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Vol. 63, Issue 2, 419-428, February 2003
7-Containing Nicotinic Acetylcholine Receptors
by Select Albumins
Neurobiology Section, Division of Biological Sciences, University of California, San Diego, La Jolla, California (W.G.C., Q.-S.L., D.K.B.); and Department of Anatomy and Neurobiology; Medical College of Ohio; Toledo, Ohio (Q.N., J.F.M.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Nicotinic receptors containing 
7 subunits are ligand-gated ion
channels widely distributed in the nervous system; they influence a
diverse array of events because of their high relative calcium permeability. We show here that nicotine-induced whole-cell responses generated by such receptors can be dramatically potentiated in a
rapidly reversible manner by some but not all albumins. The potentiation involves increases both in potency and efficacy with no
obvious differences in rise and fall times of the response. The
potentiation is not reduced by removing absorbed components; it is
abolished by proteolysis, suggesting that the albumin protein backbone
is essential. The fact that some albumins are ineffective indicates
that minor differences in amino acid sequence may be critical.
Experiments with open channel blockers indicate that the potentiation
involves increased responses from active receptors rather than
recruitment of receptors from a previously silent pool. Single channel
recordings reveal that the potentiation correlates with increased
single channel opening probability, reflected in increased frequency of
channel opening and increased mean channel open time. The potentiation
can be exploited to overcome blockade by noncompetitive inhibitors such
as 
-amyloid peptide. The results raise the possibility that
endogenous compounds use the site to modulate receptor function in
vivo, and suggest that the receptors may represent useful targets for
therapeutic intervention in cases where they have been implicated in
neuropathologies such as Alzheimer's disease.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The
ability to modulate the function of ionotropic receptors in situ offers
a potentially powerful method for therapeutic intervention. By altering
the response of a receptor to endogenous agonists, the modulator can
change the level of signaling without necessarily changing the temporal
or spatial pattern of the signaling. One of the most successful
examples of therapeutic intervention based on this strategy is the use
of benzodiazepines to enhance the response of
GABAA receptors and augment inhibitory activity in the central nervous system (Macdonald and Olsen, 1994
).
Nicotinic acetylcholine receptors (nAChRs) are widely expressed in the
nervous system and have been implicated in a variety of behaviors and
neuropathologies. One of the most abundant is a species composed of
7 subunits (7-nAChRs) that has a high relative permeability to
calcium, exceeding that of NMDA receptors. Because of this and because
of their diverse locations, 7-nAChRs can influence a wide range of
cellular functions, including enhancement of transmitter release
(McGehee et al., 1995; Gray et al., 1996), generation of synaptic
currents, activation of second messenger cascades, and regulation of
neurite extension, apoptosis, and neuron survival (for reviews, see
Broide and Leslie, 1999; Margiotta and Pugh, 2003). Recent evidence
suggests that 7-nAChRs are also involved in cognitive events (Levin
et al., 1999) and may be specifically blocked in Alzheimer's disease
(Liu et al., 2001; Pettit et al., 2001).
Most therapeutic strategies targeting neuronal nAChRs have made use of
compounds designed to act as agonists or antagonists. Examples include
compounds to treat nicotine addiction, generate nociception, and
ameliorate Alzheimer's disease (for reviews, see Francis et al., 1999;
Dani et al., 2001). Modulation of receptor function may offer an
alternative strategy, however, as suggested by the finding that plant
alkaloids can potentiate the response of 4/2-containing nAChRs
(Schrattenholz et al., 1996). Evidence that 7-nAChRs may also be
subject to potentiation comes from reports that the antihelmintic
ivermectin (Krause et al., 1998), the neuropeptide PACAP (Pardi and
Margiotta, 1999), and the hormone prostaglandin
E2 (Du and Role, 2001) can each increase the
whole-cell response generated by the receptors.
We now report a dramatic potentiation of 7-nAChR responses by
specific albumins. The effect seems to depend on the peptide sequence
of the albumin rather than on an absorbed component, and it includes
both an increase in the affinity of the receptor for agonist and an
increase in the maximum response the receptors generate. The
potentiation is mediated by an increase in channel open time rather
than recruitment of previously inactive receptors. The results suggest
that it may be possible to exploit existing sites on 7-nAChRs to
increase substantially the response they produce in vivo.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cell Cultures.
