Abstract
The physical interaction and functional cross talk among the different subtypes of neuronal nicotinic acetylcholine receptors (nAChRs) expressed in the various tissues is unknown. Here, we have investigated this issue between the only two nAChRs subtypes expressed, the α7 and α3β4 subtypes, in a human native neuroendocrine cell (the chromaffin cell) using electrophysiological patch-clamp, fluorescence, and Förster resonance energy transfer (FRET) techniques. Our data show that α7 and α3β4 receptor subtypes require their mutual and maximal efficacy of activation to increase their expression, to avoid their desensitization, and therefore, to increase their activity. In this way, after repetitive stimulation with acetylcholine (ACh), α7 and α3β4 receptor subtypes do not desensitize, but they do with choline. The nicotinic current increase associated with the α3β4 subtype is dependent on Ca2+. In addition, both receptor subtypes physically interact. Interaction and expression of both subtypes are reversibly reduced by tyrosine and serine/threonine phosphatases inhibition, not by Ca2+. In addition, expression is greater in human chromaffin cells from men compared to women, but FRET efficiency is not affected. Together, our findings indicate that human α7 and α3β4 subtypes mutually modulate their expression and activity, providing a promising line of research to pharmacologically regulate their activity.
SIGNIFICANCE STATEMENT Desensitization of nicotinic receptors is accepted to occur with repetitive agonist stimulation. However, here we show that human native α3β4 and α7 nicotinic acetylcholine receptor (nAChR) subtypes do not desensitize, and instead, increase their activity when they are activated by the physiological agonist acetylcholine (ACh). An indispensable requirement is the activation of the other receptor subtype with maximal efficacy, and the presence of Ca2+ to cooperate in the case of the α3β4 current increase. Because choline is an α3β4 partial agonist, it will act as a limiting factor of nicotinic currents enhancement in the absence of ACh, but in its presence, it will further potentiate α7 currents.
Introduction
The main objective of the present study was to investigate whether α7 and non-α7 (α3β4 in this study) nicotinic acetylcholine receptors (nAChRs) subtypes cooperate to drastically reduce their desensitization and increase their activity. Their expression and physical interaction were also investigated, as well as their regulation by Ca2+ and kinases or phosphatases. These receptors are ligand-gated cationic channels that mediate fast synaptic transmission. There are different nicotinic receptor subunits, α1–7, α9, α10, β1-β4, γ, δ, and ε in mammals, which assemble into pentamers forming different heteromeric and homomeric subtypes. At least two nAChR subtypes are expressed in the different neuronal areas of the central and peripheral nervous systems. Whether they coexist to interact and mutually cooperate to regulate their activity has not been previously investigated in the native tissue. In Xenopus oocytes, a previous report by Criado et al. (2012) showed that α7 subunits possess the capacity to stably associate with β4 or α3 subunits expressed. On the other hand, nAChRs exhibit desensitization, a characteristic kinetic property consisting of the loss of activity with repetitive agonist stimulation. In particular, the α7 subtype forms homomeric receptors that exhibit a very fast desensitization process. Heteromeric α7β2 receptors have also been reported, and show similar pharmacological sensitivities compared with the homomeric receptor. A decreased cholinergic activity has been reported for the α7 nAChR subtype in a number of pathologies such as Alzheimer's disease (AD). Therapeutic interventions aimed at reducing desensitization of this receptor subtype would be of great benefit for the treatment of these diseases.
We evaluated our hypothesis in the human chromaffin cell of the adrenal gland obtained from organ donors for the following reasons: (1) it expresses only two receptor subtypes, the α7 and α3β4 subtypes (Pérez-Alvarez et al., 2012a,b; Hone et al., 2015, 2017), which makes easier to investigate physical and functional interactions; (2) it is a native system, which allows to better understand the physiological situation; and (3) it is a human cell, which avoids extrapolation from nonhuman species facilitating translation to human pathophysiology.
