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3 AChRs: impact of 
2, 4 and 5 subunits
MS 8784 Received 28 September 1998; accepted after revision 22 January 1999.
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ABSTRACT |
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3 acetylcholine receptors (AChRs) in Xenopus oocytes and native AChRs from the human neuroblastoma cell line IMR-32. 3 AChRs exhibit channel properties that reflect subunit composition.
32 AChR open times were 0·71 ± 0·14 and 3·5 ± 0·4 ms with a predominant conductance of 26 pS. 34 AChRs had open times of 1·4 ± 0·2 and 6·5 ± 0·8 ms and a predominant conductance of 31 pS. Burst times were 0·82 ± 0·12 and 5·3 ± 0·7 ms for 32 and 1·7 ± 0·1 and 16 ± 1 ms for 34. Desensitization was faster for AChRs with the 2 subunit than for those with the 4 subunit.
352 AChRs (5·5 ± 0·3 ms) was different from those of 32 AChRs. For 354 AChRs, an additional conductance, open time and burst time (36 pS, 22 ± 3 ms and 43 ± 4 ms, respectively) were different from those for 34 AChRs.
3 AChRs were inhibited by hexamethonium or mecamylamine. The rate constants for block of 34 by hexamethonium and of 32 by mecamylamine were 1·2 × 107 and 4·6 × 107 M-1 s-1, respectively.
34 AChRs expressed in oocytes. Antibodies revealed that 5 ± 2 % of IMR-32 3 AChRs contained 5 subunits and 6 ± 2 % contained 2 subunits. IMR-32 3 AChRs are primarily 34 AChRs.
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INTRODUCTION |
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Neuronal nicotinic acetylcholine receptors (AChRs) are expressed throughout the central and peripheral nervous system where their subunits are encoded by eleven genes identified as 2-9 and 2-4 (Papke, 1993; McGehee & Role, 1995; Lindstrom, 1996). The large variety of possible subunit combinations resulting from the multitude of nicotinic genes has led to considerable functional diversity for nicotinic AChRs expressed in tissues throughout the nervous system and in heterologous expression systems. Genes for multiple AChR subunits are often transcribed within the same cell. For example, neurons of peripheral ganglia have been shown to express 3, 5, 7, 2 and 4 subunits (McGehee & Role, 1995; Lindstrom, 1996). AChRs that are expressed in ganglia represent the best-characterized source of native neuronal nicotinic AChRs at many levels including functional, structural and histological (reviewed in the following: Papke, 1993; Sargent, 1993; McGehee & Role, 1995; Lindstrom, 1996). AChRs expressed in ganglia have been attributed mainly to two types, the 7 type that may be homomeric, and the 3 type that consists of 3 subunits assembled with 5, 2 and/or 4 subunits. 7 AChRs are characterized by their sensitivity to inhibition by -bungarotoxin (-BuTX), rapid desensitization and relatively low sensitivity to activation by acetylcholine (ACh), while 3 AChRs are -BuTX insensitive, desensitize more slowly and are activated by ACh with more potency.
The subunit composition of AChRs has been shown to determine a number of pharmacological and functional properties. These include apparent affinities for both agonists and antagonists, agonist efficacy and desensitization rates (Luetje et al. 1990; Cachelin & Jaggi, 1991; Luetje & Patrick, 1991; Papke & Heinemann, 1994). Determining the subunit composition of human ganglionic AChRs is crucial to understanding the physiological roles played by these AChRs and to developing effective therapeutic agents that may target particular AChRs.
The dependence of AChR function on subunit composition has been studied extensively through expression of in vitro transcribed RNA in Xenopus oocytes coupled with electrophysiological approaches. Early studies that utilized this approach quickly established the functionality of pairwise combinations of 2, 3 or 4 with 2 or 4 subunits (Papke, 1993, and references therein). On the other hand, early attempts to establish the functionality of 5, 6 and 3 were unsuccessful when these were coinjected with any known or subunit. Much later studies established functionality for the 6 subunit when coexpressed with the 2 or 4 subunits (Gerzanich et al. 1996; Fucile et al. 1998). Recent reports suggest that the 5 subunit of chicken or human does not contribute to the agonist binding site but serves a structural role, since assembly into surface AChRs requires both an 3 and a subunit (Conroy et al. 1992; Wang et al. 1996). Assembly of the human 5 subunit into 3 AChRs altered many of the functional and pharmacological properties of the resulting AChRs (Gerzanich et al. 1998). Similarly, the 5 subunit altered the functional properties of 42 AChRs (Ramirez-Latorre et al. 1996; Kuryatov et al. 1997). Finally, the 3 subunit has recently been shown to assemble with 4 AChRs in rat brain (Forsayeth & Kobrin, 1997) and to assemble into functional AChRs when expressed heterologously with 3 and 4 subunits (Groot-Kormelink et al. 1998).
Even though the functional properties of ganglionic 3 AChRs have been well described, questions concerning the subunit composition of native 3 AChRs remain (McGehee & Role, 1995; Lindstrom, 1996). To address these questions many studies have relied on approaches that utilize pharmacological fingerprinting of heterologously expressed AChRs. Pharmacological profiles established for these subunit-defined AChRs and for AChRs studied in native tissues provide a basis for comparison. Matches between profiles substantiate subunit identity. However, no exact match has been found between heterologous AChRs and native ganglionic AChRs (Covernton et al. 1994; Sivilotti et al. 1997). This might reflect differences in the expression environment between native cells and heterologous systems.
Here, we have used single channel analysis to characterize the impact of the various AChR subunits that have been identified in ganglia on human 3 AChR function, i.e. AChRs formed from coexpression of 3, 5, 2 and 4 subunits in pairs or as triplets. We have found that for human 3 AChRs, substituting 4 for 2 resulted in an increase in open and burst times, with a slight change in channel conductance. When the 5 subunit was coexpressed with 32, the resulting 352 AChRs had a new open time that was not observed for 32 AChRs. When the 5 subunit was coexpressed with 34, the 354 AChRs had a new open time, altered burst times and an additional conductance state that were not observed for 34 AChRs. Comparison of oocyte-expressed AChR single channel properties with those of AChRs in the human neuroblastoma cell line IMR-32 suggests that the predominant functional AChR expressed in these cells is probably composed of 3 and 4 subunits. Functional properties that were linked to the presence of 2 and 5 subunits in heterologous AChRs were not observed in AChRs recorded from IMR-32 cells. This was consistent with the low level of 2- and 5-containing AChRs identified by radioimmunoassay of these cells.
