 1 mM) elicits no detectable
functional response from rat 7 receptors expressed in oocytes.
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Fig. 3.
Cocaine methiodide displacement of
125I--bungarotoxin binding. Data are normalized to
maximal control binding. Each data point represents the mean specific
binding of at least six wells from separate experiments. Binding in the
presence of 10 µM of MLA was defined as nonspecific.
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Effects of Cocaine Methiodide on Heteromeric nAChR Subtypes.
The ability of cocaine methiodide to activate other nAChR subtypes
expressed in X. laevis oocytes was also evaluated. Cocaine methiodide (10 µM-1 mM) was applied to oocytes expressing either the
42, 32, 34, or 11
subunit combinations
(Fig. 4). Cocaine methiodide elicits
<1% of the control response to ACh for each of these receptor types,
indicating that this drug is specific for the 7 receptor among
likely mammalian brain and neuromuscular junction nAChR subtypes.
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Fig. 4.
Cocaine methiodide lacks agonist properties at other
nAChR subunit combinations. Response of oocytes expressing either rat
34, 32, or 42 or mouse 11 nAChRs to
either ACh (30 µM for neuronal combinations or 5 µM for
neuromuscular junction nAChR; traces on left and right) or 100 µM
cocaine methiodide (middle trace).
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Because the concentration-response curve for cocaine methiodide
activation of 7 receptors shows features consistent with inhibition
by agonist at high concentration, we also tested whether cocaine
methiodide was an effective inhibitor of nAChR subtypes for which it
exhibited little (if any) agonist activity (Fig. 5). Coapplication of 30 µM cocaine
methiodide with 30 µM ACh to oocytes expressing either the 34
or 42 subunit combination produces similar levels of inhibition
for both receptor types (47 ± 10% and 50 ± 6%
respectively). In these simple coapplication experiments, the magnitude
of inhibition does not differ significantly between cocaine and cocaine
methiodide (we have reported previously that a short pre-equilibration
with cocaine results in increased inhibition for the 34 nAChR
subtype, Francis et al., 2000). These observations suggest that cocaine
methiodide (similar to cocaine) is a general nAChR antagonist for
heteromeric combinations. Moreover, given that cocaine is also a less
potent inhibitor of 7 nAChRs (Fig. 1), cocaine methiodide may
produce a form of 7 nAChR inhibition similar to that observed for
heteromeric nAChR subtypes in concert with the agonist effects
described above.
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Fig. 5.
Cocaine methiodide inhibits heteromeric nAChR
subtypes. Responses of oocytes expressing either rat 34 or
42 nAChRs to 30 µM ACh in the absence or presence (middle
trace) of 30 µM cocaine methiodide.
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Cocaine Methiodide Selectively Activates 7 nAChRs in Transiently
Transfected PC12 Cells.
Although oocyte expression and
two-electrode, voltage-clamp recording are powerful tools for
evaluating drug-receptor interactions, the time resolution of the
technique is limited by the requirement for perfusion of the entire
surface area of the oocyte. This requirement precludes meaningful
kinetic measurements for a rapidly desensitizing receptor such as 7.
Whereas other receptor subtypes have been amenable to efficient
heterologous expression in cultured cells, development of effective
cell culture expression systems for 7 has proven more difficult.
Although a few stably transfected cell lines have demonstrated
significant bungarotoxin binding (Puchazc et al., 1994; Quik et al.,
1996), most have not been amenable to patch clamp study (however, see
Gopalakrishnan et al., 1995). The difficulties with expression seem to
be a product of a functional requirement for the presence of
cell-specific factors for proper post-translational processing (Cooper
and Millar, 1997; Rakhilin et al., 1999; Sweileh et al., 2000) as well
as 7 sequence-specific factors impacting surface expression (Dineley
and Patrick, 2000). Consistent with other reports (Cooper and Millar,
1997; Rangwala et al., 1997), we have been unable to detect responses
to ACh in HEK 293 cells transiently transfected with human or rat 7 cDNA. In addition, we detect only small, inconsistent responses to ACh
in GH4C1 cells stably
transfected with rat 7 cDNA, although these cells do exhibit
significant bungarotoxin binding (Fig. 3). Because PC12 cells normally
express nAChRs and certain variants of this cell line have been shown
to express native as well as transfected 7 nAChRs (Blumenthal et
al., 1997; Cooper and Millar, 1997; Rangwala et al., 1997), we decided
to examine effects of cocaine methiodide on this cell line using
whole-cell recording. Our initial experiments evaluated native nAChR
expression in two separate PC12 cell line variants. Although both cell
populations gave functional responses to ACh (not shown), we chose the
variant with the lower level of intrinsic nAChR expression as the best system to evaluate effects of transfection with 7 cDNA.
