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Research report
Choline and acetylcholine have similar kinetic properties of activation
and desensitization on the
a
7 nicotinic receptors in rat hippocampal
neurons
a ,1 b a,b ,
*
Arpad Mike
, Newton G. Castro , Edson X. Albuquerque
a
Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 655 W. Baltimore St., Baltimore, MD21201, USA
b
´ ´
Departamento de Farmacologia Basica e Clınica, ICB, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-590, Brazil
Accepted 15 August 2000
Abstract
Thea7-type nicotinic acetylcholine receptor (nAChR) was recently found to be both fully activated and desensitized by choline, in addition to ACh. In order to understand the combined effects of the two agonists ona7 nAChR-mediated neuronal signaling, the kinetics of the receptor-channel’s interaction with ACh and choline was examined. To this end, whole-cell and single-channel currents evoked by fast-switching pulses of the agonists were recorded in rat hippocampal neurons in culture. Currents evoked by equieffective concentrations of choline and ACh were very similar, except that choline-evoked currents decayed more quickly to the baseline after removal of the agonist, and that recovery from desensitization was faster with choline. The conductance of channels activated by choline and ACh was 91.568.5 and 82.9611.6 pS, respectively. The mean apparent channel open times were close to 100ms, with both agonists. After a 4-s exposure to concentrations up to 80mM ACh or 600mM choline, the extent of desensitization and the cumulative charge flow carried by the channels increased in the same proportion, until reaching a maximum. At higher concentrations of either agonist, the cumulative charge started decreasing with concentration, reflecting further desensitization. Kinetic modeling suggested thata7 nAChRs have at least two non-equivalent paths to desensitized states, and that choline dissociates faster than ACh from the receptor. Our results established that the main difference between choline and ACh is of affinity, and support the concept that the switching of endogenous agonist may change the desensitization–resensitization dynamics ofa7 nAChRs. 2000 Elsevier Science B.V. All rights reserved.
Theme: Neurotransmitters, modulators, transporters, and receptors
Topic: Acetylcholine receptors: nicotinic
Keywords: Choline; Alpha7; Nicotinic receptors; Hippocampus; Patch clamp; Kinetics
1. Introduction function in the central nervous system, particularly in the
hippocampus [2–4,6,10,18,19,23,37]. These receptors are A large body of evidence has been accumulated indicat- located both postsynaptically in fresh hippocampal tissue ing that there is a direct participation of nicotinic acetyl- [4,18,22] and presynaptically, where they act by modu-choline receptors (nAChRs) in the control of neuronal lating transmitter release [1,5,7,19,20,23]. While more than one nAChR subtype is expressed in the hippocampus, sometimes with co-expression in single neurons [3,12,37],
*Corresponding author. Department of Pharmacology and
Experimen-tal Therapeutics, University of Maryland School of Medicine, 655 W. the a7-type nAChRs are by far the most abundant in
Baltimore St., Baltimore, MD 21201, USA. Tel.:11-410-706-7333; fax: cultured neurons [3,4,18]
11-410-706-3991. An important advance in understanding the function of
E-mail address: [email protected] (E.X. Albuquerque).
1 nAChRs in the CNS was the demonstration that choline, a
Present address: Department of Pharmacology, Institute of
Experimen-precursor and metabolite of ACh, is an effective agonist of
tal Medicine, Hungarian Academy of Sciences, P.O. Box 67, H-1450
Budapest, Hungary. a7 nAChRs [6,26,29]. It was recently demonstrated that
human cortical interneurons express a7 nAChRs that are 2. Materials and methods also effectively activated by choline [8]. Similar to ACh,
choline is an efficacious desensitizer, with significant 2.1. Cell culture effects observed at concentrations above 10mM [6]. This
is close to the mean extracellular concentration of choline Hippocampal neurons from Sprague-Dawley rats were in the brain, which is fairly stable around 4 to 6mM [25]. cultured as described previously [3]. Briefly, hippocampi Because of the dual activating / inhibiting effect, the phys- of 17–18-day-old fetuses were dissected; the cells were iological roles of ACh and choline must depend on the mechanically dissociated after trypsinization (30 min, detailed dynamics of their concentrations (the amount of 0.25% trypsin, Gibco) and plated on collagen-coated released transmitter, the velocity of diffusion, the rate of culture dishes. The cells were maintained in Minimum hydrolysis, and the rate of choline uptake), and also on the Essential Medium (MEM, Gibco) containing 10% heat-spatial distribution and subcellular localization of the inactivated horse serum and 2 mM glutamine. One week receptors, and their kinetics. There is novel data suggesting after plating, glial cell proliferation was inhibited with that native a7 nAChRs differ structurally from other 5-fluoro-29-deoxyuridine (2 mg / ml) and uridine (13 mg / mammalian ionotropic receptors in being homopentamers ml). Cells were used after 18 to 40 days in culture. [14,16]. This opens the possibility of there being up to five
agonist binding sites and adds considerable complexity to 2.2. Electrophysiology the kinetic behavior of the receptor-channel.
After the activation of cholinergic neurons, the con- Transmembrane currents were measured using the centration of choline is expected to rise sharply in the areas patch-clamp method [21] with a HEKA EPC-9 amplifier near ACh release sites due to the rapid hydrolysis of ACh. and Pulse software (HEKA Electronic, Lambrecht, Ger-This elevation of choline concentration surely affects the many). The resistance of borosilicate patch pipettes ranged responsiveness of the nicotinic receptors to ACh. It could from 2 to 6 MV. In whole-cell mode, 40 to 75% series isolate the nicotinic response in a spatial and temporal resistance compensation was applied. For outside-out patch manner, by causing desensitization of receptors in the recording, the pipettes were coated with Sylgard 184 (Dow neighborhood of the ACh release site. It is also possible Chemical). Only one patch was excised from each cell. that choline is the only agonist to reach a7 nAChRs Currents were recorded at 255 mV, except where stated located away from the release sites, providing a more otherwise. Experiments were performed at room tempera-prolonged (and less intense) signal that would reflect the ture (228C).