Chick cultures were prepared from embryonic
day 8 (E8) ciliary ganglion neurons and maintained for 4 to 7 days as
described previously (Nishi and Berg, 1981) before analysis. Freshly
dissociated ciliary ganglion neurons were obtained from E13 to 14 embryos and were examined 1 to 5 h later as described previously
(Liu and Berg, 1999b). Rat hippocampal cultures were prepared by
dissociating E18/19 hippocampi and maintaining the cells in culture for
8 to 18 days as described previously (Liu et al., 2001).
Electrophysiology.
All experiments were performed at room
temperature (20-23°C). Both perforated-patch and conventional
whole-cell patch-clamp recordings were performed as described
previously (Liu and Berg, 1999b, and references therein). The
composition of the bathing solution and of the pipette solutions for
perforated-patch and conventional patch-clamp recordings was as
described previously (Liu and Berg, 1999b). Agonist (nicotine) with and
without the indicated albumins was rapidly applied using a multibarrel
rapid-exchange system that achieved fluid exchange in 
3 ms (Zhang et
al., 1994). A relatively large recording electrode was used (1-1.5
M
resistance with 80% series resistance compensation). The rapid
application and low-resistance recording were required to observe the
full extent of potentiation, given the rapid desensitization of the response. When compounds were to be applied for several minutes or more
[e.g., albumins for several minutes, -bungarotoxin (Bgt) for an
hour], they were also added to the bath for the indicated times.
). Briefly,
E14 ciliary ganglion neurons were dissociated and bathed in a standard
recording solution (RS), containing 145.0 mM NaCl, 5.3 mM KCl, 5.4 mM
CaCl2, 0.8 mM MgSO4, 5.6 mM
glucose, and 5.0 mM HEPES acid pH 7.4, or in RS containing 75 µM
bovine serum albumin (BSA) and termed RS+BSA.
Outside-out membrane patches were excised from the neurons, held at
100 mV, and exposed to 0.5 µM nicotine dissolved in RS or
RS+BSA by gentle pressure microperfusion (2-5
psi). The resulting currents were digitized at 50 kHz and filtered to a
final cutoff frequency (fc) of 6.8 kHz
(3 dB), allowing resolution of channel openings with durations >98
µs [2 times the filter rise time
(tr = 0.3321/fc); Colquhoun and Sigworth,
1995]. Single nAChR channel events were detected by visual inspection
of transitions from the baseline current and manually selected such
that only those events exceeding preset thresholds for amplitude (2 times noise) and duration (98 µs) were subsequently analyzed. For
each recording, single channel currents were first compiled into
amplitude histograms (0.2-pA bins) and fit to multiple Gaussian
functions using a Simplex least-squares algorithm that minimizes the
difference between actual and fit values (pSTAT; Axon Instruments,
Foster City, CA). The sum of four Gaussian functions best fit the
amplitude distributions, as determined by eye, and in seven cases, by
comparing F statistic values obtained from fits conducted with four
versus five or three functions. In each of these cases, the fits using
four Gaussian components were significantly improved over those using
three components (p < 0.005) and not significantly
different from those using five components (p > 0.1).
Because the individual functions overlapped somewhat, the range of each
current class was determined by calculating the critical amplitudes
(Ac) between peaks in the distribution
that minimized misclassified events from adjacent ranges, as described
previously (Colquhoun and Sigworth, 1995).
To analyze BSA effects on nAChRs, the single channel conductance,
steady-state open probability, open frequency, and open duration were
compared for each current class, x. Single channel conductances (
x) were determined using
reversal potential values determined previously from patches where full
current-voltage plots were obtained (McNerney et al., 2000). The
steady-state opening probability for each class
(Popen,x) was assessed using
%Popen,x = 100[
t,x/(TLx)],
where t,x was obtained from the
summed open durations of channel class x. T
represents the total record time (ca. 40 s in all cases), and
Lx represents the number of channels of
that current class in the patch. Lx was
estimated from the number of x value current levels
visualized in a record and was usually 1 or 2. This method provides an
upper limit estimate of
%Popen,x because
Lx is likely to be an underestimate of the
actual number of channels in the patch (Colquhoun and Hawkes, 1995).
