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Introduction
Nicotinic acetylcholine receptors (nAChR) are
prototypes of the ligand-gated ion channel superfamily of
neurotransmitter receptors[1_4]. nAChR have historic importance,
as their existence as "receptive substances" in vertebrate
muscles was gleaned a century ago[5]. They have become
models for the establishment of concepts pertaining to
mechanisms of drug action, synaptic transmission, and the
structure and function of transmembrane signaling molecules.
Several nAChR subtypes have distinctive features that are
dictated in part by their composition from subunits derived
from at least 16 genes. The predominant, high-affinity,
nicotine-binding nAChR subtype in the brain contains
α4 and β2 subunits (α4β2_nAChR)[1,6]. Numbers and/or functions of
α4β2_nAChR are affected by nicotine at concentrations
found in the plasma of tobacco
users[1,7_9], and α4β2_nAChR have been implicated in nicotine self-administration, reward,
and dependence[1,2,10,11]. α4β2_nAChR also play important
roles in health and a variety of neuropsychiatric
diseases[12].
Like acetylcholine (ACh), nicotine acts acutely to cause
rapid opening of nAChR channels. However, these
responses are transient, and channel opening frequency
diminishes with protracted exposure to high concentrations of
agonists through a process (or a series of processes) termed
"desensitization"[13,14]. Depending on the duration and
concentration of agonist exposure, the rates of recovery from
desensitization can vary, but explanations of mechanisms
involved in the induction and recovery from desensitization
have been elusive. Studies done mostly using muscle-type
nAChR suggest that open-channel block by a high
concentration of agonists contributes to the loss of function, and
evidence for this has come from work showing the
production of a transient nAChR response after the removal of
applied agonists called a "hump" or "rebound"
current[15,16]. Hump currents also have been observed for other channels,
and one interpretation has been that they reflect the
transient reactivation of channel opening when agonist molecules
that had engaged in open-channel block leave the channel
pore[16_18].
Here we report on experiments that tested the
hypothesis that open-channel block by high agonist
concentrations and hump current production are attributes of human
α4β2_nAChR.
Materials and methods
Subclonal human epithelial 1 (SH-EP1)_hα4β2 cells
Established techniques[19] were used to introduce human
α4 (S452) and β2 subunits (kindly provided by Dr Ortrud
STEINLEIN, Institute of Human Genetics, University
Hospital, Ludwig-Maximillians-University, Germany)
and subcloned into pcDNA3.1-zeocin and
pcDNA3.1-hygromycin vectors, respectively) into native nAChR-null SH-EP1
cells[20] to create the stably-transfected, monoclonal
SH-EP1-hα4β2 cell line heterologously expressing human
α4β2_nAChR. Cell cultures were maintained at low passage numbers (1_26
from our frozen stocks to ensure the stable expression of the
phenotype) in complete medium[21] augmented with 0.5
mg/mL zeocin and 0.4 mg/mL hygromycin (to provide a positive
selection of transfectants) and passaged once weekly by
splitting the just-confluent cultures 1/20 to maintain cells in
proliferative growth. RT_PCR, immunofluorescence, radioligand-binding assays, and isotopic ion flux assays were
conducted recurrently to confirm the stable expression of
α4β2_nAChR as message-, protein-, and ligand-binding sites,
and functional receptors.
Patch-clamp whole-cell current recordings
Conventional whole-cell current recording, coupled with
computer-controlled U-tube fast application and the removal of agonists,
was applied in this study as previously
described[22_24]. Briefly, the cells plated on polylysine-coated 35 mm culture
dishes were placed on the stage of an inverted microscope
(Olympus iX70, Lake Success, NY, USA) and continuously
superfused with standard external solution (2 mL/min). Glass
microelectrodes (3_5 MΩ resistance between the pipette and
extracellular solutions) were used to form tight seals (>1
GΩ) on the cell surface until suction was applied to convert to
conventional whole-cell recording. The cells were then
voltage-clamped at holding potentials of _60 mV, and ion
currents in response to application of ligands were measured
(Axon Instruments 200B amplifier, Foster City, CA, USA).