cumulative cholinergic activity of the area. It appears that
the neuronal a7 nAChRs have two effective endogenous 2.3. Solutions agonists that may act simultaneously or in tandem,
com-peting for the same binding sites. Thus, to understand the The internal solution used in most experiments con-physiological significance of this condition, we investi- tained (in mM): CsCl 120, MgCl 2, EGTA 10, HEPES 102
gated the kinetics of the currents evoked by both endogen- and ATP-regenerating compounds (ATP 5 mM,
phospho-ous agonists. creatine 20 mM, creatine phosphokinase 50 U / ml); pH
Analyses of whole-cell currents mediated by a7 was adjusted to 7.3 with CsOH. For the outside-out patch nAChRs have indicated that the kinetics of receptor configuration, and in some of the whole-cell experiments, activation and inactivation are strongly dependent on ATP-regenerating compounds were not included; the com-agonist concentration [3]. Single-channel studies of both position of the solution was (in mM): CsCl 147, MgCl 2,2
native receptors [12] and ectopically expressed mammalian CaCl 1, EGTA 10, HEPES 10, pH adjusted to 7.3 with2
a7 nAChRs [9] revealed relatively very short channel open CsOH. The composition of the external solution used in times, fast rate of desensitization, and large single-channel most experiments was (in mM): NaCl 165, KCl 5, CaCl2
conductance when ACh was the agonist. However, to the 2, glucose 10, HEPES 5, pH adjusted to 7.3 with NaOH. best of our knowledge, the properties of choline-evoked Atropine (1mM) and TTX (0.3mM) were routinely added single-channel currents in neurons have not been reported to all external solutions.
before. Here, we explored the kinetic properties of a7
nAChR-gated currents recorded in whole-cell and outside- 2.4. Fast application of solutions out configurations of the patch-clamp technique,
solutions in the bath and in the U-tubes and the pulse function of time, using our own computer programs. More durations were carefully adjusted before each experiment realistic simulations of whole-cell currents with a non-to yield 10–90% solution exchange times of 4.563.7 and instantaneous agonist delivery were done by solving 6.764.1 ms for the onset and the offset, respectively, as iteratively the set of difference equations giving the measured by the change of liquid junction potential at the fractional occupancy of each state at small time steps, tip of an open pipette. The delay and slope of onset and using spreadsheet programs. The concentration-dependent offset varied from cell to cell depending on the cell’s rate constants were updated at every time step, and the geometry; therefore, the actual duration of the agonist agonist concentration was assumed to rise following the pulses was adjusted during calibration [27]. A pulse from equation:
one of the U-tubes was immediately followed by a pulse 21
A(t)5A (1` 1exp(2k(t2t )))0
from the other U-tube, without a gap between the pulses.
By mapping the solution-exchange delay times in a 50-mm where A is the concentration of the agonist solution, k is
`
radius around the position of the cell body, it could be the solution exchange rate, and t is a delay (time to 50%
0
inferred that the front of the agonist-containing solution of the final concentration). passed through the soma and the proximal part of the
dendrites within about 5 ms. This sweep time, albeit short, is expected to distort the observed risetime, desensitization
3. Results rate, and peak amplitude of the whole-cell currents,
because channel activation would not be synchronous. To
In the first part of this section we will present the avoid this artifact, we have used the current integrals (net
comparative characterization of currents evoked by the two charge transfer) for kinetic analysis of whole-cell data.
endogenous agonists ofa7 nAChRs, ACh and choline. In the second part we will attempt to explain these results in 2.5. Data analysis
terms of kinetic models: experiments described there were designed to answer specific questions about possible Whole-cell data were acquired and analyzed using the
kinetic mechanisms underlying a7 nAChR-mediated cur-Pulse software package. The net charge carried by
whole-rents. cell currents was estimated by integrating the
baseline-subtracted, digitized records, using the trapezoidal rule.
During outside-out recording the signal was filtered at 30 3.1. The effect of agonist concentration on whole-cell kHz (three-pole Bessel filter) and saved on videocassette currents evoked by ACh and choline
tape using a NeuroCorder (NeuroData), which internally
filtered the signal at 16 kHz (Butterworth). The records The kinetics of whole-cell responses to ACh and choline were played back and re-digitized at 125 kHz for analysis, were remarkably similar. When the relative concentrations using the Digidata 1200 data acquisition board and the were chosen to evoke currents with identical peak am-pClamp 6.0 software package (Axon Instruments). Addi- plitudes, the currents evoked by ACh and choline were tional digital (Gaussian) filtering at 12 kHz was applied almost exactly superimposable (Fig. 1A). The only obvious during analysis for estimation of the single-channel am- difference appeared when a relatively short agonist pulse plitudes for the I –V plot. Very brief events (,30ms) were was given, revealing that the offset of the current (the automatically rejected, and only single events longer than decay of the current after removal of the agonist) was 64 ms were included for fitting the histograms. Since no faster with choline (n54 cells). The kinetics of the a7 correction was applied for very brief openings or closings nAChR-mediated currents evoked by both agonists was that may have escaped detection, the reported channel steeply dependent on the agonist concentration, as previ-dwell times should be considered ‘apparent’. Events ously shown [3,12]. For instance, at low agonist con-evoked by repeated agonist pulses applied to the same centrations, both the activation and the decay of the patch were pooled for analysis. Dwell-time histograms currents were slower by several orders of magnitude than were fitted with the maximum likelihood estimation meth- at high agonist concentration. At 10 mM ACh, for exam-od [32]. Data are shown as mean6S.D. ple, the decay was so slow that a relatively stable plateau phase was maintained during several seconds of agonist
2.6. Simulations application (Fig. 1B). Higher concentrations of the agonist
phase of the whole-cell and single-channel currents was 0.715 (n534 cells). When the whole-cell currents were relatively small, few receptors seemed to be present in the excised patches, whereas with cells that showed large whole-cell currents, there was a higher chance of observing multiple overlapping channel openings, indicating the presence of several nAChRs in the patch. There were patches, however, that contained many fewer receptors than would be expected from the magnitude of the whole-cell current, which might be explained by the non-uniform distribution of a7 nAChRs observed in hippocampal cultures after the fourth week in vitro [35].
