Department of Pharmacology, Georgetown University School of
Medicine, Washington DC
Neuronal nicotinic acetylcholine receptors (nAChRs) are widely
distributed in the nervous system. Although there is a vast literature
on the molecular, structural and pharmacological properties of neuronal
nAChR, little is known of their pH regulation. Here we report that
rapid acidification (pH 6.0) enhances the current through the 
3/
4
recombinant nAChRs expressed stably in human embryonic kidney 293 cells
and accelerates its activation kinetics without altering selectivity.
Acidification also strongly accelerates the decay kinetics
("desensitization") of cytisine- and nicotine-evoked currents
(pKa ~6.1), but the effect is somewhat
smaller with acetylcholine and carbachol (undetermined
pKa values), suggesting that protonation of
the agonist contributes to the relaxation of the current. Transient increases of [H+]o from pH 7.4 to 6.0, during
the time course of decay of the current, enhances the current and
accelerates its decay kinetics in a manner similar to reactivation of
current by higher concentrations of agonists. We suggest that protons
interact with multiple extracellular sites on 3/4 nAChRs,
decreasing the effective EC50 values of the agonist and
accelerating gating kinetics, in part by promoting agonist-induced
block. We speculate that corelease of protons with ACh from the
secretory vesicles may induce rapid and reversible conformational
changes in the slowly "desensitizing" 3/4 nAChRs, leading to
accelerated signaling.
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Introduction |
Neuronal
nAChRs are widely distributed in the nervous system, mediating both
pre- and postsynaptic signaling. Thus far, nine (2-10) and
three (2-4) neuronal nAChR subunits have been identified,
cloned, and functionally expressed [for recent reviews, see Lindstrom
(1997)
; Lindstrom et al. (1998); McGehee (1999); Corringer et al.
(2000); Dani (2001); Elgoyhen et al. (2001); Hsiao et al. (2001)].
Recombinant neuronal nAChRs composed of different subunit combinations
expressed in Xenopus laevis oocytes or eukaryotic cell lines
provide direct evidence for the variability of their affinity to
agonists, antagonists, Ca2+ permeability, and
rate of desensitization. However, little is known about the pH
sensitivity of the neuronal nAChRs, even though there are many reports
on the pH sensitivity of the muscle and electric organ nicotinic
receptors (Trautmann and Zilber-Gachelin, 1976; Landau et al., 1981;
Palma et al., 1991; Li and McNamee, 1992). These reports show that
acidic pH generally inhibits the muscle nAChRs by reducing their
single-channel conductance. In addition, kinetic analysis suggests a
biphasic pH-dependence of the mean open time of the channel, with a
maximum near the physiological pH (Landau et al., 1981; Palma et al.,
1991). The rate of desensitization of the receptor seems also to be
accelerated at both alkaline and acidic pH values (Li and McNamee,
1992). The proton-induced effects were found to be neither
voltage-dependent nor mediated by changes in the ionic selectivity of
the receptor.
Here we have examined the effect of rapid (<20 ms) and transient
coapplication of various proton and agonist concentrations on the
recombinant rat 3/4 nAChRs stably transfected in HEK cells. The
rapid coapplication of protons and agonist was undertaken to
approximate, in part, the transient local changes in pH that might
occur as protons and the transmitter are coreleased from the secretory
vesicles (Johnson, 1987; Miesenbock et al., 1998) during synaptic
signaling. Our data suggest that protons interact with multiple
extracellular sites on 3/4 nAChRs. The coapplication of protons
enhances the agonist-induced current and accelerates its kinetics in a
manner resembling the effects of a higher agonist concentration. We
describe this as a decrease in the EC50 of the receptor, or an increase in its "apparent affinity", not to the exclusion of other mechanism. We speculate that transient changes of pH
in the synaptic cleft might play a critical role in the regulation of
synaptic signaling. A preliminary report of this work has already
appeared (Abdrakhmanova et al., 2001).
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Materials and Methods |
Cell Transfection and Culture.
Stably transfected HEK 293 cells (American Type Culture Collection, Manassas, VA) expressing rat
3/4 neuronal nAChRs (cell line designation,
KX34R2) were prepared as described
previously (Xiao et al., 1998). HEK 293 cells were maintained at 37°C
with 5% CO2 in the incubator. Growth medium for
HEK 293 cells was minimum essential medium supplemented with 10% fetal
bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin.
Transfection was conducted by calcium phosphate method (Chen and
Okayama, 1987) using a method identical to that described earlier
(Zhang et al., 1999). Stably transfected cell lines were raised in
selective growth medium containing 0.7 mg/ml of Geneticin (G418). Cells were plated in tissue culture medium (Invitrogen, Carlsbad, CA) containing bovine serum and antibiotics. ACh, nicotine, cytisine, and
carbamylcholine chloride (carbachol) were purchased from Sigma Chemical
Co. (St. Louis, MO) and were used at the indicated concentrations.
Electrophysiological Measurements.