Whole-cell access resistance was less than 20 MΩ before
series resistance compensation. Both pipette and
whole-cell current capacitances were minimized, and series
resistance was routinely compensated to 80%. Typically, data
were acquired at 10 kHz, filtered at 2 kHz, displayed and
digitized online (Digidata 1322 series A/D board, Axon
Instruments, USA), and stored to a hard drive. Data
acquisition of whole-cell currents was done using Clampex 9.2 (Axon
Instruments, USA), and the results were plotted using
Origin 5.0 (Microcal, North Hampton, MA, USA) or Prism 3.0
(GraphPad Software, San Diego, CA, USA). nAChR acute
desensitization (the decline in inward current amplitude over
the course of agonist application) was analyzed for decay
half-time (t=0.693/k for decay rate constant k), peak current
(Ip), and steady-state current
(Is), using fits to the mono (or
double) exponential expression
I=([Ip_Is
] e_kt)+Is (or
I=([Ip_Ii
] e_k1t)+([Ii_I
s] e_k2t)+Is, where
Ii is the intermediate level of
current and k1 and k2 are rate constants for the 2 separate decay
processes. Curve fitting usually was done using data
between 90% and 10% of the difference between peak and
steady-state currents. The experimental data are presented
as mean±SEM, and comparisons of different conditions were
analyzed for statistical significance using
t-tests. All experiments were performed at room temperature (22±1
oC). Concentration response profiles were fit to the Hill equation and
analyzed using Origin 5.0.
Patch-clamp single-channel recordings Outside-out
patch, single-channel recordings were performed to
compare single-channel properties of α4β2_nAChR-mediated
currents at different concentrations of ACh or at different
holding potentials. After establishing conventional whole-cell
configuration, the recording electrode was gently removed
from the cell and an outside-out patch was formed.
Single-channel signals activated by application of nicotine via a
U-tube were acquired while being filtered at 1 kHz (8-pole
Butterworth filter and digitized at 10 kHz; Digidata interface
1322A). The results were analyzed using Clampfit 9.2. Time
constants for open and closed intervals and amplitude
distributions were computed and corrected for dead time (defined
as the longest duration event that was allowed to be missed;
usually 250 ms). Channel amplitude histograms were
constructed from open-time events with durations longer than
the dynamic frequency response limit of the entire system in
order to exclude amplitude attenuation due to system
bandwidth limitation. Channel open-time histograms were based
on open events with a duration longer than 0.2 ms separated
by closed times no shorter than 0.2 ms. Open-time
constants (t) were obtained from least squares fits of the data;
the mean open time (to) was calculated as the arithmetic mean
of the sum of all open events within a record. In addition,
records were analyzed for open events shorter than or equal
to 1 ms as a percentage of all open events. Channel
conductance distribution, the mean open time, and the mean closed
time were fit using Qub software (State University of New
York, Buffalo, NY, USA).
Homology modeling and ACh docking into channel pore
Five sets of 4 transmembrane domains of the human
nicotinic receptor subunits with a subunit arrangement of
α4β2β2α4β2 (clockwise from an extracellular view) with chain
breaks were aligned with the deduced structure of
transmembrane domains from the Torpedo nicotinic receptor
(protein data bank file 1OED) using the modeler in
Discovery Studio 1.7 (Accelrys, San Diego, CA, USA; "Align
Sequence with Structure" protocol with blosum62 scoring
matrix, gap open penalty of _200, gap extension penalty of
_10, and default 2-D gap weights). The homology model was
then built using the "Building Homology Models" protocol.
The resulting model was further typed with the "CHAMm
force field" tool and energy minimized by the "minimization"
protocol with 400 steps of steepest descent cycles followed
by 1000 steps of conjugate gradient algorithm. The docking
of ACh into the ion channel pore of the α4 and β2 subunit
homology model was performed with ICM pro (Molsoft, San
Diego, CA, USA) with manual adjustment of the docking
box to cover the full length of the pore region. The docking
result was presented with Swiss PDB Viewer 3.7 (Swiss
Institute of Bioinformatics, Basel, Switzerland) and rendered by
POV-Ray 3.6 (Persistence of Vision Raytracer Pty Ltd,
Williamstown, Victoria, Australia).