In patches containing multiple nAChRs, it was possible to lower the agonist concentration until the responses consisted almost exclusively of separate openings. As seen with whole-cell currents, this lower level of activity was maintained for several seconds, thus producing a larger number of well separated single-channel openings, which allowed a reliable measurement of open times and am-plitudes. Single-channel currents evoked by 300 mM
Fig. 1. Kinetic characteristics of whole-cell currents. Currents evoked by choline are shown in the middle panel of Fig. 2. The total 750-ms pulses of 300mM ACh and 2000mM choline in the same neuron
number of single-channel openings evoked by 1-s pulses of
(A) were nearly identical, except immediately after the removal of the
10 mM ACh or 300 mM choline given at 10-s intervals
agonist: the ACh-gated current showed a longer tail. Similar results were
ranged from 17 to 71 in the first minutes after excising the
seen in four cells and also with other agonist concentrations and pulse
durations. Panel (B) shows the concentration-dependent kinetics of ACh- patch, but tended to decrease during the recording, show-evoked whole-cell currents. The initial 0.4 s of the currents show-evoked by 4-s ing a run-down. Most of the openings were well separated, pulses of ACh at 10 (the smallest amplitude), 20, 40, 80, 160, 320, and
but pairs and triads of openings were observed quite often,
3000mM (the largest amplitude) are shown as the larger traces. The inset
which seems to reflect a bursting behavior (see further
shows the entire 4-s responses scaled to the same peak amplitude to
discussion below).
highlight the concentration dependence of the decay. A similar pattern was seen in 15 cells.
3.2.1. Single-channel conductance
values very close to baseline within 100 to 1000 ms. The single-channel current amplitude showed a large Scattered channel openings still occurred until the end of variation from opening to opening (see Fig. 2), and a the agonist pulse as evidenced by the increased noise
compared to the baseline before and after the agonist pulse. The average amplitude at the end of the 1-mM pulse was 0.9260.55% of the peak amplitude. The decay of the current could often be fit better with the sum of two exponentials; the faster decay time constant, which was responsible for the larger part of the amplitude, ranged from 7 to 58 ms.
3.2. The effect of agonist concentration on ACh- and
choline-evokeda7-nAChR single-channel currents
After establishing the whole-cell configuration and before excising outside-out patches, the whole-cell re-sponse of the cell was tested by applying a brief pulse of 1 mM ACh (and also, in some of the cells, 10 mM choline). Immediately after excision of the patch, the response to the same agonist pulse was tested, in order to compare the currents evoked in whole-cell and outside-out mode. In general, the magnitude of the current recorded from the
Fig. 2. Single-channel currents evoked by pulses of ACh or choline in
outside-out patch was related to the magnitude of the one outside-out patch with many active receptors. The responses to 1 mM whole-cell currents. The correlation coefficient between ACh (top) and 300mM choline (middle) are shown, with a portion of the
single Gaussian function often did not provide a good fit of the amplitude distribution. However, the amplitude histo-grams did not have multiple peaks; thus multiple conduct-ance levels could not be unambiguously identified. To determine the single-channel conductance, only moderate filtering was applied (effective cutoff at 9 kHz) to avoid distortion of the amplitude. With further digital filtering, currents could be unmasked even at25 and 115 mV, but the frequency of events was low and their amplitudes could not be reliably measured. Therefore, only the mean amplitude values obtained at membrane potentials more negative than 230 mV were used. In three cells, the current amplitudes were measured at different membrane potentials; the current–voltage relationship could be fit with a straight line in the285 to235 mV range, yielding a mean reversal potential of 12.6 mV (Fig. 3). This value was used for calculating the chord conductances for the rest of the cells, where measurements were performed at either255 or 265 mV. Despite the large variability of the single-channel amplitude within individual patches, the mean single-channel conductance varied little between
patches (Table 1). No significant differences were found Fig. 3. Current–voltage relationship of single-channel currents evoked by between the average conductances obtained with 1 mM 10mM ACh. Single-channel currents were recorded in the same patch at
ACh (91.5 pS) and 10 mM choline (82.9 pS), or between different membrane potentials (top), and the average amplitudes of the idealized events were plotted against voltage (bottom). The regression
high and low concentrations of each agonist. No change
line for this patch gave a slope conductance of 98.5 pS and reversal
during the course of recording was observed either.
potential of 2.7 mV.
3.2.2. Open durations
Unlike the single-channel conductance, the mean chan- obtained from the rest of the patches. The arithmetic mean nel open times varied considerably between patches, of the open times pooled from these patches was ranging from 68 to 237 ms. The histograms of channel 89.5651.6 ms (n555 events) for 1 mM ACh and open times recorded from individual cells could be 85.4644.0 ms (n540 events) for 10 mM choline. In two adequately fit with a single exponential, as shown in Fig. patches where responses to three to five agonist pulses 4A, but the relatively small number of events in each could be recorded at different membrane potentials, the histogram did not allow for testing of improvement of fit open times did not show a clear voltage dependence, but with multiexponential distributions. The average open time the sample was too small to test for significance.