Functional expression of
nicotinic receptors was evaluated in the whole-cell configuration of
the patch-clamp technique using a Dagan 8900 amplifier (Dagan Corp.,
Minneapolis, MN). The patch electrodes, pulled from borosilicate glass
capillaries, had a resistance of 3 to 4 M
when filled with internal
solutions containing either 80 mM tetraethylammonium chloride, 60 mM
NaCl, 5 mM MgATP, 10 mM glucose, 10 mM EGTA, 10 mM HEPES, and 0.1 mM
cAMP (titrated to pH 7.4 with NaOH) or 110 mM CsCl, 20 mM
tetraethylammonium chloride, 5 mM MgATP, 14 mM EGTA, and 20 mM HEPES
(pH adjusted to 7.4 with CsOH). Higher
[Na+]i were used to
provide for accurate measurements of reversal potential at moderate
positive potentials in this highly rectifying nicotinic receptor (Zhang
et al., 1999). In some experiments, the pH of the internal solution was
adjusted to 6.0. About 90% of electrode resistance in the cell was
compensated electronically, so that the effective series resistance in
the whole-cell configuration was always less than 1 M. Stably
transfected HEK cells were studied for 2 to 4 days after plating the
cells on the cover slips. Generation of voltage-clamp protocols and
acquisition of data were carried out using pCLAMP software (Axon
Instruments, Inc., Union City, CA). Sampling frequency was 0.5 to 2.0 kHz and current signals were filtered at 10 kHz before digitization and
storage. All experiments were performed at room temperature
(23-25°C). The measured currents were normalized relative to the
membrane capacitance ranging between 18 and 40 pF and were quantified
as the mean ± S.E.M. for the number of cells (n). To
estimate the time constants of the decay of agonist-induced currents,
the time course of the relaxation of current in the presence of
agonists was determined by dividing the maximal slope (linear
regression) by the peak amplitude of the current. This ad hoc method
was preferred to exponential analysis because a clear monoexponential
decay often was not observed (e.g., due to our relatively brief drug
exposure times).
Application of Agonists and the Perfusion System.
Cells
plated on 15-mm round plastic cover slips (Thermanox; Nunc, Inc.,
Napierville, IL) were transferred to an experimental chamber mounted on
the stage of an inverted microscope (Diaphot; Nikon, Nagano, Japan) and
were bathed in a solution containing 137 mM NaCl, 10 mM HEPES, 1 mM
MgCl2, 10 mM glucose, 5.4 mM KCl, and 2 mM
CaCl2 (pH adjusted to 7.4 with NaOH). The
experimental chamber was constantly perfused with the control bathing
solution (1 ml/min). KCl was omitted from the control and
agonist-containing puffing solutions to suppress possible
K+ currents in the voltage-clamped cell.
The amplitude and time course of the nicotine-activated current was
highly dependent on the speed of application of nicotine. A reduction
in flow rate significantly slowed the activation, decreased the
amplitude, and slowed the desensitization of the nicotinic current
(Callewaert et al., 1991). Therefore, we used servo-controlled
miniature solenoid valves (Lee Company, Westbrook, CT) for rapid
switching between control and test solutions (Cleemann and Morad, 1991;
Zhang et al., 1999). The effective switching time was determined in rat
ventricular cells by measuring the peak Na+
currents at various times after triggering a change in
[Na+]o or by measuring
the holding current at the tip of an open patch pipette subjected to a
solution of low Cl
(Davies et al., 1988). Under
optimal conditions, such changes in solution had a delay of 6 to 8 ms
(corresponding mainly to the pull and release of the solenoid valves
and replacement of fluid in the common outlet of the perfusion
manifold) followed by a transition period in which the measured current
changed with a time constant of 5 to 10 ms. In repeated applications,
the delay was fairly reproducible, but it did show variation in
different experiments with different hydrostatic pressures and
perfusion manifolds. In experiments aiming to measure the rate of
activation of the nicotinic current (Fig. 8), fluid was applied under
high hydrostatic pressure and the transition time was typically about 20 ms. Because the rapid flow of solution tended to dislodge the voltage-clamped cell, in less critical experiments, we lowered the
hydrostatic pressure, resulting in slower fluid exchange periods (~50
ms; see Figs. 1, 5, and 10).
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Fig. 1.
Acidification enhances and accelerates the gating
kinetics of nicotine-induced current. A, superimposed traces of
currents activated by 40 µM nicotine at pH 7.4 and 6.0, recorded from
a cell voltage-clamped at  80 mV. B, exposure of the cell to pH 6.0 for 415, 830 and 1660 ms, before application of 40 µM nicotine did
not alter the magnitude or the kinetics of the current induced by
nicotine. C, step-changes of pH from 7.4 to 6.0 applied at different
times (415, 830, 1245, and 1660 ms) during the course of
"desensitization" reactivate the nicotinic current. Recordings of A
and B were obtained from the same cell (capacitance 27 pF), and those
of C from another cell (capacitance 44 pF).
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Results |
Stably transfected HEK 293 cells expressing 3/4 nAChRs
generated a rapidly activating but slowly desensitizing cationic current in response to step-changes in extracellular nicotine. Figure
1A compares the current recorded from a cell clamped at 80 mV in
response to step application of 40 µM nicotine buffered at a pH value
of either 7.4 or 6.0. The superimposed original traces show that
nicotine solution buffered at pH 6.0 not only enhanced the current but
also accelerated its decay kinetics, such that at the end of 1-s
drug application at pH 6.0 the magnitude of current was
significantly smaller. Similar enhancement of decay kinetics of the
current induced by higher concentrations of agonist have been observed
before and referred to as "rapid desensitization" (Lester and Dani,
1995) or "agonist-induced channel block" (Sine and Steinbach, 1984;
Ogden and Colquhoun, 1985; Luetje and Patrick, 1991; Maconochie and
Steinbach, 1995; Philipson et al., 2001).