In this study, we have discovered key aspects of nAChRs physiology. After activation of both receptor subtypes with maximal efficacy, a population of nAChRs increases its expression at the plasma membrane, becomes activatable, and decreases its desensitization. Expression of these nAChR subtypes is regulated by phosphatases, as it is their physical interaction. We believe these data open a new line of research in the field of nAChRs and will help to find pharmacological tools to treat diseases in which the cholinergic function is impaired.
Materials and Methods
Reagents
ACh chloride, choline chloride, amphotericin B, penicillin/streptomycin, protease type XIV, collagenase type I, poly-D-lysine hydrobromide, red blood cell lysis solution, dimethylsulfoxide, bovine serum albumin (BSA), aprotinin, leupeptin trifluoroacetate, pepstatin A, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), PNU-282987 (PNU) hydrate and all salts were purchased from Merk Sigma-Aldrich. DMEM, Glutamax, α-bungarotoxin Alexa Fluor 488 (BgTx-488) conjugate and ProLong Gold Antifade Mountant were purchased from ThermoFisher Scientific. Fetal bovine serum (FBS) was obtained from LabClinics.
Peptide synthesis
The α-conotoxin (α-Ctx) ArIB (V11,V16D; ArIB), was synthesized as previously described (Whiteaker et al., 2007). The α-Ctx TxID (TxID; Luo et al., 2013) was synthesized using an Apex 396 automated peptide synthesizer (AAPPTec) according to previously described methods (Hone et al., 2013).
Cell culture of chromaffin cells
Human adrenal chromaffin cells were isolated and cultured as previously reported (Hone et al., 2015). Briefly, glands were perfused through the vein with protease type XIV and after dissecting the gland, the medullary tissue was extracted and digested by incubation with collagenase type I. Cells were isolated after several filtrations and plated on poli-L-lysine (0.1 mg/ml) coated coverslips. Cells were maintained in DMEM medium supplemented with 1% Glutamax, 10% FBS, and penicillin/streptomycin, at 37°C in an incubator under an atmosphere of 95% air and 5% CO2 for up to 7 d. The culture medium was changed daily by exchanging ∼70% of the solution with fresh medium.
Mouse chromaffin cells of C57/B6 mice were isolated and culture following the procedure previously reported by Pérez-Alvarez et al. (2011).
Electrophysiological recordings of “patch-clamp”
To perform the recordings of nicotinic currents in the perforated-patch configuration, the extracellular solution was: 2 mm CaCl2, 145 mm NaCl, 5 mm KCl, 1 mm MgCl2, 10 mm HEPES, and 10 mm glucose (pH 7.4; 315 mOsm). When extracellular free-Ca2+ solutions were used, 5 mm EGTA was added to this solution in the absence of Ca2+. The composition of the intracellular solution was: 145 mm K-glutamate, 8 mm NaCl, 1 mm MgCl2, 10 mm HEPES, and 0.5 mm amphotericin B (pH 7.2; 322 mOsm). ACh was applied at 300 μm. An amphotericin B stock solution was prepared every day at a concentration of 50 mg/ml in dimethyl sulfoxide and kept protected from light. The final concentration of amphotericin B was prepared by the ultrasonication of 10 µl of stock amphotericin B in 1 ml of internal solution in the darkness. Pipettes were tip-dipped in amphotericin-free solution for several seconds and back-filled with freshly mixed intracellular amphotericin solution.
The electrophysiological measurements were conducted using an EPC-10 USB amplifier and the PatchMaster program (HEKA Elektronik); 2- to 3-MΩ borosilicate glass polished pipettes were used. After the formation of the seal and the perforation, only those recordings with a leakage current below 20 pA were accepted. The series resistant values were 17.7 ± 3 and 16.3 ± 2 MΩ at the beginning and the end of the experiment, respectively (n = 28). Series resistance was compensated electronically up to 70% in the voltage-clamp mode. Cell membrane capacitance (Cm) changes as an index of exocytosis were estimated by the Lindau–Neher technique implemented in the Sine+DC feature of the PatchMaster lock-in software (Lindau and Neher, 1988). A 1-kHz, 70-mV peak-to-peak amplitude sinewave was applied at a holding potential (Vh) of −60 mV. The analysis of data was done using IGOR Pro software (WaveMetrics).