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METHODS |
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Oocyte expression
cRNA from linearized cDNA templates for the human 3 (Wang et al. 1996), 5 (generously provided by Chini et al. 1992), 2 (Anand & Lindstrom, 1990) and 4 (Gerzanich et al. 1996) AChR subunits was synthesized in vitro using either T7 or SP6 RNA polymerase (depending on the expression vector) with mMessage mMachine (Ambion, Austin, TX, USA). As described previously (Wang et al. 1996), oocytes were removed surgically from Xenopus laevis under anaesthesia with 0·2 % tricaine (Sigma) in accordance with guidelines set forth by the Institutional Animal Care and Use Committee (IACUC) at the University of Pennsylvania under an approved protocol on file with that office. IACUC operates under an institutional Animal Welfare Assurance (A3079-01) on file with the Office for Protection from Research Risks at the National Institutes of Health, Rockville, MD, USA. After enzymatic dispersal oocytes were injected cytosolically with 10-25 ng of RNA per oocyte and incubated at 18°C in Leibovitz L-15 media (Gibco Laboratories) diluted 50 % in 10 mM Hepes (pH 7·3 with NaOH). Expression levels were monitored by two-electrode voltage clamp in oocytes that had been injected at least 4 days earlier. Near-saturating concentrations of ACh (usually 300 µM) were applied to the oocytes by a gravity fed glass capillary tube that was connected to a reservoir mounted above the recording chamber. ND-96 was used for recording and had the following composition (mM): NaCl, 96; KCl, 2; MgCl2, 1; CaCl2, 1·8; and Hepes, 5; pH 7·6 with NaOH and atropine sulfate (1 µM). Electrodes were filled with 3 M KCl and had resistances of 3-5 and 0·5-3 M
for the voltage and current electrodes, respectively. Oocytes clamped at -50 mV that exhibited currents greater than 5 µA were selected for attempts at single channel recording in isolated outside-out patches.
Electrophysiology - Xenopus oocytes
Xenopus oocytes were manually stripped of the vitelline membrane following osmotic shrinking with a 200 mM potassium aspartate solution (Methfessel et al. 1986). Outside-out configuration patches (Hamill et al. 1981) were formed from stripped oocytes expressing recombinant human AChRs. Single channel recordings were made in the ND-96 solution that was used for the oocyte screening. The patch pipette contained a solution consisting of (mM): CsF, 80; CsCl, 20; Cs-EGTA, 10; Hepes, 10; and MgATP, 3; pH 7·2 with CsOH. Electrodes were formed from borosilicate glass tubing (World Precision Instruments) and had resistances of typically 7-15 M.
Electrophysiology - IMR-32 cells
IMR-32 cells were obtained from American Type Culture Collection (Rockville, MD, USA) and maintained in culture medium consisting of a combination of Ham's F12 and Eagle's Minimal Essential Medium (MEM) (1:1; Sigma), and 10 % fetal bovine serum (Hyclone Laboratories, Logan, UT, USA). For recording, cells were detached by incubation in 5 mM EDTA in standard phosphate-buffered saline and then resuspended in normal culture medium followed by plating onto bare 12 mm diameter glass coverslips. After 1 day, the serum in the medium was reduced to 1 % to increase the number of surface AChRs as measured by binding of 125I-mAb 210, a monoclonal antibody that binds to 3 and 5 subunits. Recordings were attempted 3-7 days after plating to allow for adequate cell attachment. For IMR-32 cells, the same solutions that were used for recording oocyte-expressed AChRs were used with the addition of 50 mM dextrose (Sigma) to the external (bath) solution to increase the osmolarity, which seemed to promote better seal formation. Recordings for IMR-32 and heterologous 34 AChRs were also performed under 'high sodium conditions', reflecting the higher concentration of sodium in the external solution, which contained (mM): NaCl, 165; KCl, 5; CaCl2, 2; Hepes, 5; and dextrose, 10; pH 7·3 with NaOH. Patch pipettes under high sodium conditions contained a solution consisting of (mM): caesium gluconate, 145; Cs-EGTA, 10; and Hepes, 10; pH 7·2 with CsOH.
Data acquisition and analysis
Single channel currents were activated in patches by application of ACh flowing from one barrel of a two-barrel glass capillary tubing. Prior to the application of agonist, the patch was isolated in a continuously flowing control solution from the other barrel. Drug solutions were selected between by a 6-way valve. All drugs were prepared by diluting from stocks that were stored either frozen (acetylcholine chloride and hexamethonium bromide; both from Sigma) or at 4°C (mecamylamine; Merck Sharp & Dohme Research Laboratories, Rahway, NJ, USA). Recordings were obtained with an Axopatch-1D amplifier (Axon Instruments) and sampled with a VR-10A digital data recorder (Instrutech Corp., Great Neck, NY, USA) onto video media (Sharp Corp., Osaka, Japan; VHS model VC-A206C) for later analysis. Signals were sampled off-line at 10 kHz (for dwell times) or 15 kHz (for all-points amplitude determinations and channel block experiments) (Axoscope 2.0, Axon Instruments) and were usually filtered at 3 kHz, while some patches required 2 kHz filtering (Frequency Devices, Inc., Haverhill, MA, USA; Model 902; 8-pole Bessel, -3 dB) for analysis, due to higher noise levels. All single channel analyses and fitting were performed with pCLAMP 6.0.3 (Axon Instruments). For channel open time determinations, events list files were formed by visual inspection of the data and manually accepting or rejecting putative events. The data in the events list files were then log binned into histograms using seven to nine bins per decade and fitted with exponential functions using the method of maximum likelihood of the estimator and a Simplex algorithm. Events having durations of less than two sample intervals were ignored during fitting which would concatenate the events flanking those which were expunged. The number of components that best described the fits was evaluated by visual scrutiny of the goodness of the fit and by the natural logarithm of the ratio of the maximum likelihood for each model defined as follows: ln(maximum likelihood of the estimator for a model with n + 1 components/maximum likelihood of the estimator for a model with n components). The log of the likelihood ratio was usually necessary only when the improvement in the estimate was questioned between two and three components. In this case, values less than 3 for the ratio were considered insufficient improvement to justify the greater number of parameters.
Burst durations were estimated by determining a critical gap time that would establish a delineation between intraburst closed times and interburst closed times so that the theoretical rate of misclassification of each as the other would be equal. The probability density functions describing the fits (see Fig. 5) of the brief and long closed times were integrated, set equal, and then solved (Maple V, release 3; Waterloo Maple, Waterloo, Ontario, Canada) for the critical time at which the areas of overlap were equal (e.g. Magleby & Pallotta, 1983; Jaramillo & Schuetze, 1988). Estimates for the critical times were evaluated further using the test burst interval that is part of pSTAT (pCLAMP 6.0.3) by optimizing both burst duration and the number of closings per burst. The procedure involves incremental increases in the critical time value while analysing the relationship to mean burst duration (or closings per burst) until a plateau reveals possible burst structure from events list analysis files. Mean channel burst times defined by the resulting critical time approximate the activation period of the AChR, i.e. the rate of channel closures that are followed by agonist dissociation and/or the rate at which AChR desensitization occurs. Typical critical gap times usually ranged between 1 and 3 ms. Closed times shorter than the critical gap were considered intraburst closed times and those longer than the critical gap were considered interburst closed times. Misclassification rates of closed times according to intrabursts erroneously classified as interbursts or interbursts erroneously classified as intrabursts were sometimes as high as 5 % of all closed intervals. Misclassification occurred due to the overlap in distributions of the inter- and intraburst closed intervals (see Fig. 5).