Figure 1B explores the time course and the magnitude of
nicotine-activated current, when the proton concentrations were
elevated before the application of nicotine. There was no significant
difference in the magnitude or the kinetics of the nicotine-activated
current whether the proton concentrations were elevated before or
simultaneously with the application of nicotine. Furthermore, this
experiment suggests that elevation of
[H+]o does not activate a
significant current by itself.
In another series of experiments, we also tested the possible effects
of step elevation of proton concentrations after the current was first
activated by application of 40 µM nicotine at pH 7.4. Figure 1C shows
superimposed traces of repeated application of nicotine at pH 6.0, at
various intervals after the activation of current by 40 µM nicotine
buffered at pH 7.4. Note that significant current is induced during the
course of "rapid desensitization" by step elevation of proton
concentration (pH 6.0). The effect of elevation of
[H+]o was accompanied not
only by transient enhancement of the current but also by acceleration
of its decay kinetics resulting in smaller steady-state current (see
also Fig. 1A). Thus, irrespective of the timing of step-increase of
[H+]o, the
nicotine-activated current was enhanced and its gating kinetics were accelerated.
pH Effect on the Amplitude of Agonist-Induced Current.
Quantification of the whole-cell currents induced by different nicotine
concentrations (10, 40, 200, and 1000 µM) showed that increasing the
[H+]o generally enhanced
the current and accelerated its decay kinetics. The pH effect on the
magnitude of nicotine-evoked current was more pronounced at lower
nicotine concentrations, becoming smaller at 200 µM and negligible at
1 mM (see legend of Fig. 2). Figure 2A
compares the effect of elevation of
[H+]o at different
agonist concentrations. Quantification of the data suggests a shift of
concentration response curve, such that the proton effects are
minimized at higher nicotine concentrations. The data points for
activation of nicotine-induced current were fit with the empirical Hill
equation y = 1 / [1 + (EC50 /
[protons]nH)] yielding an apparent Hill
coefficient (nH) of ~1.5. This finding suggests that acidification may increase the apparent affinity of the
3/4 nAChRs to nicotine.
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Fig. 2.
Increasing [H+]o enhances
the apparent affinity of 3/4 receptor to nicotine and ACh. A,
dose-response curves for the peak currents induced by nicotine ( , 10 µM;  , 40 µM;  , 200 µM; and  , 1000 µM) at different pH
values (8.0, 7.4, 6.7, and 6.0) in cells voltage-clamped at 80 mV.
The amplitude of the currents was normalized relative to the peak
current at pH 6.0. Each symbol is labeled with the number of cells
tested and a vertical error bar indicating the S.E.M. The maximal
currents induced by 1 mM nicotine were 247 ± 8 pA/pF at pH 7.4 and 258 ± 11 pA/pF at pH 6.0 in paired measurements on four
cells. B, dose-response curves for the peak currents induced by ACh at
two different pH values ( , 7.4;  , 6.0) in cells voltage-clamped
at 80 mV. The currents recorded from each cell were normalized
relative to the current induced by 300 µM ACh. The data points were
fit with least-squares determination of EC50 and normalized
to give a maximum response at one. The maximal currents induced by 1 mM
ACh measured in two sets of cells were 259 ± 18 pA/pF at pH 7.4 (n = 5) and 304 ± 36 pA/pF at pH 6.0 (n = 4). Each symbol is labeled with a vertical
error bar indicating the SEM (n = 4-10). The
continuous curves represent a fit to Hill equation
(nH ~ 1.85).
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To examine more specifically whether protonation enhances the apparent
affinity of 3/4 receptor to ACh, we measured the dose dependence
of ACh-induced current at two different pH values. In this set of
experiments, individual cells were exposed either to pH 7.4 or 6.0, while varying the ACh concentrations. Figure 2B shows that reduction of
pH from 7.4 to 6.0, decreased the EC50 of
ACh-evoked current from 105 to 67 µM, but had little effect on the
cooperativity factor (nH ~ 1.85). As for
nicotine, acidification had no significant effect on the amplitude of
the currents measured at saturating ACh concentrations (see legend of
Fig. 2). Because ACh is unlikely to be protonated at pH 6.0, the data
suggest a direct effect of protons on the 3/4 receptor.
It is well known that agonists of nAChRs differ in their
pKa values. Some, like nicotine or
cytisine, are protonated [pKa = 6.16 and 6.11, respectively (Windholz, 1983)], whereas others (e.g., ACh or
carbachol) have no pKa in the
physiological range and are permanently ionized. To distinguish whether
changes in pH modulate the channel function by acting at a proton
sensing site on the receptor or by altering the ionization state of the ligand, we tested the pH effects of these four well-known agonists with
different affinities to the receptor. The agonists were applied at
concentrations approximating their EC50 values
[for instance, 40, 60, 100, and 450 µM for nicotine, cytisine, ACh,
and carbachol, respectively (Zhang et al., 1999; Meyer et al., 2001)].
Figure 3 shows that acidification causes
an enhancement of the agonist-induced current for all four agonists in
a similar range of pH values. The effect on the decay kinetics of the
agonist-induced current, however, varied greatly depending on the type
of the agonist. That is, in those with
pKa values around 6.1 (nicotine and
cytisine), elevation of
[H+]o strongly
accelerated the decay kinetics of their current, whereas those
permanently ionized (ACh and carbachol) were less affected by acidic pH
values (see also Fig. 6). These findings suggest that although the
enhancement of the current by nicotinic agonists was not significantly
dependent on the ionization state of the agonist, the decay kinetics of
the current seemed to be modulated in part by the protonation of the
agonists.