for currents evoked by 1 mM ACh was 108.6650.1 ms
(n58 cells); results for 10 mM ACh, 10 mM choline, and 3.2.3. Closed durations
300mM choline did not differ significantly (Table 1). In Proper conditions for analysis of the entire closed time four patches where both high and low concentrations of the distributions could not be met due to desensitization and same agonist (two each with ACh and choline) were run-down, which prevented the system from reaching an applied, there was no apparent change in mean open time apparent steady state, to the obvious presence of multiple with concentration. In the 11 patches where the number of channels in most of the patches, and to the low event events was small, the amplitude and duration of single- counts. However, brief closed times recorded in conditions channel currents seemed not to differ from the values of low opening frequency are still informative of fast
Table 1
a
Characteristics of single-channel openings evoked by different concentrations of ACh and choline
ACh 1 mM ACh 10mM Choline 10 mM Choline 300mM
(n58) (n52) (n56) (n54)
Single channel 91.568.5 89.8 82.9611.6 84.4610.9
conductance (pS) (78.6–104.0) (86.3, 93.4) (71.2–105.0) (71.5–97.9)
Mean channel 108.6650.1 151.0 92.7614.1 106.0654.8
open time (ms) (68.0–237.0) (78.0, 223.0) (73.0–110.0) (69.0–185.0)
a
currents could be distinguished. Simultaneous openings of two channels were occasionally observed, but the maximal current amplitude never exceeded this level. The average amplitude of the whole-cell currents in these cells was
2107761086 pA, and the total charge was 44.4653.9 pC. The charge carried by the initial group of single channel openings was 3.362.6 fC (calculated from the first agonist-evoked response after patch excision), which means ap-proximately six single-channel openings per agonist pulse, considering that the calculated charge carried by an average single-channel opening was 0.57 fC from the parameters of single-channel currents mentioned above. The number of single-channel openings showed a consid-erable fluctuation between individual agonist pulses ap-plied to a patch. Many of the agonist pulses failed to evoke visible openings at all. Traces without openings seemed to occur randomly in a sequence of agonist pulses, indicating that these failures were not due solely to run-down, but possibly to a low maximum opening probability. In patches from the remaining 20 cells, many channels opened at the same time at the beginning of the agonist pulse, but the frequency of openings quickly decreased, allowing the measurement and statistical evaluation of single-channel parameters. Fig. 2 illustrates currents evoked by both ACh and choline in the same patch from one of these cells. The figure illustrates currents evoked by
Fig. 4. Dwell-time histograms. The open times distribution from a single
high and low concentrations of the agonists (1 mM ACh
patch exposed to pulses of 300mM choline could be fitted with a single
and 300mM choline, respectively). Whole-cell currents in
exponential curve with a time constant of 210ms (A). The closed times
the 20 cells had an average amplitude of 24416974 pA,
distribution from another patch exposed to 10mM ACh had two clearly
distinct components, shown in two histograms with different binwidths to with a total charge flow of 148.36100.3 pC. In the
include short (B) and long (C) closed times. outside-out patches taken from these cells, the initial group
of superimposed single-channel openings lasted for 5.465.3 ms, carrying 86.7683.4 fC charge, which corre-conformational transitions of the receptor-channel. Thus, sponds to 151 single-channel openings on average (ranging currents evoked with the lowest agonist concentration, 10 from 28 to 441). The maximal amplitude of the current
mM ACh, were evaluated with regard to closed times. At reached 16.0 to 101.1 pA at 255 mV, which means that this concentration, although openings were scarce, paired three to 19 channels opened simultaneously. In some and triple openings were relatively frequent, revealing a patches overlapping channel openings could be seen after bursting behavior of the channel. In the experiment shown the initial group, but the average opening probability was in Fig. 4B and C, 30 out of 161 openings (18.6%) were still small. The average current during the 500-ms interval separated by closed times shorter than 0.3 ms, while the after the first opening was 15.4613.2 pA, ranging from 7.2 average closed time was 22.2 ms. In five patches, the to 33.1 pA. In 10 of these 20 patches the total current was closed times distributions showed at least two exponential large enough to form a distinct peak, so the decay phase components, which differed by about two orders of could be fit with an exponential curve. The decay rates magnitude. The fast component, with a time constant of seen with 1 mM ACh and 10 mM choline in the same approximately 0.1 ms, was similar in all patches and patch were very similar, so they were averaged together. reflected fast intra-burst closings. The grand mean of the decay time constants from 10 patches was 1.2660.42 ms, considerably shorter than that 3.2.4. The number of receptors in a patch of the whole-cell currents.
In three of 34 cells no single channel openings at all
were observed after excision of outside-out patches. The 3.2.5. The relationship between opening and average peak whole-cell currents in these three cells was desensitization of thea7 nAChR channel
concentration-dependent, in such a way that the more the chosen to use the total charge carried during the test-pulse channels open, the more they desensitize or inactivate. To as an indicator of the number of available channels, investigate the relationship between desensitization and because these values were more stable than the peak prior channel opening as a function of time and agonist amplitudes. The total time that all channels spend in the concentration, we have used a double-pulse protocol (Fig. open state is proportional to the total charge movement, 5). A pre-pulse of different concentrations of agonists was which could be calculated by integrating the current traces. applied from one of the U-tubes, followed by a 1-s pulse of Before integration, the baseline was subtracted from the 10 mM choline, which should activate maximally the current traces and a correction for run-down was applied. remaining non-desensitized channels and will be referred The current and charge traces for 4-s pre-pulses of a wide to as test-pulse. Challenges with two different concen- range of ACh concentrations and a test-pulse of 10 mM trations of agonist were always separated by at least two choline are shown in Fig. 5A and B. The total charge control pulses (test-pulse after a pre-pulse of external movement during the test-pulse corresponds to the differ-solution); these yielded the maximum response of the cell ence in total accumulated charge between the end of the and also served as a basis for the correction for run-down. test-pulse and the end of the pre-pulse.