Open level amplitude histograms (mean amplitude of samples during individual channel openings) were formed by conventional binning of accepted events that were longer in duration than 2·5 times the filter rise time (Tr = 0·3321/fc, where fc is the effective cut-off frequency of the system). The resulting distributions were fitted with a Gaussian function of the appropriate number of components. Conductances were determined from the slope of a linear fit of channel amplitudes determined at several holding potentials. The slopes at negative voltages were extrapolated to estimate their reversal potentials. This reversal potential was used to calculate a chord conductance for channels with insufficient activity to determine a true slope conductance by using the amplitude at -80 mV and then dividing by the difference between the holding potential and the reversal potential. All conductances were then corrected for liquid junction potentials that were measured for the appropriate recording conditions (Neher, 1992). For patches recorded with ND-96 conditions, the liquid junction potential was 9 mV and for patches recorded with high sodium conditions, the liquid junction potential was 13 mV. For graphical representation, histograms with fits were exported to Origin (Microcal Software, Inc., Northampton, MA, USA). Representative single channel traces were constructed by opening data files in Axograph 3.55 (Axon Instruments) and exporting data to Canvas 5.0 (Daneba Software, Inc., Miami, FL, USA).
Radioimmunoassays (RIA)
IMR-32 cells were grown as described above until just before confluence in T-175 (175 cm2) tissue culture flasks (Falcon 3112, Becton Dickinson, Franklin Lakes, NJ, USA) then detached with 5 mM EDTA in phosphate-buffered saline, pelleted by centrifugation, and frozen after removal of media. Previously frozen IMR-32 cells were suspended in a 2 % Triton X-100 buffer (50 mM Na2HPO4-NaH2PO4 pH 7·5, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 5 mM benzamidine, 15 mM iodoacetamide, 2 mM phenylmethylsulfonyl fluoride and 2 % Triton X-100) at a 3 : 1 to 6 : 1 ratio (buffer volume : cell volume) and placed on a rotating mixer for 1 h at 4°C. Cellular debris was removed by centrifugation and supernatant extract was collected.
For solid phase RIA, AChRs solubilized in Triton X-100 buffer were incubated in Immulon 4 microwells (Dynatech Laboratories, Chantilly, VA, USA) coated with monoclonal antibody mAb 210 (specific for 3 and 5 AChRs; Wang et al. 1996) or mAb 295 (specific for 2 AChRs; Whiting & Lindstrom, 1988) with 5-15 nM 3H-epibatidine for 12 h at 4°C. Unbound material was removed from wells by washing 3 times with cold 0·5 % Triton X-100 in 100 mM NaCl. Bound AChRs were eluted from wells with 2·5 % SDS containing 5 % -mercaptoethanol. The amount of bound 3H-epibatidine was determined by liquid scintillation counting. Background was determined using wells coated with bovine serum albumin or by including 2 mM nicotine in antibody-coated wells.
For immunoprecipitation assays, the IMR-32 AChRs were solubilized in Triton X-100 buffer as above and then incubated with excess mAb 210 or rat antiserum to human 5 (Wang et al. 1998) in the presence of 5-15 nM 3H-epibatidine for 12 h at 4°C. mAb 299 (Kuryatov et al. 1997) or a rat antiserum to human 4 (antigen was a bacterially expressed human 4 construct; A. Kuryatov & J. Lindstrom, unpublished data) were used to test for the presence of 4 AChRs in IMR-32 extracts. Rat antiserum to 6 AChR subunits (antigen was a bacterially expressed human 6 construct; A. Kuryatov & J. Lindstrom, unpublished data) was also tested on IMR-32 AChR extracts. Excess sheep anti-rat IgG was then added to precipitate antibody-bound AChRs for 2 h at 4°C. The mixture was then diluted 1 : 10 with 0·5 % Triton X-100 in 100 mM NaCl and centrifuged to pellet the precipitate. The pellet was washed two more times with the 0·5 % Triton X-100 solution and then suspended in 2·5 % SDS-5 % -mercaptoethanol. The amount of epibatidine bound was determined by liquid scintillation counting. Background was determined by parallel assays containing normal rat serum instead of the antibody used for testing.
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RESULTS |
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The subunit determines the single channel conductance of 3 AChRs
We investigated the contributions of 2, 4 and 5 subunits to human 3 AChRs expressed in Xenopus oocytes and compared the channel properties with those of AChRs expressed by IMR-32 human neuroblastoma cells.
Application of ACh to outside-out patches from oocytes expressing AChRs resulting from combinations of these subunits evoked single channel activity that exhibited differing kinetic behaviour. Examples of channel activity reflecting these AChR subunit combinations are shown in Fig. 1.
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3 AChR single channel currents
Each panel depicts representative single channel recordings for outside-out patches from oocytes that were injected with the indicated combination of human AChR subunit transcripts. ACh concentrations were 1, 5, 10 and 5 µM for | ||
32 AChRs activated by ACh (0·5-1 µM) exhibited channel openings that had two different conductance levels. Open level histograms revealed amplitudes of -1·6 ± 0·1 (mean ± S.E.M.; n = 6 patches) and -2·1 ± 0·1 pA (n = 7; Fig. 2) at a holding potential of -80 mV. The slope conductance of these channels was determined from their current-voltage relationship using all-point amplitude histograms fitted to Gaussian functions having the appropriate number of components. The slope conductance of between -100 and -40 mV for the higher conductance type of 32 AChRs was 26 pS. This represented the predominant (78 %) conductance during prolonged recording (not shown). The extrapolated reversal potential was not significantly different from 0 mV (P > 0·05, Student's t test; n = 5) as would be expected for a non-selective cation channel. At positive holding potentials, a slight outward rectification in the slope conductance was observed between +40 and +60 mV (not shown). Although not as prevalent as the 26 pS channel openings, lower conductance openings were often observed (in 6 out of 7 patches) that had sufficient activity to determine a slope conductance of 21 pS. This lower conductance channel opening also displayed a slight outward rectification at positive holding potentials. The lower conductance channel openings were more prevalent during the beginning of the agonist applications, which is when the amplitudes were measured for the slope conductance.
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3 AChRs expressed in oocytes
For each subunit composition, open level amplitude histograms were constructed from events list files created to include all event amplitudes that could be discerned for a particular patch (either one or two conductance levels). The fits to Gaussian functions with the appropriate number of components are also shown. For the | ||
34 AChRs activated by ACh (5 µM) exhibited channel openings that had predominantly a single conductance. At -80 mV, the amplitude of these channel openings was -2·3 ± 0·1 pA (n = 4; Fig. 2). A lower amplitude channel opening (-1·6 ± 0·1 pA; n = 3) was sometimes present, but at low frequency. The slope conductances of these AChRs were 31 and 24 pS, respectively. The extrapolated reversal potentials were not significantly different from 0 mV (P > 0·05; n = 4). At positive holding potentials, the currents showed slight outward rectification, similar to that observed for 32 AChRs.