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Fig. 3.
Comparison of the pH effect on the amplitude of the
current activated by different agonists. A, superimposed traces of
currents evoked at pH values 7.4 and 6.0 by nicotine (, 40 µM),
cytisine (, 60 µM), ACh (, 100 µM), and carbachol (, 450 µM). The concentrations chosen approximate the EC50 value
for each agonist, and the duration of agonist application is indicated
by the horizontal bar. B, pH-dependence of the current amplitude.
Agonist-activated current was recorded in each cell at pH 8.0, 7.4, 6.7, and 6.0 (n = 4-5). The amplitude of the
currents activated at different pH values, when normalized relative to
the peak current measured at pH 7.4, produced a similar relationship
for different agonists.
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Comparison of Reactivation of the Current by Higher Agonist
Concentration or Lower pH.
Consecutive step-changes of pH from 7.4 to 6.0 during the course of decay of the current induced by 20 µM
nicotine resulted in transient enhancement of the current and
acceleration of its relaxation kinetics (Fig.
4A). To test whether this effect also resulted from increased apparent affinity of the receptor to the agonist (see Fig. 2), we compared the effects of step-changes of
elevation of [H+]o with
step-increases of the agonist concentration (20 to 60 µM) in the same
cell. Step-increase of nicotine from 20 to 60 µM augmented both the
current and its relaxation (Fig. 4B) in a manner similar to step
elevation of [H+]o (Cf.
Figure 4A). As the step-changes of either pH 6.0 or 60 µM nicotine
were done repeatedly at different times during the course of decay of
the current activated by 20 µM nicotine, we compared the magnitude of
reactivated current measured in five cells in which both proton and
nicotine concentrations were increased (Fig. 4C). The magnitude of
reactivated currents at later times (Fig. 4C, traces 2, 3, and 4) were
measured relative to the first reactivated current (Fig. 4C, trace 1).
Figure 4C shows that current reactivated by step-change to pH 6.0 remained almost constant (within 6%) during the course of
"desensitization", whereas the current reactivated by step-increase
in nicotine concentration decreased significantly (38%) in 1245 ms.
These data demonstrate that the effects produced by acidification and
increased agonist concentration may look similar but, in fact, are
qualitatively different. Thus, it seems that step elevation of proton
concentrations, in addition to increasing the apparent receptor
affinity to agonist, may partially protect the receptor against events
that lead to suppression of the current in the presence of agonist
("rapid desensitization").
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Fig. 4.
Reactivation of nicotinic current by acidification
and higher nicotine concentration during the time course of
"desensitization". Consecutive step-changes in
[H+]o to pH 6.0 (A) or to 60 µM nicotine
(B) were applied to the same cell (holding potential 80 mV). The
reactivated current was evoked induced at time intervals of 415 (1),
830 (2), 1245 (3), and 1660 (4) ms, after the initial application of 20 µM nicotine. Bar graph in C represents the mean ± S.E.M.
(n = 5 cells) of normalized reactivated currents at
the indicated times evoked either by step-changes to pH 6.0 () or by
step-increases of nicotine concentration at the indicated times
().
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pH Modulation of Decay of Agonist-Induced Current, "Rapid
Desensitization".
One of the consistent observations of this
study was that the decay kinetics of nAChR current were significantly
accelerated by rapid elevation of
[H+]o, irrespective of
the nature of the agonist. Figure 5
compares the effect of application of 40 µM ACh, carbachol, nicotine,
and cytisine at four different pH values (8.0, 7.4, 6.7, and 6.0). Simultaneous application of any of the four agonists at higher proton
concentrations not only enhanced the current through the receptor, but
also accelerated its relaxation.
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Fig. 5.
Agonist-dependent pH response. Each shows
superimposed current traces of the indicated agonist coapplied with
proton concentrations of pH 8.0, 7.4, 6.7, and 6.0 recorded on
different representative cells (holding potential, 80 mV). Each
agonist was applied at 40 µM concentrations. Acidification uniformly
enhanced the current activated by every agonist.
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Figure 6 quantifies the effects of
changes in pH on the kinetics of relaxation of the currents induced by
four different agonists: nicotine, cytisine, ACh, and carbachol. In
this set of experiments, the concentration of agonist chosen was close
to the EC50 of each drug. The analysis is based
on records similar to those shown in Fig. 3A. Such analysis shows that
the rates of decay of cytisine- and nicotine-activated currents (A)
were strongly enhanced at acidic pH values (~10-fold from pH 6.0 to
8.0), whereas those of carbachol- and ACh-evoked currents (B) were less
strongly modified (~4 fold from pH 6.0 to 8.0). This finding is
consistent with the idea that increased ionization of cytisine and
nicotine (pKa ~ 6.1) at lower pH
values provides the agonist with better access to the mouth of the
channel, or to sites that regulate "desensitization". On the other
hand, the significant effect of protons on carbachol and ACh-induced
currents (Fig. 6B) supports the notion that pH-dependence of the decay
kinetics is not determined exclusively by the ionization state of the
agonist but may be directly mediated by the receptor.
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Fig. 6.