The open frequency for each class
(Fopen,x) was determined using Fopen,x = Nx/(TLx),
where Nx represents the total number of
events in that conductance class. Average channel open times (Topen,x) were estimated from
the average durations of events in each class using
Topen,x = (t,x/Nx) tmin where
tmin is the minimum resolvable interval
(100 µs). In patches where Nx exceeded
50, logarithmically binned histograms were constructed for the open
durations of accepted events in each conductance class (Sigworth and
Sine, 1987; Colquhoun and Sigworth, 1995) and fitted by maximum
likelihood methods using Intrv5 [Interval Analysis 3.12 (1994),
generously provided by Dr. Barry S. Pallotta, University of North
Carolina, Chapel Hill, NC]. Such histograms revealed heterogeneous
open duration distributions for the 25- and 40-pS events requiring up
to three time constants [brief
(
x,b 0.20 ms), intermediate
(0.20 < x,i 1.0 ms), and
long (x,l > 1.0 ms)] to optimize
the fits. Because a detailed kinetic analysis was not possible for each
patch, however, and long-duration events were relatively rare,
%Popen,x,
Fopen,x, and Topen,x were calculated using an
arbitrary cutoff of 200 µs to separate brief from longer 25- and
40-pS events. All kinetic parameters are expressed as mean ± S.E.M., and the statistical significance (p < 0.05) of
comparisons determined using Student's unpaired, two-tailed
t test, conducted assuming equal or unequal variances where applicable.
Biochemical Procedures. BSA was extracted with the organic solvents chloroform, methanol, or acetonitrile. BSA (1 g) was suspended in 50 ml of solvent, shaken vigorously for 2 h, centrifuged to collect the precipitate, and dried by a stream of nitrogen. The BSA was then resuspended in recording solution and tested. Dialysis of denatured BSA was done by dissolving BSA in 8 M urea to yield 10 mg/ml, adjusting the pH to 8.0 with sodium hydroxide, and then adding iodoacetic acid to a final concentration of 20 mM and incubating the solution at room temperature for 30 min to alkylate-free sulfhydryls. The BSA solution was then dialyzed against 500 ml of 8 M urea for 15 h followed by dialysis against 4 × 2 liters of recording solution for 24 h and then tested for activity. BSA at 10 mg/ml in recording solution was also treated with 10 mM dithiothreitol for 30 min at room temperature, alkylated with 20 mM iodoacetic acid, dialyzed for 2 h against recording solution, and then tested.
Protease treatments of BSA were conducted by reducing and alkylating BSA disulfides with dithiothreitol and iodoacetic acid as described above, and then incubating with 0.25 ml of-chymotrypsin-agarose or
N-tosyl-L-phenylalanine chloromethyl
ketone-trypsin-agarose for 15 h at 37°C with shaking. The
proteases were removed by centrifugation, and the BSA solutions were
diluted with recording solution and tested. Reduction and alkylation
were required to fully inactivate the BSA activity by the proteases.
Pepsin digestion of BSA was conducted by dissolving BSA in 1% acetic
acid to yield 10 mg/ml, adding 0.2 ml of pepsin-agarose, incubating
15 h at 37°C with shaking, removing the pepsin-agarose by
centrifugation, lyophilizing the peptide solution, and resuspending in
recording solution for testing. Controls for the pepsin digestion
included either omission of the pepsin-agarose or conducting the
experiment as described but adjusting the pH of the BSA solution to 7.0 before adding the pepsin-agarose.
Limited pepsin digestion of BSA and purification of peptide fragments
were conducted as described previously (Feldhoff and Peters, 1975). The
carboxyl terminal (amino acids 308-583) and amino terminal (amino
acids 1-307) halves of BSA obtained from a 20-min pepsin digestion in
the presence of octanoic acid were purified by a combination of ion
exchange and size exclusion chromatography. Additional smaller peptides
were purified by size-exclusion and reverse-phase chromatography. These
included peptides corresponding to amino acids 308 to 386 and 506 to
583. Peptide identities were confirmed by mass determinations by
electrospray ionization mass spectrometry.
Materials.
White Leghorn chick embryos were obtained locally
and maintained at 37°C in a humidified incubator. Bgt was
purchased from Biotoxins (St. Cloud, FL). -Amyloid
peptide1-42 (A) was obtained from Calbiochem
(La Jolla, CA) and prepared as described previously (Liu et al., 2001).
N-tosyl-L-phenylalanine chloromethyl ketone-trypsin-agarose and pepsin agarose were purchased from Pierce
Chemical (Rockford, IL). All other drugs, including ivermectin, BSA,
other albumins, and -chymotrypsin-agarose were purchased from
Sigma-Aldrich (St. Louis, MO). GenBank accession numbers for albumin
sequences analyzed were as follows: bovine, ABBOS; horse, ABHOS; sheep,
ABSHS; dog, CAA76841; rabbit, P49065; cat, JC4660; pig, P08835; human,
ABHUS; rat, P02770; mouse, CAA09617; and chicken, ABCHS.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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BSA-Mediated Enhancement of 7-nAChR Responses.