Channel open times of 32 AChRs are faster than those of 34 AChRs
Open time histograms of 32 AChR activity were best fitted by double exponential probability density functions with time constants of 0·71 ± 0·14 and 3·5 ± 0·4 ms (n = 6; Fig. 3). All patches had both components. The area under the components of the fitted function correlates with the number of openings that each time constant represents. Thus, 65 % of the channel openings were of the faster type and 35 % were the slower type.
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3 AChRs expressed in oocytesFor each subunit combination, log-binned histograms were constructed from events list files for the predominant conductance for that channel. As shown, the histograms were fitted with exponential functions of the appropriate number of components using the method of maximum likelihood. Time constants reflect the mean values observed for all patches including components that may not have been present in the representative example. Percentages reflect the mean areas from the fits that correspond to each exponential component when present. The holding potential was -80 mV. | ||
Open time histograms for 34 AChRs were also best fitted by double exponential functions having time constants of 1·4 ± 0·2 and 6·5 ± 0·8 ms (n = 5; Fig. 3). All patches contained both components. Each component represented 50 % of the distributions, which indicated that the gating of the 34 channel favoured the two types of openings equally.
32 AChRs exhibit shorter bursts than 34 AChRs
The tendency of 3 AChRs to run down during recording complicates the evaluation of burst durations since channel activity cannot be monitored for extended periods to distinguish unequivocally between desensitized and resting states. The problem of run-down has previously been reported for nicotinic AChRs (Fenwick et al. 1982; Alkondon et al. 1994). Attempts to counter run-down by altering the recording electrode solution in ways that have proven useful in other systems were unsuccessful. Changes that were tested included addition of phosphocreatine, creatinine phosphokinase, Mg-ATP and/or the catalytic subunit of protein kinase A or changing to a purely chloride-based recording solution. It is possible that the problem of run-down might be avoided under cell-attached recording, but our experience has been that endogenous stretch-activated channels in oocytes (Methfessel et al. 1986) present a bigger problem when using this configuration.
Even though the actual values of burst durations might be adversely affected by less than optimal conditions for burst analysis, values obtained in this way provide insight into gating mechanisms for these AChRs. Furthermore, burst times broaden our understanding of the overall impact that various AChR subunits have on 3-type nicotinic AChR gating and help correlate single channel behaviour with macroscopic currents. Burst durations were defined by evaluating closed time distributions (Fig. 5) for the appropriate critical gap time that would be likely to encompass channel closures that occur during the time that agonist is bound by the AChR (see Methods).
Histograms of burst durations for 32 AChRs were best described by double exponential functions. The time constants for the two components were 0·82 ± 0·12 and 5·3 ± 0·7 ms (n = 6; Fig. 4). Each component was present in all patches and represented 57 and 43 % of the openings, respectively. The mean number of openings in the longer duration bursts was two, based on the open times. Increasing the burst critical closed time up to fivefold had little effect on time constants that described the burst distributions, which supports the idea that the definition of intra- and interburst closed times was adequate.
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3 AChRs expressed in oocytesFor each subunit combination, log-binned histograms were constructed from events list files for the predominant channel conductance. The burst criterion times were evaluated as described in Methods. The histograms were fitted with exponential functions of the appropriate number of components using the method of maximum likelihood. The time constants reflect the mean of values observed for all patches for that AChR. The percentages reflect the mean areas from the fits that correspond to each exponential component when present. The holding potential was -80 mV. | ||
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3 AChRs expressed in oocytes
Examples of closed time distributions were fitted with exponential functions as described in Methods. For these patches, four components could adequately describe the data. The time constants determined from the fits are given for each patch. These values were used to set the critical times for burst definitions. For | ||
34 AChR burst time histograms were also best described by double exponential functions, having time constants of 1·7 ± 0·2 and 16 ± 1·0 ms (n = 5; Fig. 4). All patches contained both components and they represented 54 and 56 % of the bursts, respectively. The mean number of openings per long burst was approximately two. The duration of the shorter burst was the same as the shorter open time, which reveals a distinction between the transition that accounts for terminating these openings and the transition that accounts for terminating longer duration bursts. The longer burst duration of 34 channels compared with 32 channels reflects a slower rate of channel closure in combination with slower desensitization resulting in longer activation during agonist binding to the AChR. This difference correlates with the slower time course in the decay of 34 AChR macroscopic currents compared with the decay of 32 AChR macroscopic currents (Wang et al. 1996; Chavez-Noriega et al. 1997; Gerzanich et al. 1998).
32 AChRs desensitize and inactivate faster than 34 AChRs
At similar concentrations of ACh, 32 AChRs had a greater tendency to desensitize than 34 AChRs. For example, when 32 channels were activated with 5 µM ACh, high levels of channel activity that desensitized relatively quickly were observed. Histograms of averaged channel opening frequency over the first 100 s of the recording could be fitted with a double exponential function having time constants of 1·1 and 37 s (data not shown). By contrast, when 5 µM ACh was applied to patches containing 34 AChRs, the average frequency histogram of channel activity was best fitted by a function having time constants of 3·2 and 127 s (data not shown). This indicated that 32 AChRs desensitized faster than 34 AChRs. Additionally, both channel types exhibited permanent inactivation or 'run-down' during recordings. 32 AChR channels exhibited run-down faster than 34 AChRs, even when recorded at agonist concentrations that favoured less desensitization. Although not determined precisely, permanent inactivation typically occurred for 32 AChRs within about 4-5 min during continuous recording, while 34 AChRs remained active 3-4 times longer. These results are consistent with the faster decay of macroscopic currents for human 32 compared with 34 AChRs when expressed in oocytes (Wang et al. 1996; Chavez-Noriega et al. 1997; Gerzanich et al. 1998).
5 subunits have little effect on 32 AChR single channel properties in contrast with the large effect of 5 on 34 AChR single channel properties
Coexpression of 5 with the 3 and 2 subunits yielded single channel behaviour that had subtle differences compared with that observed for 32 AChRs (Fig. 1). Amplitude distributions for 352 AChRs exhibited two conductance levels. These conductances were essentially the same as those seen for 32 AChRs (Fig. 2). The amplitudes were -1·5 and -2·0 pA at -80 mV. The proportions of channel openings for each conductance type, when present, were 50 and 60 % for the lower and higher conductance channels, respectively. Four out of five patches had the lower conductance channel openings, while all patches had the higher conductance channel openings. The slope conductances for these AChRs were not determined due to difficulties in achieving adequate expression levels during coexpression of the 5 subunit with the 3 and 2 subunits. This problem was unique to coexpression of 5 with the 2 subunit, and was not found with the 4 subunit (see below). However, assuming the same reversal potential for 352 as was found for 32 AChRs, the slope conductances were estimated to be 22 and 26 pS, which were not different from the values estimated for 32 AChRs. Similar to what was observed for 32 AChRs, the channel activity from coexpression of 3, 2 and 5 subunits inactivated within 2-3 min after onset of activity during continuous recording and within about 5 min after pulling the patch, even if not continuously in agonist.