Solutions and drug application The standard external
solution contained (in mmol/L): 120 NaCl, 3 KCl, 2
MgCl2, 2 CaCl2, 25 D-glucose, and 10 HEPES, and was adjusted to pH
7.4 with Tris-base. In the experiments, ACh was applied as
an agonist without atropine since our preliminary data
showed that 1 µmol/L atropine sulfate did not affect
ACh-induced currents[22], and also atropine itself was reported to
block nAChR[25]. For most conventional whole-cell
recordings, K+ electrodes were used and filled with solution
containing (in mmol/L): 140 KCl, 4
MgSO4, 0.1 EGTA, 4 Mg-ATP, and 10 HEPES, and adjusted to pH 7.2 with Tris-base.
In other experiments, Tris+ electrodes were used and filled
with solution containing (in mmol/L): 110 Tris phosphate
dibasic, 28 Tris-base, 11 EGTA, 2 MgCl2, 0.1
CaCl2, and 4 Mg-ATP; pH
7.3[26]. For studies in
Ca2+-free external solutions, external 2 mmol/L
CaCl2 was replaced with 4 mmol/L NaCl, and
1 mmol/L EGTA was added to the solution.
To initiate whole-cell current responses, nicotinic
agonists were delivered into the bath medium near to the cell
being recorded via a computer-controlled U-tube system so
that solution exchange occurred within 30 ms (based on
10%_90% peak current rise times). Intervals between drug
applications (3 min) were adjusted specifically to ensure the
stability of nAChR responsiveness (without functional
rundown), and the selection of pipette solutions used in
most of the studies described here was made with the same
objective. The drugs used in the present study were (_)
nicotine, ACh, dimethyl-phenyl-piperazinium (DMPP), epibatidine
(EPBD), cytisine, lobeline, and dihydro-β-erythroidine
(DHβE); all were purchased from Sigma (St Louis, MO, USA).
Results
Heterologous expression of human α4β2_nAChR in
SH-EP1 cells SH-EP1 cells exhibited a range of morphologies
before (data not shown) or after (Figure 1A) transfection
with nAChR α4 and β2 subunits. The RT_PCR analyses
showed the expression of human nAChR α4 and β2 subunit
messages in the SH-EP1_hα4β2 cells (Figure 1B). In contrast,
there was no such expression in the absence of the reverse
transcription step or in the untransfected cell host despite
successful amplification of GAPDH message in all of the
cells (Figure 1B). Apparent levels of α4 and β2 subunit
transcripts assessed using mRNA fluorescence in
situ hybridization did not seem to fluctuate as a function of cell
confluence or between grouped or solitary cells (data not
shown). Functionally, nicotinic agonists (nicotine and ACh)
failed to induce detectable currents in the untransfected cells
(Figure 1C), but induced inward currents in α4- and
β2-transfected cells (Figure 1C). These results indicated the
appropriate expression of nAChR subunit cDNA as messages in
stably-transfected SH-EP1 cells and the generation of
functional nAChR as a consequence.
Concentration-dependence of hump current production
Open-channel block by nicotinic agonists at high
concentrations has been previously reported for several
muscle-type nAChR, and one of its manifestations is the production
of hump currents[16,27,28]. Our initial studies examined
whole-cell current responses of human α4β2_nAChR stably
expressed in SH-EP1_hα4β2 cells to ACh applied as 4 s pulses
at 3 min intervals at concentrations between 1 µmol/L and 10
mmol/L (Figure 2A). The peak whole-cell current responses
of α4β2_nAChR to nicotine or ACh were achieved rapidly
after the application of the agonist and showed a
concentration dependence that was well fitted to the Hill equation,
yielding a half-maximal effective concentration
(EC50) and a Hill coefficient of 21 µmol/L and 0.6 for ACh (see figure 2
legend for fits to 1- or 2-site models). In this study, we
defined whole-cell current responses that typically decay
during ACh exposure to approach steady-state levels as acute
desensitization (ie loss of function during acute exposure to
agonists) of α4β2_nAChR function. The dose dependence
for the absolute magnitude of steady-state currents was
bell-shaped because steady-state inward current amplitudes fell
from their highest levels at intermediate concentrations (~100
µmol/L) of ACh to lower levels at or above 1 mmol/L ACh
(Figure 2B). Inward hump currents evident during drug
washout were observed for whole-cell currents induced by either
nicotine (1 mmol/L) or ACh (10 mmol/L), and the
concentration dependence for this effect had features of the early phase
of a sigmoid log concentration_response profile (Figure 2B).