Acceleration of decay kinetics of the currents by
acidic pH is influenced by the pKa of the
agonists. Rate constants of decay of the whole-cell currents induced
by: 40 µM nicotine () and 60 µM cytisine () (A) or 100 µM
ACh () and 450 µM carbachol () (B) are compared at pH values of
8.0, 7.4, 6.7, and 6.0. The rates of decay, in pH range from 8.0 to
6.0, accelerated markedly for the nicotine
(pKa 6.16)- and cytisine
(pKa 6.11)-induced currents (A). The
acceleration of the rates of decay was smaller for ACh- and
carbachol-activated currents (with no known
pKa; n = 4-5 for each
agonist). For each agonist, paired t test showed that
the rate constants of decay of the currents at pH 6.0 were
significantly faster than that measured at pH 8.0 (**,
P < 0.002; *, P < 0.02).
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Figure 7 quantifies the effect of
elevation of [H+]o on the
kinetics of decay of the currents measured in the experiments
illustrated in Fig. 2. Figure 7A quantifies the pH effect at two
different nicotine concentrations, 40 µM
(~EC50) and 1 mM (saturating concentration). Analysis of the data in 5 to 6 cells per concentration of the agonist
at pH values from 8.0 to 6.0 shows that the rate constants of decay of
the current were dependent on the agonist concentrations, such that at
1 mM nicotine increases in
[H+]o had little or no
effect on the time course of relaxation, consistent with the finding of
Fig. 2 and 4 that elevation of proton concentration mimics the effect
of increased agonist concentrations. Figure 7B compares the dose
dependence of the rate constants of decay of current when pH is changed
from 7.4 to 6.0. Similar to the effect of pH 6.0 on ACh-induced peak
current in the same set of cells (Fig. 2B), acidification decreased the
mean EC50 value for the rate of decay of the
ACh-induced currents (from 371 to 139 µM). Furthermore, in a manner
similar to the nicotine-induced responses (Fig. 7A), the rates of decay
of the ACh-activated currents were not significantly altered by
acidification at saturating ACh concentrations.
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Fig. 7.
Rate of decay of current at different pH values and
agonist concentrations. A, the pH effect on the rate of decay of
nicotine-induced current is dependent on concentration of the agonist.
Bar graph represents the rate constants of decay of the currents
induced by 40 µM nicotine () and 1 mM nicotine ()
(n = 5-6) at pH values 8.0, 7.4, 6.7, and 6.0. At
1 mM nicotine concentration, the rate of decay is not significantly
altered by the acidic pH values. * indicates statistically
significant difference between rate constants of decay compared at pH
values 8.0 to 6.0 for currents induced by 40 µM nicotine
(P < 0.002). B, acidification accelerates and
shifts the rate of decay of the ACh-induced current toward smaller ACh
concentration (holding potential, 80 mV). The data points
(n = 4-10) were fit by the Hill equation
(nH = 2.0) with EC50 values
of 371 µM at pH 7.4 and 139 µM at pH 6.0.
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pH Effect on Rate of Activation of Agonist-Induced Current.
Elevation of [H+]o also
seemed to enhance the rate of activation of the receptor. This effect
was most pronounced when using carbachol as an agonist (Fig.
8). Similar, but less pronounced effects
were also observed with nicotine when comparing the rate of activation
of the current at pH 8.0 and 6.0. Consistent with this idea, comparison
of rise time of responses induced by nicotine (high-affinity agonist)
and carbachol (low-affinity agonist) induced responses at pH 7.4 and
6.0 (using different agonist concentrations), indicated that for both
agonists, currents activated markedly faster in acidic pH values, and
the effect was more pronounced at lower agonist concentrations. The
original traces in Fig. 8, A and B, illustrate representative examples
of the pH effect on the rate of activation of nicotine- and
carbachol-evoked currents when the agonists were applied at comparable
effective concentrations of 20 and 200 µM, respectively. The data
from a number of cells, quantified in lower graphs of Fig. 8, show that
even though the rise times (10-90%) of nicotine-induced currents at
pH 7.4 and 6.0 were similar for 100 or 200 µM nicotine
concentrations, the current accelerated on average by 26% (from 38 to
28 ms) for 40 µM, and 39% (from 80 to 49 ms) for 20 µM nicotine
(Fig. 8, lower left). For carbachol-induced currents, on the other
hand, the pH effect on the rise time was minimal only at 1000 µM
concentration but became larger at smaller carbachol concentrations at
acidic pH values. For instance, current accelerated ~4 fold for
responses induced by 200 and 40 µM carbachol (from 120 to 28 ms and
from 261 to 99 ms, respectively). The pronounced effect of changes in
pH on activation kinetics of carbachol-induced current may result, in
part, from the lower affinity of this drug for 3/4 nAChRs.
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Fig. 8.
The effect of acidification on the rate of activation
of currents evoked by high- and low-affinity agonists. Effect of pH 6.0 on rise time of currents activated by nicotine concentrations of 20 to
200 µM (A), and by carbachol (40-1000 µM) (B). Top, representative
superimposed traces of rise time of currents induced at pH 7.4 and 6.0 by 20 µM nicotine (A) and 200 µM carbachol (B) in the same cell
(holding potential, 80 mV).
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The Site and Mechanism of Proton-Induced Modulation.
To
determine the site of the proton effect on the nicotinic receptors, a
number of cells (n = 9) were dialyzed with pipette solutions buffered at pH 6.0. Figure 9A
compares nicotine-induced currents in two representative cells
dialyzed, respectively at pHi values of 7.4 or
6.0. There was no significant difference in the kinetics of
nicotine-activated current between cells dialyzed with
pHi values of 7.4 and 6.0 (Fig. 9B). Furthermore,
elevation of extracellular proton concentrations continued to enhance
the current and accelerate its decay kinetics, irrespective of
[H+]i (Fig. 9B). These
data are consistent with those of Fig. 1 and suggest that protons
interact primarily with the extracellular domains of the receptor.