Incorporation of the 5 subunit caused the appearance of a new component in the open time distributions compared with those observed for 32 AChRs. The distributions of open times were fitted by double exponential functions for all patches. After pooling the results from the fits of all patches, three time constants were identified, which were 0·50 ± 0·10, 2·1 ± 0·3 and 5·5 ± 0·3 ms (Fig. 3). Four out of five patches had the fastest component, while each of the other components was present in three out of five patches. The percentages of openings for each component when it was present were 76, 42 and 34 %, respectively.
The burst distributions for 352 AChRs were all fitted best by double exponential functions. The mean time constants were 0·90 ± 0·28 and 5·0 ± 1·0 ms. All patches (n = 5) had both components and they represented 56 and 44 % of the bursts, respectively (Fig. 4).
Coexpression of 5 with 3 and 4 subunits caused a considerable change in the single channel properties compared with 34 AChRs. The 354 AChRs had an additional larger conductance channel, a longer channel open duration and increased burst duration. Application of ACh (5 µM) to patches containing AChRs formed from coexpression of 3, 5 and 4 subunits evoked high levels of well-sustained channel activity. Desensitization occurred over a period of 2-3 min, but channel activity could be recovered by periodically moving between control and agonist solutions.
Three separate channel amplitudes were observed, which were -1·7, -2·2 and -2·6 pA at -80 mV (Fig. 2). The lowest amplitude channel appeared in three out of ten patches, the intermediate channel appeared in nine out of ten patches, and the largest amplitude channel appeared in three out of ten patches. The proportions of channel openings for each channel level were 39, 81 and 51 %, when present, respectively. Only the intermediate amplitude channel type had sufficient activity to obtain recordings at enough holding potentials to determine a slope conductance, which was 31 pS. This conductance was not significantly different from the conductance determined for oocytes expressing 34 AChRs. The extrapolated reversal potential was not significantly different from 0 mV (P > 0·05; n = 5). By assuming the same reversal potential, the slope conductances were calculated to be 24 pS for the lower amplitude channel and 36 pS for the higher amplitude channel.
The open time distributions for the AChRs formed by coexpression of the 3, 5 and 4 subunits were usually best fitted by double exponential functions. Only three out of ten patches exhibited the presence of all three components. The channel kinetics appeared to be similar for all conductance levels. The time constants for the three different open times were 1·6 ± 0·2, 6·4 ± 0·7 and 22 ± 3 ms (Fig. 3). The components were present in seven, seven and eight out of ten patches, respectively, and represented 52, 57 and 26 %, respectively, of the open times when present. We considered the possibility that agonist concentration might influence the observed kinetic properties by differentially activating (or by failing to activate) AChRs of different compositions that had differing agonist affinities, but this was not evident from the data, which was recorded mostly at concentrations of 5 or 10 µM ACh. However, if this was the case, one might expect to see a change in kinetic properties over the time course of a recording. Such instability would be evident in scatter analysis of open time versus elapsed time of recording. Such an analysis (Fig. 7) indicated that both short and long duration openings occurred throughout a recording of 354 activity, which only differs from 34 activity in the magnitude of the longer duration openings. Although some of the single channel properties found for 354 AChRs resemble those of 34 AChRs, the emergence and prominence of the longest duration channel type (Fig. 6) leads to the conclusion that it reflects a novel population of channels. These channels can be attributed logically to AChRs that have incorporated the 5 subunit. However, there was not an exclusive correlation between the largest conductance and longest duration opening, since the intermediate conductance channel exhibited long duration openings as well. This point raises the possibility of multiple conductance levels for 5 AChRs and shows that conductance alone may be inadequate to identify the presence of the 5 subunit in AChRs that also contain 3 and 4 subunits.
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354 AChRs
This recording was made at -80 mV and has been scaled so that it can be compared directly to the recordings presented in Fig. 1. Note the dramatic increase in duration of channel openings and bursts compared with | ||
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34 AChRs and 354 AChRs in oocytes
These plots demonstrate the persistence of channels of all kinetic types throughout the duration of the recordings. Note the difference in ordinate magnitudes for the two channel types and the much longer duration channel openings for | ||
The burst kinetics for AChRs formed from 3, 5 and 4 subunits were distinct from those observed for 34 AChRs. The mean burst times were 6·2 ± 0·4 (n = 9) and 43 ± 4 ms (n = 8; Fig. 4). The two components were present in nine or eight out of ten patches, respectively, and represented 64 and 39 %, respectively, of bursts when present. Examples of the longer duration bursts observed for 354 AChRs are shown in Fig. 6. Judging from the open durations, the bursts for 354 AChRs reflected both single and multiple openings (2-4 on average) of the channel, assuming that the 6 ms burst included 2 and 6 ms channel openings and the 43 ms burst included the 22 ms openings as well as the others. The burst criterion time could be increased up to fivefold without a significant difference in the burst distributions. The persistence of the longer duration openings throughout the recording (Fig. 7) supports the conclusion that incorporation of the 5 subunit is reflected in the longer duration bursts.
Sensitivity of 32 and 34 AChRs to inhibition by hexamethonium or mecamylamine
Single channel recordings from patches excised from Xenopus oocytes can sometimes be contaminated by spontaneous activity from an endogenous non-selective cation channel that is mechanosensitive (Methfessel et al. 1986). Although this 'stretch-activated' channel activity is less common in the outside-out patch configuration, it was sometimes observed during attempts at recording AChR activity for the present study. Patches that clearly exhibited these spontaneous stretch-activated channels were not used for analysis. To demonstrate the nicotinic nature of the channels analysed for the present study, and to distinguish them from the possible stretch-activated channels, activity recorded in some patches was blocked by the traditional nicotinic antagonists mecamylamine and hexamethonium. Channel activity evoked by application of 1 µM ACh to an outside-out patch from an oocyte expressing 32 AChRs was blocked nearly completely by mecamylamine (50 µM) or hexamethonium (100 µM; Fig. 8A). 352 AChRs were also blocked by mecamylamine (Fig. 8A). The channels that remained in the presence of this high concentration of mecamylamine had shorter duration open times and much reduced open frequency, consistent with a high affinity channel block mechanism. Channels that were blocked by hexamethonium were of shorter open duration, but their open frequency appeared less affected, consistent with a lower affinity channel block.
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3 AChR single channel currents by hexamethonium or mecamylamine
A, all | ||
The channel open durations for 32 AChRs were measured in the presence of a series of mecamylamine concentrations (5-25 µM) to quantify the inhibition of this AChR. The channel open duration decreased as the concentration of mecamylamine was increased. Fitting the slope of the relationship between the reciprocal of the channel open time (1/
open) and the mecamylamine concentration allowed the second order rate constant to be approximated (Fig. 8B). Using this approach, the rate of block of 32 AChRs by mecamylamine was 4·6 × 107 M-1 s-1. As stated above, the channel open frequency in the presence of mecamylamine was reduced considerably, which indicated that once the channel became blocked by mecamylamine, the duration of block was relatively long. The duration of the blocked state was in the range of closed times that occur between activations by agonist and could not be readily distinguished from these closed but unblocked states. Because of this, and the fact that the channel activity was diminishing due to channel run-down, the duration of the channel blocked state was not estimated. The concentration-dependent reduction in channel open duration, however, indicated that the mechanism of inhibition was probably through open channel blockade. A 2 min wash of the patch followed by reapplication of 1 µM ACh alone showed that the channel activity recovered fully.