Typically
7-nAChRs produce rapidly activating and rapidly desensitizing
currents (Zorumski et al., 1992; Alkondon and Albuquerque, 1993; Zhang
et al., 1994), although some variants can produce a slowly
desensitizing response (Cuevas and Berg, 1998; Yu and Role, 1998).
Diagnostically, 7-nAChR responses can be blocked by nanomolar
concentrations of either Bgt (Couturier et al., 1990) or
methyllycaconitine (Alkondon et al., 1992).
7-nAChRs as measured with
125I-Bgt binding and exhibit spontaneous
excitatory synaptic potentials generated in part by 7-nAChR
activation (Chen et al., 2001). Whole-cell patch-clamp recording from
E8 ciliary ganglion neurons grown 1 week in culture, however, failed to
detect significant nicotine-induced responses that could be attributed
to 7-nAChRs (Fig. 1A). Unexpectedly,
the inclusion of BSA in the perfusion solution allowed a large, rapidly
activating and rapidly desensitizing nicotinic response to be seen. The
response was not seen after treatment with Bgt (Fig. 1B), indicating
that it was generated by 7-nAChRs (Fig. 1C).
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7-nAChR response when
challenged with nicotine. Nonetheless, BSA had an enormously
potentiating effect on the response. The effect was so great that the
7-nAChR currents could not be accurately measured when recorded in
neurons voltage-clamped at 60 mV. At a holding potential of 20 mV,
75 µM BSA increased the peak nicotine-induced whole-cell current by
an order of magnitude (Fig. 2A), and the increased response was blocked by Bgt as expected for 7-nAChRs (data not shown). The temporal profile of the response, including both
rise and fall times, were similar to those of 7-nAChRs in untreated
cells. Thus, at 20 mV, rise times (10-90% of peak) for control and
BSA-treated neurons were 4.8 ± 0.3 and 5.4 ± 0.4 ms,
respectively, whereas decay time constants for the large, rapidly
decaying component of the response were 21.3 ± 2.8 and 15.4 ± 1.4 ms, respectively (mean ± S.E.M.; n = 10).
The similarity in decay time suggests that the BSA-mediated
potentiation of the 7-nAChR response is unlikely to result from a
dramatic reduction in the rate of receptor desensitization.
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7-nAChR
response indicated an EC50 value of about 10 µM
(Fig. 2B). The BSA effect was completely reversible: less than 20 s was required for maximal potentiation and less than 20 s for
return to control levels (Fig. 3, A and
B). Reapplication of BSA reproduced the potentiation (data not shown).
The potentiation seemed to include both an effect on agonist affinity
and an effect on the maximal whole-cell response. Thus, in the presence
of BSA, the dose-response curve for nicotine was shifted slightly to
lower agonist concentrations but also reached significantly greater maximum values (Fig. 4). Interpreting
changes in the dose-response curve for 7-nAChRs, however, is
complicated by the fact that it is influenced by receptor
desensitization, which can limit response amplitude and varies with
agonist concentration (Zhang et al., 1994).
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Specificity of the Potentiation.
The potentiation
preferentially affected 7-nAChRs and was produced by albumins
obtained from several, but not all, species. Little potentiation was
seen in either the non-7 nicotinic response or the
GABAA response in E13 ciliary ganglion neurons
treated with BSA (Fig. 5A). BSA had no
significant effect on either the AMPA- or NMDA-induced responses from
rat hippocampal neurons, although it did potentiate 7-nAChR
responses from the neurons as it did in chick ciliary ganglion neurons
(Fig. 5B). Over half of the albumins tested on chick neurons produced
substantial potentiation, whereas four, including chick and rat
albumin, had no effect (Fig. 5C). Rat albumin also failed to potentiate
significantly the rat hippocampal 7-nAChR response: peak values
induced by 1 mM ACh in the presence of 10 µM rat albumin were
1.3 ± 0.2-fold that of control responses (mean ± S.E.M.;
n = 8 cells). The results suggest that "competent"
albumins either share a critical amino acid sequence or have an
absorbed component necessary to produce the potentiation.
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Mechanism of the Potentiation.