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Fig. 9.
Comparison of the effect of external acidification on
the nicotine-activated current in cells buffered with two different
intracellular pH values. A, representative superimposed traces of
currents evoked by 40 µM nicotine coapplied with either pH 7.4 (trace
1) or 6.0 (trace 2) and return to pH 7.4 (trace 3) in two different
cells dialyzed by pipette solutions of either pH 7.4 (left) or pH 6.0 (right). B, average effects of external acidification on
nicotine-induced currents recorded from cells dialyzed by pipette
solution of pH 7.4 (n = 3) or pH 6.0 (n = 9). Holding potential, 80 mV.
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Whether pH-induced altered cationic selectivity of nAChRs could be
responsible for changes in the magnitude and kinetics of the
agonist-induced current was also tested. Figure
10 shows data from two cells in which
the effects of nicotine and ACh at EC50 values on
the voltage-dependence of the activated current were quantified at pH
6.0, 7.4, and 8.0. Currents induced by ACh or nicotine showed strong
rectification between 0 and 100 mV, a characteristic of 3/4
receptor (Zhang et al., 1999; Haghighi and Cooper, 2000), but there was
no measurable change in the reversal potential of the current activated
at different [H+]o
(n > 20). Note that both ACh and nicotine
significantly enhanced the current and its kinetics as the pH was
changed from 8.0 to 7.4 and 6.0. The data are consistent with the idea
that enhancement of the current and acceleration of its kinetics is
brought about by a mechanism independent of the change in selectivity
of the channel.
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Fig. 10.
Voltage-dependence of nicotine- and ACh- induced
currents at different pH values. Each shows data obtained from a single
representative cell. Current was quantified at its maximal value by
activating it with either 40 µM nicotine (A) or 100 µM ACh (B).
Reversal potentials for nicotine- (26.6, 26.7, and 28.5 mV) or
ACh-evoked currents (33.3, 34.6, and 37.6 mV) were not significantly
different at pH 8.0, 7.4, and 6.0. The three insets in A and B
represent superimposed traces of the evoked currents at the indicated
pH values.
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Experiments of the type shown in Fig. 10 were analyzed in greater
detail to test whether the pH effect was modulated by the holding
potential. The top of Fig. 11 shows the
manner in which the peak currents at different potentials evoked by 40 µM nicotine (A) or 100 µM ACh (B) were altered by pH 6.0 and pH 8.0 relative to the currents measured at pH 7.4. The effects of pH on the
current amplitude was not significantly voltage-dependent (Fig. 11). A similar analysis was performed for the decay kinetics of the current. The rates of decay of the current induced by nicotine (C) and ACh (D)
were independent of voltage at pH 6.0. Consistent with our finding that
protons increase the affinity of 3/4 receptors to the agonists
(see Fig. 2), it is possible that at pH 6.0, the current activated by
40 µM nicotine mimics that induced by saturating nicotine
concentrations at which the effects of protons on the decay kinetics of
the current are markedly reduced or negligible (see Fig. 7). At pH 7.4, the rates of decay of the nicotine- and ACh-induced currents (C and D)
were accelerated at very negative holding potentials, consistent with
the idea that protons or positively charged agonist may modulate the
rate of "desensitization" slightly by moving through a small
fraction of the membrane field. This effect was quantified in terms of
a Boltzmann factor, exp(FVm/RT) [where
Vm is the membrane potential, R
is the gas constant, T is the absolute temperature (RT/F = 25 mV),
and is the fraction of membrane field seen by monovalention]. The
values of , calculated between 120 mV and 0 mV (0.18 for nicotine,
0.11 for ACh), in combination with the apparent Hill-coefficient (~2;
Fig. 7B), suggest that H+ moves through only a
small fraction (<9%) of the electric field of the membrane. This
fraction is even smaller if the rate constants measured at +120 mV are
included in the analysis (<6% for nicotine, < 2% for ACh). These
approximate calculations support the notion that protons do not move
appreciably into the channel pore but modulate the nAChR from
extracellular sites.
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Fig. 11.
Analysis of voltage-dependence of agonist-induced
current and its "desensitization". A, mean (± S.E.M.) amplitude of
the currents activated by 40 µM nicotine (n = 4)
at pH 8.0 () and at pH 6.0 ( ), and that activated by 100 µM ACh
(B; n = 4) at pH 8.0 () and at pH 6.0 (squlo])
measured at different holding potentials and normalized to pH 7.4. Similarly, in the same set of cells, the voltage-dependence of mean
(±S.E.M.) rate constants of decay ("desensitization") of the
currents activated by 40 µM nicotine (C; n = 5)
at pH 7.4 () and at pH 6.0 () are compared with those activated
by 100 µM ACh (D; n = 6) at pH 7.4 () and pH
6.0 (). Because few if any currents were activated at 40 and 80 mV,
these data points were excluded from the analysis.
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Discussion |
The major finding of this report is that rapid coapplication of
agonists and protons first enhances and then suppresses the evoked
current through the 3/4 nAChR. The eventual suppression of the
current is most likely caused by acceleration of the decay kinetics of
the agonist-induced current in acidic pH values, depending, in part, on
the pKa value of the agonist. The pH
effect was specific to the extracellular site of the receptor, was
mostly independent of potential, and was not accompanied by significant
changes in the cationic selectivity of the 3/4 receptor. In this
respect, the response of 3/4 nAChR to acidification was similar
to that described for non-neuronal nAChR in frog neuromuscular junction (Trautmann and Zilber-Gachelin, 1976; Landau et al., 1981), the N-methyl-D-aspartate (Tang et al.,
1990), and GABAA receptor (Krishek et al., 1996).