To conduct the experiments, cells were gravity perfused with the extracellular solution delivered through a polyethylene tube placed 10 µm to the cell of interest. To ensure rapid solution exchange, a multibarrel pipette was used for agonist delivery. Agonist pulses (500-ms pulses) were applied using a valve controller triggered by the amplifier. The extracellular solution without the agonist was perfused once the agonist application ended. The fluid level was continuously controlled by a home-made fiber optics system coupled to a pump that removed excess fluid.
Cell treatments
In the electrophysiological recordings and image experiments, ArIB and TxID were perfused at 100 and 300 nm, respectively, 5 min before and then during the whole protocol of the experiment. Okadaic acid (OA) was incubated for 24 h at 5 nm. Pervanadate (1 mm stock solution) was prepared daily by adding 50 μl of H2O2 0.3% at 950 μl of orthovanadate 0.1 m and then used as a standard concentration of 50 μm for 40 min. Genistein at 200 μm was perfused for 10 min to evaluate its effect alone, or before the addition of pervanadate or OA, and then during the rest of the experiment.
The concentrations used for the different agonists and antagonists were chosen from previous studies performed in our laboratory in human chromaffin cells (Pérez-Alvarez and Albillos, 2007; Pérez-Alvarez et al., 2012a,b; Hone et al., 2015, 2017). Pervanadate and genistein conditions were taken from Charpantier et al. (2005). We tested different conditions for the OA (5, 10, and 20 nm) to find a concentration that provokes minimal cell death but sufficient effect on protein phosphatase (PP)2A. That range of concentrations was chosen to selectively target PP2A, given that higher concentrations also affect PP1.
Fluorescence
Cells plated on 12-mm glass coverslips were rinsed with PBS and then fixed with 2% paraformaldehyde (PFA) for 20 min. After that, three washing steps of 5 min with PBS were performed. Incubation of fluorescent labeled toxins was performed for 30–40 min on ice in a buffer containing 0.1 mg/ml BSA to block nonspecific binding and 10 μg/ml each of aprotinin, leupeptin trifluoroacetate, and pepstatin A to prevent peptide degradation by proteases. This was followed by extensive wash-out with PBS. Finally, coverslips were mounted in ProLong, a liquid curing mountant applied to the microscope slides in direct contact with the cells of study, to protect fluorescencent dyes from fading.
To study the interaction and expression of α3β4 and α7 receptor subtypes, α-Ctx BuIA labeled with Cy3 (BuIA-Cy3; 300 nm), and BgTx-488 (1.2 μm) were used, respectively. To define nonspecific binding, control coverslips were treated with 2 nm nicotine 15 min before and during the fluorescent toxins incubation. As a negative control, some coverslips were labeled with only one fluorescent toxin. As an additional negative control, Förster resonance energy transfer (FRET) was also performed in nonchromaffin cells of the same coverslip, and in chromaffin cells of two-month-old mice in a different set of experiments, obtaining a FRET efficiency of zero in both cases (data not shown).
After performing FRET, the energy transfer efficiency could be quantified as:
Microscope image acquisition and analysis
Microscopy images were collected using a spectral confocal microscope (Leica TCS SP5) equipped with an argon laser (wavelength, 488 nm), DPSS laser (wavelength, 561 nm) and helium-neon laser (wavelengths, 594 and 633 nm) for the excitation of fluorescent probes. It has a 63× oil immersion objective with 1.4 of numerical aperture. Images were acquired in a medial plane (width of 0.772 µm) in each cell under identical conditions to facilitate statistical analysis across conditions.