Hump currents induced by different agonists
To determine whether nicotinic agonists differ in their abilities to
induce hump currents, whole-cell current responses were
ascertained for several agonists applied at concentrations
near to those that produced maximal responses of human
α4β2_nAChR[19,24]. Whole-cell current responses from 6_10
cells showed that hump currents were induced by 10
mmol/L ACh, 1 mmol/L nicotine, 0.1 mmol/L DMPP, and 0.1 mmol/L
lobeline, but not by 1 μmol/L EPBD or 0.1 mmol/L cytisine.
Figure 3 summarizes the ratio of hump/peak currents induced
by different agonists (Figure 3B) and the rising time of peak
and hump currents induced by 10 mmol/L ACh, 1 mmol/L
nicotine, 0.1 mmol/L DMPP, and 0.1 mmol/L lobeline. This
finding discounted the possibility of a technical artifact due
to the poor removal of drug and/or wash-back reperfusion of
the cell with agonists because not all agonists produced
hump currents. ACh, nicotine, DMPP, and EPBD are full
agonists, whereas cytisine and lobeline are partial agonists
at human α4β2_nAChR, indicating that hump current
production was not an attribute of only full or partial agonists.
The rates of acute desensitization were faster for ACh,
nicotine, EPBD, and lobeline than for DMPP or cytisine,
indicating that hump current production was not obviously
related to the acute desensitization rate. ACh and DMPP are
positively-charged, quaternary ammonium ions, whereas the
other compounds are not. Lobeline and ACh are the largest
and smallest of these compounds, respectively. Thus, the
ability to induce hump currents is not simply and solely
attributable to agonist size, charge, or acute functional potency.
Conductance change associated with hump current
production When hyperpolarizing pulses (10 mV, 50 ms) were
applied at different times during the recording of whole-cell
current responses to monitor cellular membrane conductance,
evidence for conductance increases proportional to inward
current amplitude was obtained during both peak and hump
current phases (Figure 4). In contrast, conductance during
the steady-state phase of the whole-cell response was
similar to that prior to the application of agonists, suggesting
that the receptor desensitized to a non-functional status.
Current-voltage relationship for hump currents
To test the hypothesis that hump currents reflect the reactivation of
nAChR recovering from open-channel block after agonist
removal, current-voltage relationships for peak and hump
currents were assessed. When whole-cell current responses
to 10 mmol/L ACh were measured using cells voltage clamped
at different holding potentials, both peak and hump current
responses showed similar current-voltage relationships and
reversal potentials (~0 mV) and evidence for inward
rectification (Figure 5A_5C). These findings are consistent with
current transit through the same channel during peak or hump
currents mediated by α4β2_nAChR.
Pharmacological blockade of hump currents
To further characterize the pharmacological features of hump currents,
the effects of DHβE were determined. DHβE is a potent
antagonist at α4β2_nAChR, as it reduces whole-cell peak
current responses to 3 µmol/L nicotine with a half-maximal
inhibitory concentration (IC50) of _6.05±0.03 mol/L and a Hill
coefficient of _1.45±0.16 (Figure 6A). DHβE appears to
operate as a competitive antagonist[19] because its presence at
a concentration of 300 nmol/L shifted nicotine-induced peak
current response curves to the right (the nicotine
EC50 of 3.1 µmol/L in the absence of
DHβE was shifted to 9.2 µmol/L in the presence of 300 nmol/L
DHβE; Figure 6B). The effects were measured on whole-cell current responses to 10
mmol/L ACh when 100 µmol/L DHβE was applied at different times
relative to the application of agonists (Figure 6C). Under
conditions where ACh application alone induced both peak
and hump currents (Figure 6C), DHβE application
simultaneous with ACh application dramatically reduced peak
current responses and eliminated hump currents (Figure 6C).