Similarly, mecamylamine (50 µM) blocked 34 AChRs. They were also blocked by hexamethonium (50 µM; Fig. 8A). Channels were first activated by application of agonist alone. Subsequent co-application of mecamylamine (50 µM) resulted in channel activity exhibiting shorter channel open times and much reduced open frequency. Application of hexamethonium with ACh evoked channels having reduced channel open durations. Similar sensitivity to block by hexamethonium was observed with 354 (Fig. 8A).
To quantify the interaction of hexamethonium with 34 AChRs, a series of concentrations (5-50 µM) was tested for the extent of reduction in channel open duration. As the concentration of hexamethonium was increased, channel open duration was reduced (Fig. 8B), which was consistent with open channel block. Based on the concentration dependence of the reduction in channel open duration, the rate of block of 34 AChRs by hexamethonium was estimated as 1·2 × 107 M-1 s-1. Application of ACh alone after approximately 2 min recovery in the absence of hexamethonium showed the reversibility of this blockade. Thus, the mechanism by which hexamethonium inhibited 34 AChR activity was through a channel block mechanism. The potency of inhibition of 34 open times by hexamethonium was at least threefold lower than the inhibition of 32 AChRs by mecamylamine.
Single channel properties of IMR-32 AChRs resemble those of 34 AChRs expressed in oocytes
Application of ACh (5-10 µM) to outside-out patches from IMR-32 cells evoked moderate levels of single channel activity that remained for approximately 2-4 min in the continuous presence of agonist (Fig. 9), which was similar to heterologous 3 AChRs having the 4 subunit, but not those with the 2 subunit. The channel activity recovered partially after approximately 1 min in agonist-free solution, but it eventually inactivated completely due to run-down. Fits of open level amplitude histograms revealed a unitary amplitude of -2·2 ± 0·1 pA (n = 5) at -80 mV (Fig. 9). The chord conductance was estimated to be 32 pS. Fits of open duration histograms to double exponential functions revealed mean open times of 1·5 ± 0·3 (n = 4) and 9·6 ± 1·2 ms (n = 4) with the two components representing 48 and 52 % of the distribution, respectively. Histograms of channel burst times were fitted to reveal durations of 1·6 ± 0·4 (n = 4) and 25 ± 3·0 ms (n = 4). The components represented 54 and 46 %, respectively, when present. To aid comparison, the single channel properties of IMR-32 AChRs are summarized along with those of oocyte-expressed AChRs in Table 1. Although not exact, the channel properties of IMR-32 AChRs resemble most closely those of 34 AChRs.
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3 AChR single channel currents recorded from IMR-32 neuroblastoma cellsRepresentative examples are shown for single channel activity recorded from outside-out patches from IMR-32 cells for the distributions of amplitude, open duration and burst duration. Time constants and percentages were determined as described for figures depicting heterologously expressed AChRs. All data were recorded at -80 mV. | ||
Table 1. Human 3 AChR single channel properties
| Conductance (pS) |
Mean open time (ms) |
Mean burst time (ms) |
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| AChR |  1 |
2 |
3 |
1 |
2 |
3 |
1 |
2 |
3 |
| 32 |
21 ± 1 | 26 ± 1 | - | 0·71 ± 0·14 | 3·5 ± 0·4 | - | 0·82 ± 0·1 | 25·3 ± 0·7 | - |
| (22 ± 6 % | (78 ± 6 % | - | (65 ± 4 % | (35 ± 4 % | - | (57 ± 4 % | (43 ± 4 % | - | |
| 6/7) | 7/7) | - | 6/6) | 6/6) | - | 6/6) | 6/6) | - | |
| 352 |
22 ± 1 | 26 ± 1 | - | 0·50 ± 0·10 | 2·1 ± 0·3 | 5·5 ± 0·3 | 0·90 ± 0·28 | 5·0 ± 1·0 | - |
| (50 ± 19 % | (60 ± 18 % | - | (76 ± 1 % | (42 ± 15 % | (34 ± 18 % | (56 ± 14% | (44 ± 14 % | - | |
| 4/5) | 5/5) | - | 4/5) | 3/5) | 3/5) | 5/5) | 5/5) | - | |
| 34 |
24 ± 1 | 31 ± 1 | - | 1·4 ± 0·2 | 6·5 ± 0·8 | - | 1·7 ± 0·2 | 16 ± 1·0 | - |
| (50 % | (90 ± 10 % | - | (50 ± 7 % | (50 ± 7 % | - | (54 ± 4 % | (56 ± 4 % | - | |
| 1/5) | 5/5) | - | 5/5) | 5/5) | - | 5/5) | 5/5) | - | |
| 354 |
24 ± 1 | 31 ± 1 | 36 ± 1 | 1·6 ± 0·2 | 6·4 ± 0·7 | 22 ± 3 | - | 6·2 ± 0·4 | 43 ± 4 |
| (39 ± 6 % | (81 ± 6 % | (51 ± 24 % | (52 ± 8 % | (57 ± 9 % | (26 ± 5 % | - | (64 ± 7 % | (39 ± 7 % | |
| 3/10) | 9/10) | 3/10) | 7/10) | 7/10) | 8/10) | - | 9/10) | 8/10) | |
| IMR-32 | - | 32 ± 1 | - | 1·5 ± 0·3 | 9·6 ± 1·2 | - | 1·6 ± 0·4 | 25 ± 3·0 | - |
| - | (100 % | - | (48 ± 7 % | (52 ± 7 % | - | (54 ± 7 % | (46 ± 7 % | - | |
| - | 5/5) | - | 4/4) | 4/4) | - | 3/4) | 4/4) | - | |
IMR-32 AChRs and 34 AChRs were also recorded and compared under different conditions to gain insight into the effects of ionic conditions on AChR function. For these experiments, the bath solution was very similar to the ND-96 solution used for all previous experiments, but contained a higher sodium concentration and will be referred to as high sodium conditions (see Methods). The pipette solution for the high sodium conditions differed substantially from that used for all previous experiments and contained caesium gluconate instead of caesium fluoride and caesium chloride (see Methods). As can be seen in Fig. 10, the IMR-32 and 34 AChR channels had a remarkably similar appearance under these conditions. The mean amplitude of IMR-32 channels and that of the main conductance for 34 AChRs were identical. The presence of the lower conductance channel for 34 AChRs in oocytes was not affected by the different recording conditions. All channels had larger amplitudes under the high sodium conditions, which was consistent with the higher sodium concentration in the bath compared with the recordings made in ND-96. The presence of clear direct transitions from the main conducting state of 34 AChRs to the lower conductance state became evident in some patches under the high sodium conditions. The reason for the appearance of direct transitions under the high sodium conditions remains unclear. Whether the change reflects a modification of the AChR that was different for these recordings compared with the earlier data or whether the difference reflects the effects of holding potential or ionic conditions will require further investigation. The presence of the direct transition between conductance levels leads to the conclusion that the lower conductance level reflects the same population of channels giving rise to the higher conductance levels and not a structurally (with respect to subunit composition) unique population of AChRs. It is interesting to note that subconductance level transitions were not observed for IMR-32 AChRs under either recording condition.