It is known that albumins can
act as scavengers to absorb lipids and other components from blood
(Peters, 1985). One family of candidates is ivermectin-like components
that might have been present in the feed of some but not all animal
species supplying the albumins; ivermectin has been reported to produce
significant potentiation of heterologously expressed 7-nAChRs
(Krause et al., 1998). Treating E13 ciliary ganglion neurons with 10 to
30 µM ivermectin for 2 to 5 min, however, had no effect on the peak 7-nAChR response (data not shown). Moreover, extracting BSA with a
variety of organic solvents, including chloroform, methanol, and
acetonitrile, to remove absorbed components, had no effect on the
ability of the BSA to potentiate 7-nAChR responses (Fig. 6A). Nor was the potentiation diminished
when the BSA was partially denatured with either urea or dithiothreitol
in the presence of iodoacetamide and then dialyzed to remove absorbed
components (Fig. 6A). In contrast, extensive proteolysis to destroy the
BSA protein backbone eliminated the potentiation. Pepsin, chymotrypsin, and trypsin each reduced the potentiating ability of BSA to background levels, whereas negative controls, e.g., pepsin incubation at a
nonpermissive pH, had no effect (Fig. 6B). The results are most consistent with the BSA itself generating the potentiation, and using
specific domains of the protein not common to albumins from all
species.
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) yielded a 30-kDa carboxy
terminal half of the protein that remained competent. The fragment was
isolated by using a combination of size exclusion and ion exchange
chromatography. At 10 µM, the fragment produced 560 ± 80%
(mean ± S.E.M.; n = 9) potentiation of the peak
nicotine-induced response. This was indistinguishable from potentiation
by intact BSA (p > 0.5). Further proteolysis, followed
by high-performance liquid chromatography fractionation to separate
individual fragments, failed to identify active components. Either the
proteases used (pepsin or trypsin) destroyed the active site, or
protein conformation, rather than sequence alone, was important for the potentiation.
The BSA-mediated potentiation was not voltage-dependent. The ratio of
peak nicotine-induced responses obtained at +40 mV to those seen at
20 mV in the same neurons under control conditions was 9.2 ± 1.2% (n = 5). The ratio seen in the presence of 10 µM BSA was identical: 9.2 ± 1.3% (n = 6).
The BSA effect was not mediated via common second messenger pathways
such as those depending on intracellular calcium or G protein-coupled
receptors. Dialyzing the interior of the neuron from the patch pipette
either with BAPTA to chelate calcium or with a stable GDP analog had no
effect on the BSA-induced potentiation (Fig.
7). Neither did the compounds nor a
stable GTP analog have any effect on control responses in the absence
of BSA. Blockers of protein kinases A, C, and G also seemed to be
without effect on the potentiation (Fig. 7). The results suggest a
direct interaction between albumin and 7-nAChRs or possibly one
mediated by surface components on the neurons; intracellular signaling
cascades are not likely to be required.
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Properties of 7-nAChRs Affected by the Potentiation.
It has
been suggested that only a small fraction of the 7-nAChRs present on
chick ciliary ganglion neurons may be functionally available (McNerney
et al., 2000). This assessment may require revision because it relied
on Bgt binding to quantify 7-nAChRs; the stoichiometry of Bgt
binding may be greater than previously thought, if the results obtained
with soluble acetylcholine binding protein (Brejc et al., 2001) can be
extended to 7-nAChRs. Nonetheless, it was important to consider the
possibility that the potentiation of the whole-cell 7-nAChR response
by BSA represented a recruitment of receptors from a previously
"silent" pool to a pool that was now functionally available for
nicotine-induced activation. This was tested by using an open channel
blocker. We reasoned that if the potentiation represented silent
receptors being recruited de novo by the BSA treatment, then previous
exposure to agonist in the presence of an open channel blocker (while
the receptors were in silent mode) would have no effect on the ability
of BSA subsequently to produce a large nicotine-induced response. The protocol would require removing agonist and unbound open channel blocker before adding the BSA so that no blockade of newly recruited receptors would occur. Alternatively, if the open channel blocker was
able to attenuate the BSA effect as it did control responses even
though the blocker was only available before the BSA incubation, the
results would suggest that the same receptor population produced both
the control and potentiated whole-cell responses.