Expression of the different nAChR subtypes was determined by measuring the integrated density (IntDen), which is the product of the area and the mean gray value, by using Fiji-ImageJ software. Also, the Fiji-ImageJ colocalization Colormap plugin was used to calculate the colocalization correlation index values (Icorr). To perform and analyze FRET experiments, wizard AB (LAS AF, Leica) was used. The protocol consisted of 20 laser passes to bleach the acceptor (BuIA-Cy3). During the bleaching of the acceptor, only the 555 laser was on. The fluorescence of the donor (BgTx-488) was measured with a 488 laser before and after AB. Only experiments in which AB was larger than 80% were considered for analysis. Red images were converted to magenta, to ensure legibility to color-blind people.
Experimental design and statistical analysis
The number of data n refers to the number of cells unless otherwise stated. Each experiment was performed in at least five donors. Data are given as mean ± SEM. Statistical analysis was performed using IBM SPSS Statistics version 24.0. A Kolmogorov–Smirnov normality test was first performed. Then, paired or unpaired Student's t test, Mann–Whitney's U test, or Wilcoxon signed-rank tests were used and reported as t value and degrees of freedom, U value, sum of signed-ranks (W) and number of pairs, respectively. The exact p value was provided for each test. Differences were accepted as significantly different when the p value was under 0.05 (plot with an asterisk *), 0.01 (**), 0.001 (***), and 0.0001 (****). All the data values are shown in the dot plots.
Study approval
The human adrenal glands used in this study were collected from 30 organ donors (19 women and 11 men, from 23 to 82 years old) in three hospitals in Madrid (12 de Octubre, Fundación Jiménez Díaz, and Hospital Clínico San Carlos). Written consents were signed by donor's relatives. The protocol was approved by the Ethics Committee of the Autonomous University of Madrid and by review boards of each hospital.
The mouse adrenal glands were collected from seven female C57/B6 mice two months old, provided by the animal facility of the Autonomous University of Madrid. All animals were housed on a 12/12 h light/dark cycle, with food and water ad libitum. All procedures used in this work were conducted according to the protocols approved by the Ethics Committee of the Autonomous University of Madrid and attention was put into ensuring that the animals experience the minimal suffering possible.
Results
Repetitive pulses of ACh elicit an increase of nicotinic currents
Repetitive pulses of ACh (300 μm, 500 ms, applied every 90 s) elicited currents of increasing peak amplitude and charge. The r Pearson's correlation coefficient was 0.97 (R2 = 0.9496, p < 0.0001). The number of ACh pulses to reach the maximal increase of current was 7.1 ± 1.1 (n = 29; Fig. 1A). Initial peak current and charge values amounted to 813.1 ± 103.4 pA and 892.2 ± 176 pC (n = 29), respectively. After the application of ACh pulses, the maximal ACh peak current and charge increased to 1638 ± 167.8 pA (W(29) = −427, p < 0.0001) and 1976 ± 318.7 pC (W(29) = −401, p < 0.0001; n = 29), respectively, which normalized with respect to their own controls represents 2.1 ± 0.2-fold and 2.5 ± 0.3-fold of increment, respectively. The elicited-ACh currents exhibited faster activation kinetics with repetitive pulses, as shown by the representative traces and the activation time constant (τactivation) values in Figure 1B, left and right, respectively. Control experiments in mouse chromaffin cells were performed to exclude the possibility that the results in Figure 1B are an artifact of the perfusion system. Following the same protocol as in human cells, repetitive pulses of ACh elicited nicotinic currents with τactivation values that do not exhibit faster activation kinetics. The r Pearson's correlation coefficient was 0.32 (ns, n = 23; Fig. 1C).