Although most nontransfected (undifferentiated) PC12 cells exhibited
small but measurable peak responses (273 ± 80 pA) to application
of 1 mM ACh (69%, 18 of 26 cells tested; Fig.
6, A, top, and B), only a single response
to 300 µM cocaine methiodide was observed (each of the drug
concentrations tested should be saturating based upon the oocyte data
in Fig. 2C). Cells transfected with GFP alone also showed only small
ACh responses (132 ± 55 pA, in three of six cells tested) and a
single response to cocaine methiodide. In contrast, nearly all
GFP-positive PC12 cells cotransfected with human 7 cDNA exhibited
rapid current responses to ACh (92%, 24 of 26 cells tested, Fig. 6A,
center). The peak response amplitudes of nontransfected and transfected
PC12 cells for which both drugs were tested are summarized in Fig. 6B.
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Fig. 6.
Cocaine methiodide is a specific agonist for 7
nAChRs in transiently transfected PC12 cells. A, whole-cell responses
of nontransfected PC12 cells (top) and PC12 cells transiently
transfected with the human 7 cDNA (center and bottom) to either 1 mM
ACh or 300 µM cocaine methiodide. Solutions were applied by u-tube
with an exchange time (10-90%) of 1 to 3 ms. Solution exchange time
(10-90%) was measured experimentally by both open-tip recording of
voltage response to application of 500 mM CsCl (0.9 ms; bottom, dashed
line) and u-tube application of a saturating concentration of inhibitor
during the plateau phase of the whole-cell response of a slowly
desensitizing nAChR subtype (2.9 ms; thin black line). The baseline
noise apparent in the whole-cell solution exchange trace reflects
open-channel noise before application of inhibitor. We take these
measures to reflect the lower and upper limit of whole-cell solution
exchange time. The trace representing the open-tip response was
recorded using the same methods as the whole-cell recording and
therefore could be superimposed in the time domain. The trace
representing whole-cell solution exchange by application of inhibitor
was necessarily acquired using a different experimental protocol and
therefore was aligned according to the initial falling phase of the
open-tip response. Responses to cocaine methiodide were completely
inhibited by application of 100 nM MLA (lower right). B, scatterplot
summarizing the peak responses of all transfected and nontransfected
cells for which responses to both cocaine methiodide (300 µM) and ACh
(1 mM) were recorded. Because some current rundown was observed between
responses, transfected cells are classified according to the order of
drug application. Because no significant differences between cells
transfected with GFP alone and nontransfected cells were observed,
these two classes are grouped together in the figure. Cells which had
peak responses of >5 nA to cocaine methiodide or ACh were not
included.
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Of the transfected cells responding to ACh, 95% of cells tested also
had fast desensitizing responses to cocaine methiodide (Fig. 6A, 21 of
22 cells tested), consistent with expression of 7 nAChR. The ACh
responses of transfected cells could be divided qualitatively into two
classes: those that desensitized to a steady-state level (65% of cells
tested, example in Fig. 6A) and those that desensitized completely
during the time course of the application (35% of cells tested,
example in Fig. 7). In contrast, cocaine methiodide responses showed rapid and complete desensitization in all
cells tested. Moreover, currents elicited by cocaine methiodide were
inhibited completely by application of 100 nM MLA (Fig. 6A, bottom,
n = 4).
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Fig. 7.
ACh responses of some transfected cells desensitize
rapidly with kinetics similar to cocaine methiodide responses. A,
whole-cell responses to either 300 µM cocaine methiodide (top), 1 mM
ACh (center), or ACh in the presence of 100 nM MLA are shown (bottom).
B, the ACh (thin line) and cocaine methiodide (thicker line) responses
can be superimposed when scaled to the same peak amplitude (to correct
for the effects of receptor rundown). Although considerable
cell-to-cell variability was observed, rundown between consecutive
applications of different agonists was on average 50% (due to time
required for switching solutions, ~8 min).
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For both classes of cells, we observed an apparent response-rise time
to cocaine methiodide of ~1 ms and an apparent desensitization time
constant (
d) in the range of 1 to 2 ms. With
our application system, we observed solution exchange (10-90%) within
1 to 3 ms (Fig. 6A, bottom and legend). Thus, the peak of the response
to cocaine methiodide occurs before complete whole-cell solution exchange (Fig. 6A, bottom, thin black line), indicating that
desensitization probably occurs on a more rapid time scale than
whole-cell agonist application in this system. This effect impacts our
measurements in several ways. At the peak of the response, the maximal
agonist concentration is probably not achieved across the entire
surface area of the cell (Papke and Thinschmidt, 1998). In addition,
rapid desensitization relative to solution exchange limits our ability to make accurate and independent measures of the activation and desensitization rates. Therefore, the apparent activation and inactivation rates may be underestimates of the rates that would be
observed with a uniform step change in agonist concentration across the
entire surface area of the cell.