The fraction of non-desensitized channels after a pre-pulse As the concentration of ACh during the pre-pulse was of agonist could be estimated from the ratio between the elevated, the peak amplitude of the evoked current in-peak response given by the test-pulse and the in-peak creased throughout the entire concentration range tested response given by a control pulse. However, we have (10 mM to 3 mM). The cumulative charge movement
Fig. 5. Progression of desensitization monitored by a double-pulse protocol. The neurons were exposed via one U-tube to a pre-pulse of various agonist concentrations during 0.3 to 4 s, then a 1-s test-pulse of 10 mM choline was delivered via the other U-tube. Panels (A) to (C) show the whole-cell currents (downward traces, scaled by the left vertical axis) and the corresponding integrals below, depicting the cumulative charge flowing into the cell (scaled by the right vertical axis). Effect of pre-pulses of low ACh concentrations (A): responses to zero (external solution, labeled as1 ), 10mM (2 ), 20mM (3 ), 40
mM (4 ), and 80mM (5 ) ACh are indicated. The peak amplitude of the pre-pulse response grew with the concentration of ACh, while that of the test-pulse decreased. The cumulative charge flow reached a maximum between 40 and 80mM. Effect of pre-pulses of high ACh concentrations (B): responses to 80
during the 4-s pre-pulse, however, seemed to reach a or choline would have contributed to the acceleration of maximum between 40 and 80 mM ACh. In this low whole-cell current decay seen at high agonist concen-concentration range, the fraction of available receptors trations, such an effect should be voltage-dependent, as has (assessed by the test-pulse) was inversely proportional to been established for other nAChR subtypes [28,33]. To test the cumulative charge movement of the pre-pulse. The this hypothesis, the voltage dependence of the amplitude concentration at which the test-pulse current disappeared and decay of whole-cell currents evoked by 300mM and 3 was exactly the concentration where the cumulative charge mM ACh was investigated. Current traces and the current– movement reached its maximum (between 40 and 80mM, voltage plots are illustrated in Fig. 6. The traces were see Fig. 5A), suggesting that almost the total population of normalized and superimposed as shown in the insets of receptors had reached a desensitized state during the period Fig. 6A and B to allow visual comparison of the current of 4 s. As the concentration of ACh was raised beyond 80 time course obtained at different potentials (only currents
mM, the cumulative charge movement decreased, in spite evoked at negative potentials are shown). The decay of the increase in peak amplitude (Fig. 5B). The same kinetics of whole-cell currents was constant and the peak phenomenon was observed with different pre-pulse dura- current varied linearly in the 2144 to 224 mV range at tions, and also when the agonist was choline (0.1 to 10 both concentrations of ACh in two cells (Fig. 6C), and mM) in the pre-pulses, in three cells. with 10 mM choline in another cell. Thus, there was no With low agonist concentrations, the degree of prior evidence of a significant voltage-dependent channel block channel opening was proportional to the degree of de- by ACh or choline even at high concentrations and very sensitization irrespective of the duration of the pre-pulse, negative membrane potentials.
as illustrated in Fig. 5C. In this experiment, the duration of
the pre-pulses was 300, 500, 1000, 2000 or 4000 ms, all 3.4. Recovery of the a7 nAChR from desensitization with 40mM ACh. As the pre-pulse was made shorter, the
response to the test-pulse increased in amplitude and in The recovery from desensitization in whole-cell record-total charge. When the record-total charge flow evoked by the ings was studied using dual U-tubes, which ensured a very test-pulse was plotted against the total charge flow evoked fast and complete removal of the agonist. Since the by the pre-pulse, the relationship was apparently linear removal was still not instantaneous, the following results (Fig. 5D, open symbols). Fig. 5D also shows data corre- underestimate the actual speed of recovery. Two 800-ms sponding to the traces in Fig. 5A and B, with a fixed pulses of either 1 mM ACh or 10 mM choline were pre-pulse length of 4 s and ACh concentrations ranging applied with varying time intervals (0, 200, 500, 900, from 10mM to 3 mM. Both protocols showed that, at low
agonist concentrations, the response to the test-pulse decreased linearly depending on the prior cumulative charge flow, indicating that the degree of desensitization was directly correlated with the total time spent in the open conformation. At concentrations above 40mM for ACh, or 300mM for choline, the test-pulse responses stabilized at a minimum near zero, but the pre-pulse charge started decreasing, bending the line to the left (Fig. 5D, filled symbols). This suggests that the relative likelihood of desensitization versus activation changes with concentra-tion, and might be taken as evidence that different levels of agonist occupancy lead to the desensitized state(s) through kinetically distinct pathways.