However, in sharp contrast to the effect of acidification on other
ligand- (Traynelis, 1998) or voltage-gated channels (Tombaugh and
Somjen, 1996), the cationic current through the 3/4 receptor was
transiently enhanced by acidic pH values, suggesting that protons may
rapidly and reversibly alter nAChR gating without interfering with the
permeation path of the receptor. The acceleration of decay kinetics in
acidic pH values led eventually to suppression of steady-state evoked
current consistent with the idea that protons may increase the rate and
extent of desensitization or block of the channel either by interfering
with its permeation site (Imoto et al., 1988; Prod'hom et al., 1989)
and/or by allowing the agonist to serve as an open channel blocker
(Sine and Steinbach, 1984; Ogden and Colquhoun, 1985; Luetje and
Patrick, 1991; Maconochie and Steinbach, 1995; Philipson et al., 2001).
One critical procedure used in the present study that may bear on
comparing the present results with those of other investigators was
that the agonist and the higher proton concentrations were coapplied
rapidly (~ 20 ms) for periods less than 2.0 s, making it
possible to quantify both the transient and quasi-steady-state responses to extracellular acidification. In this respect, it was noted
that the potentiating effects of acidification on the 3/4
occurred within 50 ms, allowing the nicotinic agonists to both
transiently enhance and then suppress the current (Fig. 3). In part,
some of the differences between pH modulation of 3/4 neuronal
nAChR reported here and those reported for the muscle and Torpedo
californica nicotinic receptors may be related to much slower
application of agonists used in the data published previously.
Does [H+]o Change the Affinity of
3/4 nAChR?
Figs. 1, 2, 3, 5, and 8 clearly show that
coapplication of agonists with higher proton concentrations enhanced
the magnitude and accelerated the rate of activation of the current.
These effects were more pronounced at lower agonist concentrations
(Fig. 2 and 8) and/or when using lower affinity agonists (Fig. 5). In
fact, at nicotine concentrations between 0.2 to 1 mM, the amplitude and
the rate of rise of the current were only minimally modified by
elevation of [H+]o (Figs. 2A and 8A). This
finding suggests that as the receptors become fully saturated, their
gating can no longer be modified by elevation of
[H+]o, as if increasing the
[H+]o emulated the increase in agonist
concentration. For carbachol and ACh immunity to pH modulation occurred
at saturating concentrations of ~1 mM (Figs. 2B and 8B). In the case
of acidic agonists, such as nicotine and cytisine
(pKa ~ 6.1), the effects of pH may,
in part, reflect preferential binding of the protonated agonist to receptor sites. In this respect, it is interesting that the natural neurotransmitter ACh, which is ionized and not further protonated at pH
6.0 to 8.0, has a highly pH-sensitive dose-response curve (shifting its
EC50 from 105 µM to 67 µM; Fig. 2B) exhibiting similar pH-induced changes in the amplitude and kinetics of the current.
The fast activation kinetics of AChRs have been analyzed previously in
considerable detail, making it possible to distinguish, for instance,
between ligand-binding and channel opening (Maconochie et al., 1994;
Maconochie and Steinbach, 1995). The role of protons in such multistate
schemes could be important in evaluation of their potential role as
modulators of fast neuronal signaling. In this context, it should be
noted that the resolution of faster (<20 ms) activation times often
observed with high affinity agonists or at higher drug concentrations
(Fig. 8), maybe limited in our study by the inherent delays (~10 ms)
in the application speed of the solutions.
The possibility that elevation of
[H+]o may increase the
apparent affinity of the receptor for the agonists is not supported by
studies on frog, chick, and T. californica non-neuronal
nAChRs, where acidification has been shown to decrease both the
conductance and kinetics of the channels without affecting the agonist
binding affinity (Huang et al., 1978). In muscle nAChR, elevation of
[H+]o decreases both the
single-channel conductance and its mean open time, consistent with the
idea that carboxylic side chains near the vestibule of the channel were
protonated (Imoto et al., 1988). Similarly, elevation of
[H+]o, suppresses the
voltage-gated Ca2+ channels by reducing their
single-channel conductance (Prod'hom et al., 1989; Tombaugh and
Somjen, 1996). The mean open time of non-neuronal nAChR channel,
however, shows bell-shaped dependence on pHo,
with a maximum around pH 7.4, consistent with the idea that protonation
may also affect the alkaline pKa sites
(e.g., histidine moieties) (Landau et al., 1981). On the other hand, somewhat similar to our finding, in GABAA
receptor of cerebellar granule cells or in the recombinant GABA
receptor 11, enhancement of the anionic current at low pH values
seems to be mediated by the higher affinity of the receptors to the
agonist (Robello et al., 1994). Thus, the proton-induced modulation of
3/4 nAChR seems to be qualitatively different from that reported
for the muscle or T. californica nAChRs (Trautmann and
Zilber-Gachelin, 1976; Landau et al., 1981; Palma et al., 1991; Li and
McNamee, 1992) and more consistent with the possibility that
step-increases in [H+]o
induce a transient conformational change of the receptor, resulting in
higher apparent receptor affinity.