For cells that exhibited incomplete desensitization to ACh (Fig. 6,
middle trace), responses to cocaine methiodide desensitized more
rapidly than responses to ACh; this process could be fit by a single
exponential with an average d value of
1.4 ± 0.2 ms. For cells in which only a rapidly desensitizing
response component to ACh was evident, d of
the ACh response did not differ significantly from the same measure for
cocaine methiodide (Fig. 7) in either cell type, again consistent with
specific activation of 7 nAChRs by cocaine methiodide. Furthermore,
ACh responses that desensitized completely were also sensitive to MLA
(Fig. 7A). These data suggest that only 7-containing nAChRs are
expressed in significant numbers as functional receptors in the
subpopulation of cells exhibiting only a rapidly desensitizing response
to ACh.
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Discussion |
The present study describes the conversion of cocaine from an
antagonist of neuronal nAChR subunit combinations to a specific agonist
of the 7 nAChR by the addition of a methyl group, converting the
amine group from tertiary to quaternary. Cocaine methiodide inhibits
binding of 125I--bungarotoxin to rat 7
receptors (Fig. 3), whereas cocaine has no detectable effect on
125I--bungarotoxin binding at a concentration
10 µM. Cocaine methiodide activates the rat and human isoforms of
the 7 receptor with nearly 6-fold greater potency than acetylcholine
(Fig. 2) and is specific for the 7 subtype in both oocyte expression
studies (Fig. 4) and in studies of transiently transfected PC12 cells
(Fig. 6), indicating that cocaine derivatives could have importance as
subtype-selective agonists of the 7 receptor. Although cocaine
methiodide does seem to exhibit some inhibitory effects at a high
concentration, low affinity for an antagonist site (presumably the same
site to which cocaine binds) allows cocaine methiodide to have full efficacy (compared with ACh) at the 7 receptor. Finally, we show that transient transfection of 7 nAChRs into PC12 cells used in
combination with an 7-specific agonist provides a convenient and
effective method to study 7 nAChR function.
The desensitization time (1-2 ms) we report in our studies of PC12
cells transfected with 7 nAChR is faster than that reported previously for responses of human 7 nAChRs, stably expressed in HEK
293 cells, to nicotine (~12 ms, Delbono et al., 1997). A few studies
have reported longer d value in cultured
neurons as well (ranging from 8 to 27 ms; Zorumski et al., 1992;
Alkondon and Albuquerque, 1993; Zhang et al., 1994), but these
measures may reflect low-level activation of other nAChR subtypes in
addition to 7. The very rapid desensitization we observe in our
experiments does not seem to be caused by differences in application
rate as the open-tip solution exchange time in this study [~1 ms
(10-90%), Fig. 6A, bottom] is comparable with the faster solution
exchange times (as measured by open-tip recording) reported in previous studies. Moreover, using the same application system as described in
the present study, we observe kinetic properties for another rapidly
desensitizing receptor type, the homomeric glutamate receptor GluR6,
that are comparable with literature values for that receptor (d ~ 8 ms at saturating agonist
concentration; B. G. Kornreich and R. E. Oswald,
unpublished observations). Secondary inhibition by cocaine methiodide
could result in more rapid apparent desensitization. However, for cells
that exhibit only a completely desensitizing response component to ACh
(suggesting the presence of only 7-containing receptors), the
desensitization time of the ACh response does not differ significantly
from that of the response to cocaine methiodide. Because the choice of
expression system has been shown previously to influence single-channel
properties for other nAChR subtypes (Lewis et al., 1997), we cannot
rule out differences in desensitization as a function of host cell type.
PC12 cells have been shown previously to express 3, 5, 7,
2, and 4 nAChR subunits (Rogers et al., 1992; Henderson et al.,
1994; Fanger et al., 1995). We cannot exclude the possibility that
other nAChR subunits combine with 7 subunits to form heteromeric receptors in our transfected cells. However, a previous report has
concluded that native 7 nAChRs in PC12 cells are homomeric (Drisdel
and Green, 2000). In our cells, we observe rapidly desensitizing responses (compared with ACh responses in the same cell), to an 7-specific agonist, which are inhibited by MLA. If 7-containing heteromeric combinations are present, they are not distinguishable on
the basis of obvious differences in kinetics or MLA sensitivity in our experiments.
Previous studies of quaternary local anesthetic nAChR inhibitors have
indicated that quaternization affects the state dependence of
inhibition, in large part limiting inhibitory effects to the open-channel form of the receptor (Neher and Steinbach, 1978). In
contrast, quaternization of cocaine confers affinity for the agonist
binding site of the 7 nAChR. Although many quaternary amines exhibit
some degree of affinity for the acetylcholine binding site, the
efficacy and specificity of cocaine methiodide agonist effects for the
7 nAChR are novel. Because cocaine (pKa = 8.5) is largely protonated at physiological pH (>90%), the tertiary amine group effectively contains a partial positive charge.