Brief pretreatment with DHβE, followed by exposure to both
ACh and continuing DHβE, fully blocked both peak and
hump currents (Figure 6C). If DHβE was applied after peak
current production by ACh, but before ACh was washed
out, a complete block of the hump currents occurred (Figure
6C). If DHβE application preceded ACh application, the
peak current responses were blocked, but if the removal of
DHβE occurred prior to ACh washout, hump current
production became evident (Figure 6C). These results indicate that
DHβE can block both peak and hump currents or can block
either peak or hump currents separately, depending on the
timing of drug applications, which is consistent with the
production of hump currents by reactivation of α4β2_nAChR
after the washout of agonists applied at high concentrations.
Hump current amplitude is smaller for desensitized
nAChR Acute desensitization of nAChR responses
during seconds of agonist exposure is reversible if nAChR
are allowed to recover before being challenged again with
agonists. However, if agonist exposure continues, then
recovery from loss of function is slower, apparently reflecting
conversion to more deeply desensitized states. To test for
the sensitivity of hump current production to nAChR
desensitization, 2 approaches were applied to manipulate
the rates and extents of desensitization and to determine the
consequences for hump current production. First, the
ability to manipulate the rates and extents of nAChR
desensitization by conducting whole-cell current recording using
pipettes filled with different solutions was exploited. The rates
and extents of acute desensitization occurring during
agonist exposure were higher if whole-cell current recordings
were derived using K+ electrodes, rather than using
Tris+ electrodes (Figure 7A). When peak whole-cell current
amplitudes in response to 10 mmol/L ACh were normalized, the
magnitudes of hump currents also were lower when recorded
using K+ electrodes than when using
Tris+ electrodes (Figure 7A). Thus, the higher rate and greater extent of nAChR
desensitization observed when the cytosolic space was
perfused with K+ electrode solution was associated with
eliminated hump current magnitude. In contrast, a lower rate and
extent of desensitization occurred when recordings were
made using Tris+ electrodes, and more nAChR were
available to participate in hump current production. Second, the
ability to manipulate levels of nAChR desensitization by
altering the duration of ACh exposure was exploited. When
normalized to peak whole-cell current response to 10 mmol/L
ACh, hump current amplitude was greatest for the shortest
duration of ACh exposure and smallest for the longest ACh
exposure time (Figure 7B). Figure 7B shows that hump
currents declined with prolonged agonist exposure time. This
suggests that desensitization of nAChR also desensitizes
hump current production, consistent again with hump
current production by nAChR capable of responding to
agonists during removal from blocked channels.
Extracellular Ca2+ eliminates hump currents
We altered external Ca2+ concentrations to ascertain the effects on
hump current production. At a concentration of 1 mmol/L
ACh, little hump currents were induced when the external
medium contained 2 mmol/L Ca2+ (Figure 8A), while obvious
hump currents were produced after external solution exchange
to remove extracellular Ca2+ (ie using medium that contained
no Ca2+, but was supplemented with 1 mmol/L EGTA; Figure
8A). Figure 8B summarizes the effects of external
Ca2+ on ACh-induced currents. Across 7 tested cells, the peak
components for 1 mmol/L ACh-induced currents were not
significantly different as a function of external
Ca2+ concentration. However, hump current amplitudes were
significantly increased upon removal of external
Ca2+. Interestingly, the rate of acute desensitization (inverse of
the decay half-time) was significantly increased upon
removal of external Ca2+. Thus, although these findings
suggest that external Ca2+ at physiological concentrations may
slow the rate of α4β2_nAChR desensitization, it still inhibits
hump current production.