; Hicks et al., 2000). At 20 µM
chlorisondamine quickly produced a dramatic reduction in the peak
whole-cell nicotinic response (Fig. 8A),
which is dominated by 7-nAChRs (Liu and Berg, 1999b). It also almost
completely eliminated the slower non-7 nAChR response. Agonist was
applied for 1 s at 30-s intervals to allow full recovery from
receptor desensitization between trials, and perforated patch-clamp
recording was used to minimize rundown of the response during the
experiment (Liu and Berg, 1999a). After even a few applications of
agonist, the chlorisondamine reduced control responses by about 80%
(Fig. 8B). The reduction was consistent with an open channel blockade because it did not occur if the chlorisondamine was applied
continuously during the 29-s intervals between nicotine applications
but excluded during the 1-s nicotine applications themselves (Fig. 8B).
|
7-nAChRs, we conducted recordings from excised outside-out neuron
patches to determine which functional single channel properties might
be altered to account for the potentiation. Previous studies on chick
ciliary ganglion neurons identified single nAChR channel events of 25, 40, 60, and 80 pS activated by 0.5 µM nicotine in excised outside-out
neuron patches (Margiotta and Gurantz, 1989; McNerney et al., 2000).
The 60- and 80-pS events were brief (<0.2 ms) and were blocked by
Bgt, indicating that they probably resulted from activation of
7-nAChRs. The 25- and 40-pS events were of longer duration and not
blocked by Bgt, suggesting they were produced by the non-7 nAChRs
on the neurons. Recent studies have further distinguished brief 25- and
40-pS events (<0.2 ms) that can be blocked by Bgt, marking them as
additional candidates for 7-nAChR responses (J. F. Margiotta
and Q. Nai, unpublished observations). BSA did not detectably change
the amplitudes of the single channel events induced by 0.5 µM
nicotine (Fig. 9, A and B), indicating no
change in single channel conductance. In contrast, BSA treatment
significantly increased the steady-state opening probability
(%Popen) of both the 60- and 80-pS
events with about 5-fold increases evident for both after a 10-min
exposure to 75 µM BSA (Fig. 9C; see legend for
%Popen values). This effect was
accompanied by approximately 2- and 3-fold increases in the average
open duration (Topen) and frequency of
opening (Fopen), respectively (Fig.
9D). The increase in Topen seems to
result from a larger fraction of longer duration openings in patches from BSA-treated neurons, as shown for the 60-pS class of events (Fig.
9, E and F). Similar results were obtained for the 80-pS events (data
not shown). No single channel events were seen during comparable
recording times in the absence of nicotine.
|
Bgt-sensitive events in the sense that the
treatment had no significant effect on the long duration 25- and 40-pS
events attributable to non-7 nAChRs in the same patches (Fig. 9C).
The fact that BSA also had no significant effect, however, on the short
duration, Bgt-sensitive 25- and 40-pS events (Fig. 9C), suggests
that these latter events may be produced by a distinct subclass of
7-nAChRs or a different class of Bgt-sensitive receptors. About
5% of the Bgt binding sites in ciliary ganglia are associated with receptors lacking any known gene product (Pugh et al., 1995); their
cellular source and functional responses remain unknown, but they may
contribute to some of the single channel events observed. Overall, the
single channel results are consistent with the whole-cell recordings
presented above, and suggest that the BSA-dependent potentiation of
7-nAChR responses is caused by increases in
%Popen, Fopen, and
Topen for two prominent classes of
channel events.
BSA-Mediated Compensation for Inhibition of 7-nAChRs by
A.
A specifically and potently inhibits 7-nAChR function
by a noncompetitive mechanism, suggesting the possibility that the receptors represent an important early molecular victim of Alzheimer's disease (Liu et al., 2001; Pettit et al., 2001). The ability to potentiate 7-nAChR function could provide a mechanism for partially overcoming such inhibition and helping to alleviate symptoms of the
disease involving the receptors. We evaluated this by examining the
inhibition of 7-nAChRs by A in the presence and absence of BSA.