α7 and α3β4 receptor subtypes require mutual and maximal efficacy of activation to avoid their desensitization and to increase their activity
In an additional set of experiments, we found that the increases of peak current achieved with repetitive ACh pulses were abolished if: (1) the α7 nicotinic current was desensitized previously at the beginning of the experiment by applying three pulses of 3 mm choline, an α7 receptor agonist; or (2) cells were perfused with the selective α7 receptor blocker ArIB (Whiteaker et al., 2007; Hone et al., 2015) at 100 nm during 5 min before the application of ACh pulses and then during the whole experiment. The protocol of these experiments was the same as that performed in Figure 1A. The graph of the ACh peak current at the different pulses and the corresponding representative recordings under both conditions, desensitizing previously the α7 receptor subtypes or after ArIB treatment, are drawn in Figure 2A,B, respectively. The r Pearson's correlation coefficients obtained were −0.98 (R2 = 0.9635, p < 0.0001) and −0.92 (R2 = 0.8525, p = 0.0002) for desensitized α7 receptor and ArIB conditions, respectively. Thus, the increase of current peak achieved with repetitive ACh pulses in control conditions was due in part to α7 receptor activation, since the observed changes were abolished in α7 desensitized receptors or ArIB-treated cells. On the other hand, the τactivation of the nicotinic currents elicited with the successive ACh pulses in panels A, B are shown in Figure 2C,D, respectively. The r Pearson correlation coefficients were 0.94 (R2 = 0.8918, p = 0.0014) and 0.45 (R = 0.1985, p = 0.3165), respectively, for the desensitized α7 receptor and ArIB conditions, respectively. This means that the recruitment of fast α7 receptors was essential for the decrease of the rate of activation observed in the control situation after repetitive ACh stimulation. Thus, during the repetitive stimulation with ACh α7 receptors not only do not desensitize but also activate faster and increase their amplitude.
However, the increase of currents cannot be attributed exclusively to α7 receptors recruitment, because the charge of the currents was also largely enhanced with successive agonist pulses. Therefore, activation of α3β4 receptors is also increased, as it was later confirmed by the mutual requirement of their activation to obtain an increased expression of both receptor subtypes (see below). We posed the hypothesis that full and increased activation of α3β4 receptors avoids α7 receptor desensitization, increasing their activity. We performed the experiments shown in Figure 2E,F to prove it. First, three pulses of 3 mm choline, an α7 receptor agonist that also activates non-α7 receptors in these cells (Pérez-Alvarez et al., 2012b), were applied successively every 90 s. The first peak of the current triggered by choline is because of α7 receptor stimulation, while the slow current activated after it is because of partial activation of non-α7 receptors (Pérez-Alvarez et al., 2012b; Hone et al., 2015). Under that stimulation, a significant decrease of the choline peak current occurred. In these experiments, an initial peak current value of 164.2 ± 38.4 pA was decreased to 60.3 ± 12.6 pA. This corresponds to a decrease of 51.5 ± 13.8% in 11 cells tested (t(10) = 2.704, p = 0.0222; Fig. 2E). However, if successive pulses of ACh were applied after the first choline pulse, and later on another choline pulse was applied again, the second choline pulse elicited now an increased peak current (α7 receptor potentiated currents; Fig. 2F). On average, peak currents increased from 242.2 ± 78.7 to 689.5 ± 119.1 pA after the ACh pulses. This meant a 2.8-fold increase in the 27 cells tested (t(26) = 4.696, p < 0.0001). These α7 receptor potentiated currents could be again desensitized if repetitive pulses of choline were applied. Choline elicited currents now decreased from 636.3 ± 94.4 to 347.3 ± 63.3 pA, which represents a 45.3 ± 4.8% (n = 48; t(47) = 6.889, p < 0.0001; Fig. 2G). All these data are summarized in the dot plot of Figure 2H. To search whether the same process occurs with a different α7 receptor agonist, 30 μm PNU was perfused instead of choline, and the same protocol was performed. Three sequential PNU pulses caused a decrease of the peak current of 66.6 ± 8.1%, from 348.3 ± 93.3 to 172.2 ± 51.4 pA (n = 11; t(10) = 3.989, p = 0.0026; Fig. 2I). Also, a different α3β4 and α7 agonist was now applied, DMPP, instead of ACh. The decrease of current triggered by PNU could be prevented if pulses of DMPP (100 μm) were applied until the maximal current was achieved (5.2 ± 0.8 pulses). PNU-elicited peak currents increased now from 256.8 ± 88.4 to 720.8 ± 123.8 pA. This corresponds to a 2.8-fold increase of the α7 receptor potentiated currents (n = 8; t(7) = 4.783, p = 0.002; Fig. 2J). Data are summarized in the dot plot of Figure 2K. Thus, the α7 receptor subtype requires the activation of the α3β4 receptor with full efficacy to avoid its desensitization.