3.3. Is thea7 nAChR channel blocked by the agonist?
The results suggest that high agonist concentrations evoke current flow less efficiently than low agonist con-centrations. Besides desensitization, one explanation for this could be that the receptor is blocked by high
con-centrations of the agonist. No obvious signs of open- Fig. 6. Whole-cell currents evoked by 300mM ACh (A) and 3 mM ACh (B) at various membrane potentials: 2143.5, 2113.5, 283.5, 253.5,
channel block (shorter open duration or lower
single-223.5, 6.5, 36.5, 66.5 and 96.5 mV. After being scaled to the same peak
channel amplitude at high concentrations) were observed in
and superimposed (insets), the five currents recorded at negative
mem-single-channel records, but a simultaneous concentration- brane potentials had similar rise and decay rates. In this cell, the peak dependent increase in open duration might have obscured amplitude (divided by the value at 253.5 mV) increased with
1500, 2400, 3700, 5400 and 10 000 ms), and the extent of also because the native hippocampal nAChR might contain recovery was expressed as the ratio between the peak up to five agonist-binding alpha subunits, if it is assembled amplitudes of the responses to the second and the first as ana7 homomer [14,16]. For simplicity, only the results pulse. When the percent recovery was plotted as a function with three binding sites are shown. Second, either there of the duration of the interval between the two pulses, the was a single open state, or each of the open states had the
21
points followed approximately sigmoid curves (Fig. 7). same overall exiting rate of about 10 000 s , because The times to 50% recovery were 7436237 and 18646556 there was only one population of single-channel open ms when the first pulse contained 10 mM choline and 1 times with mean around 100 ms, which did not vary mM ACh, respectively; 90% recovery was reached at significantly with voltage, recording time, agonist con-33246978 ms (choline) and 526361263 ms (ACh) (n5 centration or even between ACh and choline. Third, 8). The differences between the recovery times for choline desensitization was assumed to occur only at the same and ACh were significant (paired t-test, P,0.005). In level(s) of receptor occupancy associated with channel outside-out patches the recovery from desensitization was opening [11]. Other simplifying assumptions were that the even faster. For these experiments, a single U-tube was agonist binding sites were kinetically equivalent, that used, and the removal of the agonist was relatively slow activation and desensitization gates operated in a concerted and variable. In three patches, 250-ms pulses of 1 mM manner, and that the agonist could not dissociate from the ACh or 10 mM choline were given at intervals ranging open channel. The overall apparent rates of desensitization from 100 to 2000 ms, and 50% recovery was reached and recovery were chosen to be of the order of 1000 and
21
within 200 ms with both agonists. Since the recovery times 1–10 s , respectively, to match the single-channel data. were of the same order as the agonist wash-out times, these The rate constants were also chosen to roughly reproduce experiments show that thea7 nAChRs in excised patches the bursting behavior and the low maximum open-channel can recover from desensitization induced by ACh or probability. We have experimented with two distinct choline in less than 200 ms, if the agonist is removed fast connectivity paradigms that could yield a correlation
enough. between the degree of desensitization and the degree of
channel opening (Fig. 8). In one, desensitized and open 3.5. Simulations of mass kinetic behavior states were directly connected, that is, desensitization could proceed from the open state, as in the classical We performed simulations to help to identify possible model by Katz and Thesleff [24]. In these models the mechanisms underlying the observed effect of agonist agonist can dissociate from the desensitized receptor, concentration on the activation and desensitization rates allowing recovery without channel reopening and closing a and on the desensitization–charge flow relationship. Sever- circle. In the other paradigm, open and desensitized states al constraints were taken into account when devising a were connected to common short-lived ligand-bound suitable kinetic scheme. First, at least two agonist binding closed states. In these bifurcated models, receptor oc-sites were included to account for the Hill coefficient cupancy can lead to channel opening or desensitization, greater than one in the concentration–response curves and yielding correlation without causality between the two processes. Both models allow for reopening after removal of the agonist, but the chance of reopening can be made much smaller in the circular models.
With a single open state, both the circular and bifurcated schemes yielded linear relationships between the fraction of available receptors (fraction non-desensitized) and the cumulative open time, estimated as the integral of the open-channel probability, P(o). The models were then extended by adding another set of open and desensitized states, as shown in Fig. 8A and B. When the rate constants leading to and from the A D and A D desensitized states2 3
were the same (d125d13, d225d23), both the circular and the bifurcated models behaved as if there was a single open state. Fig. 8C shows plots of the concentration dependence of the peak open-channel probability (maxi-mum activation) and the steady-state probability of all
Fig. 7. Recovery from desensitization, measured with a paired-pulse desensitized states, corresponding to the desensitizing protocol. The peak amplitude of the second response relative to the first is effect of the agonist. The mean effective concentration for plotted against the interval between the pulses. Data points and error bars
the activating and desensitizing effects were in the same
are mean and S.D. of values from eight cells. Both agonists, ACh (1 mM,
proportion of the published values for the hippocampala7
filled symbols) and choline (10 mM, empty symbols), were tested in each
those in Fig. 5D and show that the fraction of available To achieve deeper desensitization at higher concen-receptors decreased linearly as a function of the cumulative trations with the circular model, we first added a direct prior opening of the channels at all concentrations (0.1 to path of desensitization from the state of highest agonist 10 000 mM) and at different time points (100 and 4000 occupancy, removing one open state. As shown in Fig. 8D, ms). The cumulative charge carried by a long pulse of this yielded the intended curvature in the plot on the right, agonist would rise with concentration, approaching an but the activation curve on the left was distorted. This asymptotic maximum, instead of peaking and falling as model was not favored because it predicted that the peak
agonist concentration was increased, which was not in covery are the A D–A R transitions, which would thenn n
agreement with the experimental observations. It is inter- occur at different rates depending on the level of receptor esting to note that, if the agonist concentration was made occupancy. In the circular model, recovery is essentially to rise slowly to a high value (see equation in Materials concentration-independent as long as the desensitized and methods), the channel could spend more time in the states are equivalent in terms of agonist binding, because intermediate A R states, from which it was likely to open,n dissociation of the agonist is what controls recovery. This before all the binding sites were occupied and it desensi- difference can be used to test the adequacy of one model tized. A rising time of 0.1 ms (10 to 90%) sufficed to yield against the other.