A at 100 nM produced a substantial inhibition of the whole-cell
7-nAChR response in E13 ciliary ganglion neurons as reported
previously. Coapplication of BSA at a concentration near the
EC50 value for potentiation returned the response
amplitude to near control levels even in the continued presence of A
(Fig. 10). A higher concentration of 75 µM BSA produced a much larger response (Fig. 10), consistent
with the concentration dependence of the BSA effect on control
responses (Fig. 2). Thus, BSA-mediated potentiation can, in effect,
compensate for inhibition by A even though the latter was previously
shown to be noncompetitive with respect to agonist (Liu et al., 2001).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The results demonstrate that native 7-nAChRs can be
significantly potentiated, and that the potentiation can produce up to an order of magnitude increase in the whole-cell response. The potentiation includes an increase in agonist affinity and an increase in the maximum amplitude of the response. The mechanistic basis for the
potentiation seems to be an increase in the steady-state channel
opening probability produced both by an increased frequency of channel
opening and an increased mean channel open time. No change is seen in
single current amplitude, and no evidence suggests the potentiation
depends on the recruitment of a previously silent pool of receptors. No
change is seen in the temporal profile of the whole-cell response that
could account for the potentiation.
The ability of certain albumins to produce the potentiation seems to
depend on information contained in the protein backbone rather than on
absorbed components. Neither extensive extraction with organic solvents
nor unfolding followed by exhaustive dialysis diminished the ability of
BSA to induce the potentiation. Proteolysis, on the other hand,
completely abolished it. The finding that the carboxyl half of BSA was
sufficient to produce the potentiation focused attention on the amino
acid sequences therein. A comparison of the active albumins with those
of the inactive ones indicated six distinct sites at which the two
groups diverged by at least one amino acid. Examining the
three-dimensional X-ray crystallographic structure of human serum
albumin indicated that five of the six sites were likely to reside on
the surface of the protein where they could interact with other
molecules (Curry et al., 1998). One or more of these, possibly in a
conformation-dependent manner, may be responsible for causing the
potentiation of 7-nAChR responses.
The properties of the potentiation are consistent with the albumins
acting as allosteric modulators of the 7-nAChR. The modulation may
result from a direct interaction between the albumin and the receptor.
This is suggested by the finding that the potentiation was rapid,
reversible, and reproducible, and did not depend on any of a variety of
common second messenger systems. Moreover, the albumin was effective at
sustaining the potentiation when applied to excised outside-out
patches. We cannot exclude the possibility, however, that the effect is
indirect, e.g., that the albumin reversibly binds a negative modulator
tethered in the vicinity of the receptor. The potentiation was most
pronounced for 7-nAChRs, although a small potentiation could be seen
for certain other ionotropic receptors.
A previous study examined albumin effects on nicotinic receptors and
concluded that non-7 nAChR responses were potentiated in ciliary
ganglion neurons (Gurantz et al., 1993). The techniques used then would
not have revealed the rapidly decaying 7-nAChR responses normally
displayed by the neurons. The present results find little, if any,
albumin effect on ciliary ganglion non-7 nAChRs and suggest instead
that the potentiated response seen previously actually arose from
enhanced 7-nAChR currents persisting sufficiently long as to
override the much smaller non-7 responses. This possibility was not
tested at the time because 7-nAChRs had not yet been shown to
represent functional ligand-gated ion channels. In other respects the
present results are consistent with the previous study, including the
lack of changes seen in non-7 nAChR single channel properties, the
small enhancement of GABAA receptor responses by
BSA, and the fact that some albumins are effective, whereas others are
not. An additional point worth comment was a finding in the previous
study that cAMP produced a small enhancement of the whole-cell
nicotinic response and that the enhancement was nonadditive with that
produced by BSA (Gurantz et al., 1993). We found no requirement for
second messengers in the potentiation of 7-nAChR responses by BSA,
although a cAMP-dependent process can increase to some extent the
7-nAChR response (Pardi and Margiotta, 1999). The simplest
explanation is that the BSA effect on 7-nAChRs is independent from
cAMP pathways and either obscures their effects in this case or
converges on a common target.
Three components that increase 7-nAChR responses have been reported
previously. Ivermectin was found to produce a large enhancement of the
nicotinic responses generated by heterologously expressed 7-nAChRs
if supplied in advance of agonist (Krause et al., 1998). No effect of
ivermectin was seen in the present experiments, however, with native
7-nAChRs on chick ciliary ganglion neurons. Either the source of
ivermectin and associated minor components is critical for the effect,
or the source of 7-nAChRs determines the outcome. Another known
modulator of 7-nAChRs is prostaglandin E2,
which increases the opening probability and open duration of 23-pS
single channel events attributed to the receptors on chick sympathetic neurons (Du and Role, 2001). Whole-cell currents generated by such
receptors have relatively slow kinetics of activation and desensitization, different from the 7-nAChRs described here. The
prostaglandin E2 effect was thought to be
indirect, possibly relying on calcium as a second messenger (Du and
Role, 2001). A third modulator is PACAP, a neuropeptide endogenous in
the ciliary ganglion. PACAP acts through adenylate cyclase and protein
kinase A to enhance the responses of both 7- and non-7 nAChRs on
the neurons (Pardi and Margiotta, 1999). Neither prostaglandin
E2 nor PACAP, however, produced the dramatic
potentiation of 7-nAChR responses seen here with BSA.