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3 AChR single channel currents recorded from IMR-32 neuroblastoma cells resemble oocyte-expressed 34 AChRs under high sodium conditions
High sodium conditions had higher external sodium than previous experiments and no internal fluoride (see Methods). Representative examples are shown for channel activity recorded from outside-out patches along with the distributions of channel amplitudes, open durations and burst durations. Time constants and percentages were determined as described in earlier figures and reflect the means ± S.E.M. from three to five patches. All data were recorded at -60 mV. The arrow marks a clear transition to a subconductance state during the | ||
The channel kinetics were similar between IMR-32 and 34 AChRs under high sodium conditions (Fig. 10). 34 open times were unchanged, while IMR-32 channel openings were of somewhat shorter duration than was observed using the ND-96 solutions. Because the kinetics of 34 AChRs appeared to be unaffected by the recording conditions, the difference in the IMR-32 AChR properties must reflect some heterogeneity in function for these AChRs, which could be the result of different post-translational modification.
Fraction of total IMR-32 3 AChRs containing 5 or 2 subunits
Monoclonal antibodies or subunit-specific antisera can be used to select AChRs containing particular subunits from crude extracts of IMR-32 cells, and the number of AChRs containing the particular subunit can be quantified by 3H-epibatidine binding. Using this approach, antibodies were used to determine the fraction of IMR-32 3 AChRs that contained 2 or 5 subunits. To determine the fraction of AChRs that contained the 2 subunit, Triton X-100-solubilized AChRs from IMR-32 cells were adsorbed onto either mAb 210-coated microwells for determining the total amount of 3 AChRs or mAb 295-coated microwells for determining the total 2 AChRs that were present. The adsorbed AChRs were labelled with 3H-epibatidine and the labelled, bound AChRs were eluted and measured by liquid scintillation counting. The amount of 2 AChRs bound was 6 ± 2 % (n = 3) of the total 3 AChRs in IMR-32 cells (Fig. 11A).
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3 AChRs that contain 2 or 5 subunits
A, solid-phase RIAs were used to demonstrate the fraction of total IMR-32 | ||
To determine the fraction of 3 AChRs that contained the 5 subunit, Triton X-100-solubilized AChRs were bound by mAb 210 or antiserum to 5 labelled with 3H-epibatidine, and then immunoprecipitated with sheep anti-rat IgG as the secondary antibody. The amount of 5 AChRs that could be immunoprecipitated was determined by liquid scintillation counting to be 5 ± 2 % (n = 5) of the total 3 AChRs in IMR-32 cells (Fig. 11B). Antibodies to the 4 and 6 subunits were also tested, but they detected no AChRs containing these subunits in IMR-32 cells (data not shown).
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DISCUSSION |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Single channel analysis provides the most direct means of characterizing the impact of a particular subunit on the functional properties of AChRs, since each subunit contributes to the formation of the ion channel through its M2 transmembrane region. Conformational changes that lead to gating presumably involve the co-ordinated movement of all subunits (Unwin, 1995). Thus, each subunit will influence the ion channel permeability, conductance and gating kinetics (reviewed in Papke, 1993). With this in mind, we have described the single channel properties of various human 3 AChR subtypes recorded from outside-out patches from Xenopus oocytes and IMR-32 neuroblastoma cells. Both single channel kinetics and conductance properties of human 3 AChRs reflect subunit composition. When expressed in pairs, changing between 2 and 4 subunits altered the kinetics of 3 AChRs with a slight effect on channel conductance. The open and burst times for 32 AChRs were shorter than those for 34 AChRs. The 5 subunit increased the channel open duration of 352 AChRs when compared with 32 AChRs, but had little effect on channel burst duration or channel conductance. Coexpression of the 5 subunit introduced a longer bursting and larger conductance channel for 354 AChRs when compared with 34 AChRs. The single channel properties of IMR-32 AChRs most closely resembled those of 34 AChRs expressed in oocytes. Results of RIAs using subunit-specific antibodies were consistent with this conclusion, as they revealed only low levels of 2 and 5 subunits present in the AChRs expressed by IMR-32 cells.
Influence of the subunit on 3 AChR gating kinetics
We found that open and burst times were longer for human 34 AChRs than for human 32 AChRs. Mean open time values for 32 AChRs were 0·71 and 3·5 ms and for 34 AChRs were 1·4 and 6·5 ms. These open times are similar to values for the corresponding rat AChRs (Papke & Heinemann, 1991; but see Sivilotti et al. 1997). The interspecies sequence identities between human and rat for each of these subunits are 91 % for 3, 95 % for 2 and 84 % for 4 (Elliot et al. 1996). The difference in identity between human and rat was greater for the 4 subunit than the 2 subunit, showing that greater potential exists for functional differences for 34 AChRs compared with 32 AChRs across species. In contrast to the values obtained for human 34 AChRs and one earlier report for rat 34 AChRs (Papke & Heinemann, 1991), a recent study showed that rat 34 AChRs had longer open times (approximately fivefold longer) and burst durations that averaged several hundred milliseconds (Sivilotti et al. 1997). Although we have found that burst durations were longer for human 34 compared with 32 AChRs, the longer bursts were only 16 ms for 34, thus significantly less than the values reported for rat (Sivilotti et al. 1997). Even human 354 AChRs had burst durations that averaged only 43 ms for the slower component. Rat 34 AChRs also showed prevalent long-lived transitions to apparent subconducting states (Sivilotti et al. 1997) which were rare and very short lived for human 3 AChRs expressed in oocytes. The apparent functional differences between species could reflect the 16 % difference in sequence identity for the 4 subunit. Alternatively, we speculate that the functional differences could simply reflect differences in post-translational modification of the AChRs that might be influenced by the oocyte incubation medium, which consisted of Leibovitz L-15 medium in our study and was Barth's solution in the study of rat AChRs. Apparent differences between the species might also simply reflect differences between the ionic conditions used for recording in the two studies, but this seems unlikely considering the similarity in 34 AChR channel properties under two different conditions for the present study.
Differences in gating kinetics between human 32 and 34 AChRs parallel the approximately 25 % difference in the overall sequence identity between subunits. The open and burst times indicate that on a time average basis the likelihood of the channel being in the open state is greater for 34 AChRs than for 32 AChRs due to this difference. The seemingly greater preference of 34 AChRs for the open state compared with 32 AChRs indicates a major contribution of the subunit to the activation energies involved in channel gating. Differences within the M2 channel domains are not responsible since mutation of the only channel residue that differs between 2 and 4 was not sufficient to convey 32 AChR kinetics to 34 AChRs (Figl et al. 1996). Kinetic differences therefore must involve less restricted inter- or intra-subunit interactions within AChRs during gating within regions outside of the M2 domain of the subunit.