larger peak responses than the corresponding step change Although both the bifurcated and the circular model in concentration (Fig. 8D, asterisk). A better outcome was with d2n.d2(n11 ) seemed capable of reproducing the obtained with the two basic models when desensitization observed concentration–response and activation–desensi-rates were made faster at higher levels of agonist oc- tization relationships, the circular model was clearly more cupancy (d13.d12), as shown in Fig. 8E and F. However, adequate in other aspects. To achieve the observed low the circular model had the additional constraint that the opening probability after long exposures to high agonist corresponding channel-closing ratesanhad to be altered to concentrations, the d2n rate corresponding to the fully
21
keep the same open time, which was a significant con- occupied receptor had to be set under 10 s . With these straint. When the d13 rate was larger than d12 by several rates, recovery from desensitization could not be com-times and of the same order of the channel opening rateb, pleted in less than 1 s with the bifurcated model (data not the peak response tended to fall at high agonist con- shown). The circular model not only allowed a recovery centrations, but this effect was also attenuated when the rate compatible with the single-channel experiments, but agonist was applied slowly. Another drawback of changing also provided a simple explanation for the difference in tail the d1n rates was that the behavior of single channels currents after the end of equieffective pulses of ACh and could become incompatible with the observations. For choline (see Fig. 1A). The effects of choline on the a7
21
instance, when d1n was set below about 5000 s , the nAChR were simulated by assuming a single change in simulated single-channel currents showed a clear clustering affinity: that the dissociation rates from both the resting at high agonist concentrations, because when returning (A R) and desensitized (A D) conformations were 10n n
from desensitization, the channel could spend more time in times faster than with ACh. This increased 10 times both transitions to / from the open state (with rates b 510 000 the EC50 for the peak response and the IC50 for
steady-21 21
s anda 55000 s ) before desensitizing again (data not state desensitization, as expected, but the response shown). In our experiments, isolated groups of more than waveforms for ACh and choline were nearly identical three openings were rare. With both model types, the best when the concentrations were in a ratio of 10. However, results were obtained when the higher-order desensitized when choline dissociated after the end of a pulse, the states were made more absorbing by setting d2n.d2(n11 ), channels spent less time on the intermediate desensitized so that recovery or reopening were faster from the less states (A D in Fig. 8A), having a lower chance of2
occupied A D state, as shown in Fig. 8G and H. In then reopening than with ACh. The effect was clear when the
21
case of the bifurcated model with these modifications, intermediate reopening rate (d22) was about 50 s , which recovery from desensitization is predicted to be concen- was of the same order of the dissociation rate of ACh in
21
tration-dependent, because the rate-limiting steps to re- our model (20 s ), otherwise the tail currents produced
Fig. 8. Simulations performed with six different kinetic schemes. The two basic circular (A) and bifurcated (B) models were used to simulate the data in (C) to (H), as indicated. Panels (C) to (H) show two graphs: the left one shows the concentration–response curves corresponding to the agonist effect (peak opening probability, P(o),s), and the desensitizing effect (steady-state occupancy of desensitized states, s.s. P(d),d); the graph on the right shows
the relationship between the fraction of non-desensitized receptors and the cumulative open time (the integral of P(o)) for concentrations ranging from 0.1 to 10 000mM, measured at two time points after the beginning of the agonist pulse: 100 and 4000 ms (nandm, respectively). In (C), the two open states
and the two desensitized states were kinetically undistinguishable, and both circular and bifurcated models behaved as if there was only one open and one desensitized state. The relationship between degree of desensitization and prior activation was linear at all times. The data correspond to a circular model
21 21
with d125d1352000 s anda 5 a 52 3 8000 s . When desensitization was made deeper at higher agonist occupancy by adding a direct pathway to desensitization in the circular model (D), the desensitization–activation relationships were no longer linear, but the peak P(o) was reduced at higher agonist concentrations. When a non-instantaneous agonist application with 0.1 ms risetime (10 to 90%) was simulated with the same model, the fall in peak P(o) was less marked (*). The required non-linear desensitization–activation plots and a stable maximum P(o) were obtained when desensitization was made
21 21 21 21
faster from the second open state of the circular model (E), with d1251000 s , d1352000 s ,a 52 9000 s , anda 53 8000 s . A similar result was
21 21 21
obtained with the bifurcated model (F), with d1252500 s , d1355000 s , d225d2351 s . A good compromise could be achieved by keeping the
21 21
d1nrates at about 5000 s and setting different reopening rates (d2n), both in the circular model (G), with d22550d23550 s , and in the bifurcated
21
by the reopenings were too small. Fig. 8I shows the early Choline-activated channels had very brief open times, phase of responses to pulses of 30, 100, and 300mM ACh averaging around 100 ms, similar to ACh. A wide record-(or of 300, 1000, and 3000 mM choline) for the circular ing bandwidth (see Section 2.5) was essential for
appro-21 21
model with d22550 s and d2351 s . It can be seen priate measurement of these channel events. Open times of that, with time, the cumulative charge carried (proportional similar duration were measured in our previous study of to the integral of P(o)) would be larger for the response to the hippocampala7 nAChR [12] and in a recent study of the lower concentration of agonist. Fig. 8J (left panel) recombinant chick a7 homomeric receptors [9]. The shows that the maximum cumulative charge at the end of a finding that the open duration is relatively independent of 4-s agonist pulse was obtained with about 3mM ACh (or the agonist is consistent with studies of ACh analogs on 30mM choline). The right panel of Fig. 8J illustrates that muscle nAChRs, where the nature of the agonist was found the tail currents expected after 4-s pulses of ACh 300mM to affect primarily the association, dissociation and open-and choline 3 mM were of the same magnitude, but the ing rates, and not so much the channel closing rates
one with choline was much briefer. [34,36].