Single-channel evidence for agonist-induced
open-channel block of α4β2_nAChR Agonist-induced open-channel
block has been studied by single-channel recordings using
peripheral muscle-type nAChR[16,27,28]. Key evidence for
open-channel block in muscle-type nAChR relies on
concentration- and voltage-dependent reductions in mean
single-channel currents[27,28]. To determine the single-channel
properties of agonist-induced open-channel block, outside-out
patch recordings were applied. Figure 9 shows that 1µmol/L
nicotine induced inward single-channel currents with
dominant amplitude of 5.97±0.86 pA at a
VH of _100 mV (Figure 9A). However, as nicotine concentration increased 100-fold,
the mean amplitude of single-channel currents was reduced
to 3.81±0.78 pA (Figure 9B) at a
VH of _100 mV (P<0.05). Figure 9A,9B shows the distribution of the amplitude of
single-channel currents induced by 1 or 100 μmol/L nicotine
for VH=_100 mV. Figure 10 compares the channel mean open
or closed durations for single-channel activities induced by
1 or 100 µmol/L nicotine at a VH of _100 mV. When the cells
were exposed to 1 µmol/L nicotine, the mean open and closed
times (t) were 1.7±0.2 and 9.8±1.5 ms (Figure 10A,10B),
respectively, whereas exposure to nicotine at 100 µmol/L
shortened the mean open time (t=0.3±1.1 ms;
P<0.01; Figure 10C) and prolonged the mean closed time
(t=16.1±1.3 ms; P<0.01; Figure 10D). These results indicate that like
muscle-type nAChR[27], transfected human neuronal
α4β2_nAChR exhibit the same open-channel block phenomenon
represented as concentration- and voltage-dependent features in
single-channel activity.
Putative-binding site for ACh in the channel domain of
the receptor Using a homology model of the pore-forming
transmembrane domains of the α4β2_nAChR and docking
software[29] with the selected docking box covering the
entire pore region, we successfully docked ACh to a site close
to the intracellular end of the pore (Figure 11). This potential
ACh-binding site was immediately below the putative
channel gate[30]. ACh, with its longitudinal axis oriented
horizontally, interacted with residues, mainly at the 6' and 3'
positions of the second transmembrane domains of 4
subunits (2 α4 and 2 β2). Occupancy by ACh in the narrow part
of the pore clearly blocked the ion path of the channel. This
location is similar to the binding site for non-competitive
antagonists/open-channel blockers (eg picrotoxin) in the pore
of the γ-aminobutyric acid type A
(GABAA) receptor[31], another member of the cys-loop receptor family.
Open-channel blocking behavior and conservation with the binding
sites of other channel blockers in the same protein family
further support the location of the putative ACh-binding
site in the pore of the α4β2_nAChR.
Discussion
Major findings from this study This study demonstrates
that the exposure of heterologously-expressed human
α4β2_nAChR to select nicotinic agonists at high concentrations
induces acute desensitization of functional responses
during the period of agonist exposure, but also induces hump
currents during drug washout. Magnitudes of peak and hump
current responses are directly proportional to whole-cell
membrane conductance. Hump currents show voltage
dependence and sensitivity to competitive antagonist
blockade like those for peak whole-cell current responses
mediated via α4β2_nAChR. Prolonged exposure to ACh
inducing more pronounced acute desensitization of inward
currents also produces decreases in hump current amplitude.
Changes in internal ion composition that slow acute
desensitization also allow for the maintenance of higher hump
current amplitudes, although the removal of external
Ca2+ increases both the apparent rate of desensitization and hump
current amplitude. Using outside-out patch single-channel
recordings, high agonist concentrations reduced current
amplitude, shortened the channel mean open time, and
prolonged the channel mean closed time, further supporting the
agonist-induced open-channel block in human α4β2_nAChR.
Collectively, the present evidence suggests the hypothesis
that hump currents occur when the agonist producing the
blockade of open α4β2_nAChR channels is rapidly released
from the channel pore (low-affinity sites) while the agonist is
still bound to α4β2_nAChR external-binding sites
(high-affinity sites). Both open-channel block and hump current
production may occur during synaptic activity that releases
a high concentration of ACh, and therefore may underlie
some functions of α4β2_nAChR and their modulation.