Influence of subunits on channel desensitization and inactivation
In addition to the difference in channel gating kinetics between 32 and 34 AChRs, there was a clear difference in the tendency of these AChRs to desensitize. The loss of channel activity during agonist application was much more pronounced for 32 AChRs than for 34 AChRs. This activation-dependent desensitization was reversible by allowing recovery in agonist-free solution. However, permanent inactivation was also observed for all 3 AChRs. This channel 'run-down' occurred more rapidly for 32 AChRs than for 34 AChRs expressed in oocytes. Channel activity for 32 AChRs disappeared completely after about 5 min of recording and would not recover when agonist was removed. By contrast, 34 AChRs could be activated for about 10-15 min although the level of activity decreased over the duration of the patch. Attempts to counter the run-down included using a purely chloride-based pipette solution or adding ATP, phosphocreatine, creatine phosphokinase or the catalytic subunit of protein kinase A, all of which were ineffective. This tendency to inactivate showed no clear dependence on channel activation or agonist concentration. Agonist concentration seemed to influence only the rate at which reversible inactivation (desensitization) occurred. Because of this, the ACh concentration was chosen to achieve the greatest level of channel activity without causing desensitization before permanent inactivation occurred. The large cytoplasmic loop between transmembrane domains M3 and M4 accounts for the majority of differences among AChR subunit sequences and could easily alter functional properties of AChRs by intracellular regulatory mechanisms such as phosphorylation (Huganir & Greengard, 1990). This region of the subunits might be responsible for the differences in the rate of channel run-down between 32 and 34 AChRs.
Influence of the subunit on conductance and kinetic properties of AChRs
The subunit has been reported to influence the single channel properties of rat AChRs (Papke et al. 1989). Comparisons between single channel properties of human 32 and 42 AChRs (Kuryatov et al. 1997) reveal that the subunit also influences channel properties of AChRs in a manner similar to that found for subunits. Under the same recording conditions used for 3 AChRs, human 42 AChRs expressed in oocytes exhibited two conductance states having amplitudes of -1·4 and -2·3 pA at -80 mV. These values are similar to our present findings for 32 AChRs. The similar conductances for 32 and 42 AChRs correlate with the high sequence identity between 3 and 4 subunits within the channel region (M2). The only difference in conductance profiles between the two AChR types was the apparent preference of each AChR for a particular conductance state. For 42 AChRs, the frequency of occurrence of the larger and smaller conductance channels was similar, while 32 AChRs favoured the larger conductance channel.
Comparisons between channel kinetics for 42 and 32 AChRs reveal the impact of the subunit on transitions between the open and closed states of the channel. Channel durations for 42 AChRs were 2·3 and 8·0 ms; for 32 AChRs they were 0·7 and 3·5 ms. This shows that the subunit can influence gating behaviour in a manner that resembles the impact of the subunit on AChR gating behaviour, i.e. 42 AChRs have a greater likelihood of being in the open state than 32 AChRs when activated by equipotent concentrations of agonist. This also indicates that both and subunits influence AChR gating and that kinetic properties of AChRs serve as useful indicators of the presence of particular subunits within native AChRs and should be used along with channel conductances when evaluating the subunit composition of native AChRs.
Functional impact of the 5 subunit on 3 AChR single channel properties
In addition to the 3, 2 and 4 subunits, ganglionic neurons also typically express the 5 subunit (Sargent, 1993; Lindstrom, 1996). In the present study, both 352 and 354 AChRs exhibited significantly longer open times compared with 3 AChRs recorded from oocytes not injected with the 5 subunit transcript. The difference observed for 352 AChRs compared with 32 AChRs was the introduction of a third open time that was slightly, but significantly, different from those observed for 32 AChRs. The burst durations for 352 AChRs were not different from those determined for 32 AChRs. Since simple expression of triplet combinations of subunits in oocytes does not ensure predictable incorporation of all subunits into mature surface AChRs, it is unclear whether the minimal impact of the 5 subunit on 32 AChRs reflected poor incorporation, or whether 5 truly has little impact on the bursting behaviour of 352 AChRs. Previous determinations of incorporation efficiency of 5 subunits into 32 AChRs in oocytes reported that 70 % of total AChRs contained the 5 subunit (Wang et al. 1996). This level of incorporation would presumably be sufficient to reveal the functional impact, if any, on single channel properties. Attempts at improving incorporation efficiency by altering RNA ratios were unsuccessful as far as single channel analysis was concerned. Unfortunately, AChR surface expression levels measured by macroscopic current amplitudes were reduced with increasing ratios of 5 cRNA relative to 3 and 2 cRNA. This hampered single channel recording.
The effect of the 5 subunit was especially pronounced when it was coexpressed with 3 and 4 subunits. Open time distributions for these AChRs revealed a new, slower component when compared with 34 open time distributions, with a time constant of 22 ms. Half of the patches exhibited this slower channel, which represented about 26 % of the distribution of all openings in patches where present. The effect of the 5 subunit on 34 AChR burst duration was even more remarkable. The long duration bursts for 354 AChRs averaged 43 ms compared with 34 bursts which were only 16 ms.
Macroscopic properties of 3 AChRs: correlations to single channel properties
The functional impact of the subunit on the macroscopic currents of 3 AChRs has been well established (reviewed in Papke, 1993). It is not surprising that both and subunits can influence the functional properties of AChRs since substantial evidence has been reported that ACh binding sites are formed at interfaces between adjacent subunits and involve several parts of the extracellular domains of each (Karlin & Akabas, 1995). 2 AChRs compared with 4 AChRs exhibit reduced efficacy for activation by cytisine (Papke & Heinemann, 1994), increased rate of desensitization (Cachelin & Jaggi, 1991; Wang et al. 1996; Gerzanich et al. 1998), and greater sensitivity to inhibition by neuronal BuTX (Luetje et al. 1990). Macroscopic studies of human 3 AChRs expressed in oocytes have been reported recently by two independent groups (Wang et al. 1996; Chavez-Noriega et al. 1997; Gerzanich et al. 1998). The subunit alters both the pharmacological characteristics and the time course of currents significantly. Whereas 34 AChRs exhibit a slow onset of desensitization, 32 AChRs desensitize much faster. Additionally, the rank order of agonist potency for 34 AChRs differs substantially from that for 32 AChRs. Although the magnitudes of agonist EC50 values reported by the two groups are somewhat disparate, both report accelerated desensitization for 32 compared with 34 AChRs, which shows that the subunit is a major determinant of this property. Furthermore, comparisons between human 32 and 34 AChRs in permanently transfected human embryonic kidney cells also reveal dramatic differences in the desensitization rates for these AChRs when recorded with rapid agonist applications (Wang et al. 1998). The faster current decay of 32 AChRs compared with 34 AChRs correlates at the single channel level to more rapid open and burst kinetics. This probably reflects a greater rate of open channels entering the desensitized state and/or a slower rate of returning from it.
The 5 subunit is incorporated into 3 AChRs in both chicken and human (Conroy et al. 1992; Vernallis et al. 1993; Wang et al. 1996). By using subunit-specific monoclonal antibodies on human AChRs expressed in oocytes, it has been shown that when triplet combinations (