4. Discussion 4.2. The concentration dependence of the kinetics:
possible kinetic models and functional implications
4.1. Single-channel currents mediated by the two natural
agonists of a7 nAChRs A major feature of the a7 nAChR is its rapid and pronounced desensitization, which is likely to shape the The present investigation describes for the first time the receptor’s function in the brain. Our experiments showed single-channel currents activated by choline in mammalian that desensitization was proportional to channel opening at neurons. These currents had very similar properties to low agonist concentrations, but the relationship somehow those activated by ACh and, based on several arguments, changed to yield deeper desensitization at higher agonist both can be attributed to the native hippocampal a7 concentrations (above |100 mM ACh and |600 mM
nAChRs, as previously proposed [12]. Whole-cell currents choline). In fact, considering the cumulative charge carried activated by choline in hippocampal neurons were com- through the channel, the relative efficacy, when the agon-pletely blocked bya-bungarotoxin and methyllycaconitine, ists were applied for more than a few tens of milliseconds, both in isolated cultured cells [6] and in fresh slices [7], was higher at low agonist concentrations (Fig. 5A and B). and so were the currents mediated by recombinant a7 That the concentration–charge relationships of both agon-nAChRs expressed in frog oocytes [26,29]. Since this ists reached a maximum at concentrations below the EC50
pattern of toxin sensitivity defines the response of a7 for the peak current and then decreased is particularly nAChRs, if the single-channel and whole-cell currents relevant because a large portion of this charge must be
21
activated by choline were mediated by the same receptor, carried by Ca [13]. We have tried to model the channel’s then this must be, by pharmacological criteria, the a7 behavior starting from the linear relationship seen at low nAChR. Indeed, we observed a great similarity in con- agonist concentrations between the total time spent in the centration-dependent kinetics between the choline-gated open state(s) and the degree of desensitization. The open single-channel currents and their macroscopic counterparts times distribution seemed to have a single exponential recorded in the same neurons, which supports the conclu- component and did not vary significantly with agonist sion that the receptor underlying the responses in both concentration. This implies that if the channel has more modes is the same, that is, the a7 nAChR. than one open conformation, the states were kinetically The single-channel currents activated by both agonists indistinguishable within the resolution of our experiments. were very similar. The nAChR channel showed a relatively This observation was embodied in two types of kinetic high average conductance, without significant differences model in rather different ways. In the circular model (Fig. between ACh and choline. With either agonist, there 8A), if one assumes a single open state, in order to seemed to be multiple current levels in the same patch, as reproduce the deeper desensitization at high agonist con-reported in our previous study with ACh in the same centrations the model must include direct connections preparation [12], but it was not possible to separate these between the bound-closed states (A R) and the desensi-n
concentrations. This and other artifacts introduced in the reproduce most of the observations (Fig. 8B and H). Dilger
a7 nAChR responses by the limited performance of and Liu [15] and Franke and colleagues [17] also found solution delivery systems including the underestimation of that models with desensitization proceeding exclusively agonist potency and of the Hill coefficient have been from ligand-bound closed states could reproduce most of investigated by Papke and co-workers [30,31]. These the behavior of muscle nAChRs, but the latter authors authors recently proposed a multi-circle model with a disregarded these models and chose a circular model single open state similar to the scheme we have tested because no openings were observed after agonist removal (data shown in Fig. 8D), and argued that the expected in their recordings. More recently, Auerbach and Akk hill-shaped concentration–response curve (see Fig. 8D, found desensitization to be more likely from a fully open circles) was not seen due to slow solution exchange occupied recombinant muscle-type nAChR, and concluded [30]. Although this type of model eventually may be that it proceeds from the open state, in agreement with the proven right by using a faster drug delivery (e.g., flash circular model [11]. In spite of its greater parsimony and photolysis of caged agonist), we have found alternate simplicity, the bifurcated model was also inferior in our explanations for the strong concentration dependence of study, because it could not be made to yield fast and deep
desensitization. desensitization and fast recovery at the same time.
How-The circular model could be improved without intro- ever, a hybrid model, similar to that in Fig. 8B but with the ducing additional paths of desensitization from closed desensitized states interconnected, could fit the data (not states by including more than one open state with different shown) and might be an alternative to the circular model. equilibrium constants with respect to desensitization. Set- To address the question of whether a7 nAChRs are ting the difference only in the forward desensitization rates involved in the neuronal processing underlying cognition from each open state (d1n) forced the adjustment of the in the hippocampus and elsewhere in the brain it is channel closing rates (an), in order to maintain a mean essential to discuss the dynamics of receptor activation open time of 100ms. Besides this constraint, the changes during electroencephalographic theta frequency activity. in d1ncould not explain the different tail currents observed With the agonist concentration varying rhythmically at a upon removal of ACh and choline. The alternative was to main frequency of 4–8 Hz, the central question is whether set different reopening rates (d2n), making channel reopen- thea7 receptor would follow the rhythm or be inactivated ing less likely from the higher-occupancy desensitized by cumulative desensitization. In whole-cell mode, after a state. With this arrangement, the model could be adjusted 800-ms agonist pulse, it took 3 to 5 s to achieve 90% to reproduce the main features of the experimental data, recovery of the response. However, receptors recovered by including the differences between ACh and choline. Al- 50% within about 200 ms in outside-out patches, so that though we have not attempted to fit the set of rate the actual recovery time must be even shorter, because it constants to the data, the simulations suggested that a was hindered by the slow removal of the agonist. Frazier et change in the agonist dissociation rate alone might explain al. [18] reported that the refractory period of putative the differences in potency and in the tail currents observed a7-mediated excitatory postsynaptic currents was probably with the two agonists. Because the channels open only for shorter than 100–200 ms in hippocampal interneurons. In about 100ms in very brief bursts, the tail currents must be view of our results, this suggests that the concentrations of due to delayed reopening of desensitized channels, and not the two agonists, ACh and choline, probably vanish within to ongoing activity at the moment of agonist removal. less than 50 ms in the synaptic region, and that synaptical-Thus, in the proposed circular model the duration of the ly located a7 nAChRs may indeed sustain repetitive tail depends on the ratio between the reopening rate (d1n) activation during theta frequency activity. Because both and the dissociation rate from the corresponding desensit- ACh and choline are effective agonists, it is likely that ized state, and is not affected by the agonist binding rates. diffusion and reuptake are the main determinants of the In general, agonists whose difference in potency is due duration of the cholinergic response (activation and de-mainly to the dissociation rates would be predicted to show sensitization), and not ACh hydrolysis. However, our data different tail durations after pulses of equieffective con- showed that choline dissociates faster than ACh, sug-centrations. Indeed, relative to ACh and choline, nicotine gesting that the activity of cholinesterases might have a showed a more prolonged tail current (unpublished results). significant role in the recovery process, and that anticholin-A general theoretical and practical disadvantage of a esterase drugs might affecta7 nAChR-mediated synaptic circular model with more than one opening–desensitization transmission.
pathway is the difficulty in achieving microscopic re-versibility of the transitions simultaneously in all sub-cycles of the scheme. With the bifurcated model, because
activation and desensitization are not directly connected, Acknowledgements several open and desensitized states can be included
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