The significance of the present findings is 3-fold. First, they
indicate that the response of 7-nAChRs can be dramatically potentiated by increasing the steady-state opening probability of the
receptors. The previously reported low
%Popen values associated with both
the 60- and 80-pS channel events of ciliary ganglion 7-nAChRs
(McNerney et al., 2000) are not, therefore, inherently rigid features
of the receptors but rather are subject to allosteric modulation. The
site of interaction and the mechanism accounting for the potentiation
will be subjects of considerable interest for future analysis.
The second important aspect of the findings is the recognition that a
site exists on native 7-nAChRs capable of dramatic modulation. This
finding raises the possibility that endogenous compounds, possibly
neuropeptides but conceivably compounds of completely different
composition, exploit the site in vivo during normal physiological
function. Blood levels of albumin are typically about 40 mg/ml but can
be 200-fold less in cerebral spinal fluid (Curry et al., 1998).
Although even this reduced level could have some effect, we think
albumins are not likely to be the normal ligand acting at such sites.
One reason for thinking so is that the cognate albumin (e.g., chicken
albumin and chick 7-nAChRs) was ineffective. Identification of
endogenous compounds exerting the modulation and elucidation of the
physiological significance could have profound implications for
nicotinic signaling in the nervous system.
One candidate modulator is lynx1, an endogenous prototoxin that has
recently been shown to complex with 7-nAChRs when coexpressed in
transfected cells (Ibanez-Tallon et al., 2002). Lynx1 has multiple effects on the response of 42-nAChRs, increasing the proportion of large current events but also increasing the
EC50 value for agonist and the rate and extent of
receptor desensitization (Ibanez-Tallon et al., 2002). The overall
effect of lynx1 on 42-nAChRs is very different from the dramatic
potentiation of 7-nAChRs seen here with select albumins. How lynx1
or related family members might affect 7-nAChR function is not yet known.
Last, the demonstration that 7-nAChR function can be potentiated in
a way that compensates for receptor blockade by the A peptide,
encourages speculation that the receptor may be a useful target for
therapeutic strategies aiming at augmenting nicotinic signaling. The
hope would be that knowledge about the modulatory site would permit the
design of nonpeptide molecules capable of accessing and modulating the
receptors in situ. This could be germane to neuropathologies such as
Alzheimer's disease (Liu et al., 2001; Pettit et al., 2001) and
neurodisorders such as schizophrenia (Leonard et al., 2000).
| |
Acknowledgments |
|---|
We thank Dr. Russell F. Doolittle (University of California, San Diego) for advice on albumin structure and protein biochemistry.
| |
Footnotes |
|---|
Received September 6, 2002; Accepted October 21, 2002
This study was supported by Tobacco-Related Disease Research Program Grants 9RT-0058 (to W.G.C.) and 9RT-0221, and by National Institutes of Health Grants NS12601, NS35469 (to D.K.B.), and NS24417 (to J.F.M.). Q.-S.L. is an American Heart Association Postdoctoral Fellow.
W.G.C. and Q.-S.L. contributed equally to this work.
Address correspondence to: Darwin K. Berg, Neurobiology Section, Division of Biological Sciences, 9500 Gilman Dr., University of California, San Diego, La Jolla, CA 92093-0357. E-mail: dberg{at}ucsd.edu
| |
Abbreviations |
|---|
nAChR, nicotinic acetylcholine receptor;
NMDA, N-methyl-D-aspartate;
PACAP, pituitary
adenylyl cyclase-activating protein;
E, embryonic day;
Bgt, -bungarotoxin;
RS, standard recording solution;
BSA, bovine serum
albumin;
A, -amyloid peptide1-42;
BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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) and after
(
) application of 10 µM BSA. Values represent the mean ± S.E.M. of results from 7 to 14 neurons for each condition; the curves
represent the best least-squares fit of the data. BSA increased the
affinity of the receptor for agonist as reflected in the lower
EC50 value and increased the maximum whole-cell response
elicited from the neurons.
, 75 µM BSA; 