Other examples of agonist-induced hump currents
Hump or rebound currents like those described in this study of
human α4β2_nAChR have been found in studies of the effects
of high concentrations of ACh on muscle-type α1*_nAChR
at frog end-plates[15] and the effects of high (>1
mmol/L) concentrations of pentobarbital on presumed
GABAA receptor-gated Cl- currents in isolated frog sensory
neurons[32]. In the latter study, the sensitivity of hump currents to the
selective GABAA receptor blocker bicuculline was taken
as evidence for the reactivation of
GABAA receptors by pentobarbital released from blocked channels by rapid
washout[32]. Hump currents also were observed in acutely
dissociated hippocampal CA1 neurons exhibiting
strychnine-induced K+
currents[17] or sevoflurane-induced
Cl- currents[18]. More recently, rebound currents mediated by muscle-type
nAChR were detected using outside-out patch recordings
and modeled using computer simulations consistent with
the release from open-channel block[16].
Voltage-dependence, antagonist block, and
desensitization of hump currents The very similar current_voltage
relationships for peak and hump current production are
consistent with both events reflecting the opening of the same
kind of channel with the same permeability characteristics.
Although a more complete description of the pharmacology
for the blockade of hump currents could prove useful,
sensitivity to blockade by DHβE was seen for both peak and
hump currents or for peak or hump currents individually.
Prolonged exposure to ACh or manipulation of intracellular
solution composition to enhance the acute desensitization
of α4β2_nAChR during agonist exposure also produces
desensitization of hump current responses. All of these results
are consistent with hump current production by the same
channels that mediate peak currents and with reactivation of
nAChR channel opening by agonists rapidly leaving the
channel after participating in open-channel block.
Concentration-dependence of hump current production
by α4β2_nAChR Hump current production by α4β2_nAChR
only occurs after washout of higher concentrations of
selected agonists. For muscle-type nAChR, it has been
hypothesized that the existence of 2 distinct binding sites, one
with lower agonist-binding affinity perhaps within the pore
and one with higher affinity at the nAChR activation site,
may help to explain this effect[27,33]. We entertain a similar
explanation for findings in studies of α4β2_nAChR, although
it seems that the processes of nAChR inactivation must be
more complex than can be explained by a 2-site model. For
example, acute desensitization (ie loss of whole-cell inward
currents during seconds of exposure to an agonist) of
α4β2_nAChR occurs for all nicotinic agonists tested at any
concentration that shows significant production of peak
whole-cell currents, at least under some conditions of recording (ie
using K+ electrodes). However, not all agonists tested were
capable of producing hump currents. Thus, the mechanism
involved in acute desensitization and the mechanism involved
in the loss of nAChR function that is a precursor to hump
current production cannot be entirely reconciled. Perhaps a
combination of these and other features of agonists are
required for hump current production and/or the process that
enables it, but the ability to induce hump currents could not
be simply attributed to drug action as full or partial agonists,
to drug charge or size, or to drug acute potency.
Concentration_response studies indicate that a hump
current magnitude produced after exposure to 10 mmol/L ACh
is similar to that produced as peak currents by exposure to
~300 µmol/L ACh (ie approximately 35% of the peak current
produced by 10 mmol/L ACh). Because hump currents are
generally temporally broader than peak currents, perhaps
reflecting less synchrony in channel openings than on
initial rapid agonist application, the percentage of peak current
response may be underestimated. Assume for the moment
that the ability of ACh to convert nAChR at rest to the
open-channel state is equal to the ability of ACh to convert nAChR
from the open-channel blocked state to the open state and
that all nAChR are in open-channel block during exposure to
10 mmol/L ACh just before drug washout. The
concentration_response studies would then suggest that 35% of
open-channel blocked nAChR are reactivated by ACh, leaving
channels after 10 mmol/L ACh exposure, meaning that nAChR
reactivation would be quite an efficient process, all the more
so if the percentage of functionally-available nAChR was
reduced due to desensitization. The basis for this efficiency
is not presently clear. Outside-out single-channel
recordings demonstrated that a higher concentration (100 µmol/L)
of nicotine reduced current amplitude, shortened the
channel open time, and prolonged the channel closed time,
further supporting the hypothesis that open-channel block
precedes hump current production.