Voltage-dependent inhibition by cocaine suggests that the charged
species is responsible for at least some of the nAChR inhibitory
effects observed upon coapplication with ACh in our experiments (Fig.
1). Given the fact that we do not observe any agonist effects with
application of even 1 mM cocaine, it seems unlikely that the presence
of an additional methyl group in cocaine methiodide confers high
affinity for the 7 acetylcholine binding site solely as a function
of charge. Other factors such as steric constraints and hydrogen bonding differences are also likely to be important.
The backbone ring structure of cocaine (Fig. 2A) does share certain
general features with other nAChR agonists such as (+)-anatoxin-A (Swanson et al., 1986) and epibatidine (Badio and Daly, 1994). However,
although both epibatidine and (+)-anatoxin-A are potent activators of
7 nAChRs, neither compound exhibits specificity for this subtype
over other nAChR subunit combinations. Among 7-selective agonists,
AR-R 17779 (Mullen et al., 2000) and GTS-21 (de Fiebre et al., 1995)
have been reported to show limited efficacy at heteromeric nAChR
subtypes while the activity of cocaine methiodide appears specific for
activation of 7 receptors. Moreover, despite the appearance of
inhibition at high concentrations of cocaine methiodide, the drug is
fully efficacious for 7 (compared with ACh), whereas the efficacy of
GTS-21 is more limited. The primary metabolite of GTS-21,
4-hydroxy-GTS-21 (Meyer et al., 1998), has been reported to show
increased efficacy at human 7 nAChR. Finally, the endogenous
compound choline has also been shown to be a specific agonist for the
7 nAChR (Mandelzys et al., 1995; Papke et al., 1996) but exhibits
much lower potency.
Cocaine methiodide is a quaternary amine, which makes direct clinical
application of this compound unlikely because of limited brain access.
However, if affinity for the 7 agonist binding site results from
purely steric constraints conferred by the presence of an additional
methyl group, cocaine derivatives mimicking this density may have
increased therapeutic potential. A second priority for this line of
development will certainly be the identification of analogs that retain
affinity for 7 nAChR but lack affinity for other sites of cocaine
action, such as monoamine transporters and voltage-gated sodium
channels. Quaternization of cocaine does reduce affinity for the
dopamine transporter (Abraham et al., 1992). The reported
Ki value for cocaine methiodide binding to the dopamine transporter is in the range of 8 µM at physiological pH
(Xu and Reith, 1996), roughly 40-fold lower affinity than was observed
for cocaine methiodide binding to the acetylcholine site of 7 nAChR
(Ki = 190 nM; Fig. 3) in this study.
Because functional effects on both the transporter and 7 require
higher drug concentrations than are typically measured in binding
studies, direct functional comparisons will ultimately be required.
Although the agonist effects of cocaine methiodide seem specific for
7 among nAChR subtypes, antagonist activity at other nAChR subtypes
and the potential for effects at other sites of cocaine action in the
nervous system remain to be addressed experimentally. Nonetheless,
cocaine methiodide is a promising lead compound for the development of
7-selective drugs. Furthermore, direct activation of 7 nAChRs by
agonists such as cocaine methiodide should prove to be a powerful tool
in functional studies that, to date, have, for the most part, relied on
sensitivity to specific inhibitors of 7 nAChRs to evaluate
7-mediated effects.
We thank Desiree Snyder for technical support, Dr. Maryka Quik
for providing GH4C1 cells
stably transfected with rat 7 nAChR, Drs. Ken Kellar and Yingxian
Xiao (Georgetown University, Washington, DC) for providing HEK 293 cells stably transfected with rat 34 nAChR, Dr. Jon Lindstrom for
providing the human 7 cDNA, and Dr. Roger Papke for providing the
human 7/pCI-neo construct.
This work was supported by National Institutes of Health Grant
DA11643 to R.E.O. and a postdoctoral fellowship from the National Institute on Drug Abuse to M.M.F.
Robert E. Oswald, Department of
Molecular Medicine, C3 167 Veterinary Medical Center, College of
Veterinary Medicine, Cornell University, Ithaca, NY 14853. E-mail:
reo1{at}cornell.edu
nAChR, nicotinic acetylcholine receptor;
FBS, fetal bovine serum;
GFP, green fluorescent protein;
MLA, methyllycaconitine;
HEK, human embryonic kidney;
DMEM, Dulbecco's
modified Eagle's medium.
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7 Nicotinic Acetylcholine Receptor
by a Quaternary Analog of Cocaine
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Abstract
Introduction