Does pH Alter the Decay Kinetics of the Agonist-Induced
Current?
The other major observation of this study is that
elevation of proton concentrations accelerates the decay kinetics of
the 3/4 nAChRs (Fig. 1, 4, 5, 6, and 7). It is likely that
ionization state of the ligand contributes, in part, to acceleration of
relaxation of the current on increasing the
[H+]o, because the decay kinetics of
nicotine- and cytisine-activated current (pKa ~6.1) were
more strongly accelerated at pH 6.0 than those of ACh and carbachol
(with no known pKa) (Fig. 6). This finding suggests that
the protonated forms of nicotine and cytisine may have better access to
the channel pore, consistent with the idea that the decay of the
nicotinic current reflects not only receptor desensitization but also
the agonist-induced channel block (Sine and Steinbach, 1984; Webster et
al., 1999). Models for the agonist-induced channel block of AChRs
suggest that the blocked channels may isomerize into states comparable
with a drug-bound closed state or the "desensitized" state
(Maconochie and Steinbach, 1995). Alternatively, nicotine may bind to
some inhibitory site with lower off-rate (dissociation rate) that
becomes available when the receptor is subjected to
superpharmacological concentrations of the agonist (Webster et al.,
1999).
In some sets of experiments, the rate constants of decay of the
agonist-induced currents were found to be different even under identical conditions; for instance, the values presented in Figs. 9 and
11 are somewhat smaller than those in Figs. 6 and 7. Such variations in
the rate of "desensitization" were reported earlier (Zhang et al.,
1999) in this cell line, and may be related to the stoichiometry of
subunit expression.
In contrast to our observations, the bell-shaped dependence of pH
modulation in frog neuromuscular junction (Landau et al., 1981) was
interpreted to be caused by the electrostatic interaction between two
fixed and titratable ionic groups and a mobile charge in the receptor
molecule. It is likely, therefore, that different nicotinic receptor
subtypes are differentially modulated by
[H+]o, depending, in
part, on their molecular structure.
The action of protons seems to occur at extracellular sites of the
3/4 receptor, because intracellular acidification produced no
effects (Fig. 9). Similarly, the protons do not seem to penetrate sufficiently into the channel pore or membrane field to alter ionic
selectivity (Fig. 10) or voltage dependence (Figs. 10 and 11),
respectively. This finding is consistent with those reported for the
nAChR in frog neuromuscular junction (Trautmann and Zilber-Gachelin, 1976; Landau et al., 1981).
Physiological Implications.
Rapid coapplication of the
nicotinic agonists and protons provides an approximation to
physiological release of the contents of the vesicle into the synaptic
cleft. Considering the high vesicular proton concentration (pH ~ 5.5; Miesenbock et al., 1998), it is likely that significant transient
acidification of the postsynaptic receptor takes place before
activation of the current. If indeed the receptors in vivo were to
behave like the recombinant 3/4 receptors, then ACh, a
low-affinity agonist, would transiently develop the properties of a
high-affinity ligand, thus evoking a larger and faster current,
especially at lower ACh concentrations (Fig. 5). The advantage of
protons as cotransmitters includes rapid diffusion speeds, ability to
be rapidly buffered by the extracellular buffers, and allowing the
low-affinity ligand to have both fast on- and off-rates. The proposed
pH-modulation of neuronal signaling is presently rather speculative
because it will depend on the yet-to-be-determined ability of nAChR to
respond to a possible pH change in the synaptic clefts. Irrespectively, such a mechanism would be quite different from the well-studied suppression of nicotinic current by longer lasting acidification.
We also considered how the enhanced relaxation of current might
accelerate synaptic signaling. The finding that progressively longer
exposures to acidic pH values during the onset of "desensitization" of 3/4 nAChRs results in the enhancement of the reactivated nicotinic current (Fig. 4) might suggest that as protons accelerate the
relaxation of the current, they may also protect the 3/4 receptor
against "rapid desensitization". This is consistent with the
finding that the pH-induced reactivated current remained fairly constant during the time course of "rapid desensitization" compared with that induced by step-increases in nicotine concentrations (Fig. 4,
see also Fig. 1C). It is possible that as protons increase the apparent
affinity of 3/4 receptors to the agonist, they may also slow down
the processes that leads to "rapid desensitization" of the receptor
(Fig. 4). We speculate that during repetitive firing of a cholinergic
synaptic pathway, as protons and ACh are coreleased from the secretory
vesicles for a few milliseconds into the synaptic cleft, they provide
the conditions that are essential for generating rapidly relaxing
currents mediated by the slow 3/4 receptor (Fenster et al., 1997)
without causing significant "desensitization" that would preclude
its reactivation.
We thank Dr. Lars Cleemann for critical reading of the
manuscript and many helpful comments. We also thank Dr. Kenneth J. Kellar for providing the cell line expressing 3/4 nAChRs.
This work was supported by National Institutes of Health grants
R01- HL62525 and DA06486.
Dr. Martin Morad, Department of
Pharmacology, Georgetown University School of Medicine, 4000 Reservoir
Road Building D, Washington DC 20007. E-mail: moradm{at}georgetown.edu
nAChR, nicotinic acetylcholine receptor;
HEK, human embryonic kidney;
ACh, acetylcholine;
GABA, 
-aminobutyric
acid.
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3/
4 Neuronal
Nicotinic Acetylcholine Receptor by Increasing Its Apparent Affinity to
Agonists
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Abstract
Introduction