Practical exploitation of hump currents to illuminate
mechanisms of nAChR function Hump currents could serve
as important indicators of receptor conditions that might
illuminate obscure notions about receptor state during acute
desensitization, the existence of open-channel block, and
processes of functional recovery from desensitization and
open-channel block. There is still disagreement as to whether
hump current production occurring during the washout of
agonists applied at high concentrations reflects the
reactivation of desensitized receptors/channels recovering from
open-channel block[32] or rapid recovery from full
desensitization[16]. The hypothesis of rapid recovery from full
desensitization would seem at odds with the traditional cyclical
reaction scheme, in which the recovery of nAChR from full
desensitization to the basal state would require more than
one step[13,34,35], and with measures showing that such a
process requires no less than 1 s[36]. In the latter studies, which
were done using a double 0.1 mmol/L ACh pulse protocol to
measure the time constant of muscle-type nAChR recovery
from receptor desensitization, ~92% desensitization remained
50 ms after the first conditioning
pulse[36]. In the present study, the time for washout of agonists was approximately
30 ms, and hump current amplitude was approximately 35%
of peak current amplitude, even though a much higher
concentration of ACh was used. Thus, either kinetics for
recovery from desensitization differs for muscle-type and
α4β2_nAChR or hump current production does not reflect
recovery from desensitization.
The present findings that hump currents can be
desensitized with prolonged agonist exposure or with modulation of
intracellular solution also strongly suggest a distinction
between desensitization and the process that precedes hump
current production. These findings could indicate that loss
of nAChR function (whole-cell current decay) during acute
exposure to high concentrations of agonists is due to a
combination of desensitization and open-channel block. With
increased time of agonist exposure, there would be
proportionately more desensitization and smaller amplitude hump
current production. Decreased internal
K+ would decrease the rate and/or extent of whole-cell current decay, increase
the proportion of nAChR inactivated via open-channel block,
and thereby increase hump current amplitude. From this
perspective, perhaps agonist-induced open-channel block
protects against desensitization. Recently, Bianchi and
Macdonald[37] formulated an "agonist trapping" concept to
explain the slow kinetics for the decay of GABAergic
inhibitory postsynaptic potentials. They postulated that the
conversion of closed or desensitized channel states to open
states only occurs if those closed or desensitized states have
bound agonists. By analogy, the open-channel block of
nAChR may be viewed as a form of agonist trapping,
although in this case not at the active site, but through a
mechanism that would allow for the rapid release of agonists
from the channel pore, leading to apparent recovery from
desensitization, manifested as hump current production
immediately after agonist washout. However, the present study
employing the manipulation of external
Ca2+ concentrations suggests that hump current production is more complicated
than simple recovery from desensitization and that the
extent of open-channel block (revealed by hump current
magnitude) may not be simply inversely proportional to rates
or extents of desensitization. This is because both hump
current amplitude and the rate of desensitization of
α4β2_nAChR are decreased in the presence of external 2 mmol/L
Ca2+. Although there may be other ways in which
Ca2+ modulates nAChR function, perhaps this reflects the existence of
different sites on α4β2_nAChR involved in the
Ca2+ inhibition of agonist access to open-channel blocking sites and in
the Ca2+ inhibition of desensitization.
Physiological significance of hump currents
The present study and other previous studies have established
evidence for the hypothesis that hump current production
indicates the reactivation of receptor function recovering
from agonist-induced open-channel
block[16_18,32,38]. In studies of nAChR at the zebrafish neuromuscular junction, and
consistent with their simulations, Legendre et
al[16] found that the synaptic current time-course was influenced by rates
of activation and deactivation of nAChR at lower ACh
concentrations, but at higher peak concentrations of released
ACh the duration of hump (rebound) currents more strongly
influenced synaptic current temporal profiles. Thus, at sites
proximal to those involved in the release of ACh at high
concentrations, open-channel block and subsequent hump
current production could help prolong postsynaptic
responses and provide distinctive signatures for sites at
different distances from nerve terminals that could translate
temporal forms into spatial forms of coding critical for
synapse formation, maintenance, and remodeling.
Acknowledgements
The contents of this report are solely the responsibility
of the authors and do not necessarily represent the views of
the aforementioned awarding agencies. The authors thank
Dr Ortrud STEINLEIN for the gracious gifts of human
α4 and β2 subunit cDNA.
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