Interestingly, the potentiation of the α7 receptor currents was a very rapid process since a single pulse of ACh was sufficient to avoid the α7 receptor desensitization and to induce the increase of the α7 current. As a control, we applied 13 choline pulses successively, decreasing the initial current from 271.9 ± 49.1 to 27 ± 14 pA (n = 2–15; 90% decrease; Fig. 2L). The Spearman's r correlation was −0.98 (p < 0.0001). However, when seven pulses of choline were alternatively applied with ACh pulses, the choline peak current increased its amplitude by 3-fold from 305.8 ± 127 pA (n = 6) to 992.3 ± 354.8 pA (n = 3). Also, the ACh elicited current increased from 1057.6 ± 255.7 pA (n = 6) to 1626.6 ± 441.3 pA (n = 3; 1.5-fold increase; Fig. 2M). The Spearman's r correlation between ACh and choline currents was 0.69 (p < 0.0001). These results reinforce the idea that activation with full efficacy of α7 and α3β4 receptor subtypes is a requirement to increase their mutual activity.
α7 and α3β4 receptors require their mutual activation and to be in an active state to increase their expression
We examined the variation of expression of α7 and α3β4 receptor subtypes with repetitive pulses of ACh to evaluate whether it correlates with the activity of the channels. We used the BgTx-488 and BuIA-Cy3 to determine the expression of α7 and α3β4 receptor subtypes, respectively. We can observe that the application of seven pulses of ACh (number of pulses required to achieve the maximal increase of ACh current) led to an increase of expression of both receptor subtypes (+ACh condition; Fig. 3A–C). We hypothesized that if the mutual activation of α7 and α3β4 receptor subtypes avoids their desensitization leading to an increase of currents activation, it might well be that also the increase of expression of both receptor subtypes requires their mutual activation.
Different treatments were applied before stimulating the cell with seven pulses of ACh. These treatments included the application of three pulses of choline to desensitize the α7 receptor, the α7 receptor selective blocker ArIB, the α3β4 receptor selective blocker TxID, or ArIB, and TxID simultaneously. The ArIB does not block non-α7 receptors (α3β2, α3β4, β3α6β2α3β4 and α6M211L, cytα3β4) according to Hone et al. (2015). Besides, TxID does not block α7 receptor subtypes (Hone et al., 2017). Original fluorescence images are shown in Figure 3A and the summaries of the normalized integrated density of BgTx-488 and BuIA-Cy3 are plotted in Figure 3B,C, respectively. These experiments revealed that successive ACh pulses increased α7 and α3β4 receptors expression by 1.5 ± 0.1-fold (U = 631, p = 0.0019 and t(87) = 3.886, p = 0.0002, respectively; n = 44). α7, and surprisingly, also α3β4 receptor expression increases were abolished when α7 receptors were desensitized (t(53) = 2.457, p = 0.0173 and U = 100, p = 0.0021, respectively; n = 11 with respect to ACh potentiated currents), or blocked with the selective α7 receptor antagonist ArIB (t(60) = 2.185, p = 0.0328 and U = 214, p = 0.0042, respectively; n = 18). The increased expression of α7 or α3β4 receptors was also prevented by the treatment of the cells with the α3β4 receptor blocker TxID (t(59) = 3.123, p = 0.0028 and U = 218, p = 0.0114, respectively; n = 17). When cells were treated with both toxins simultaneously, α7 and α3β4 receptors' expression was not increased (t(60) = 3.323, p = 0.0015 and U = 256, p = 0.0296, respectively; n = 18). These experiments reflect that α7 and α3β4 receptor subtypes must be in an active, nondesensitized state to contribute to their increased expression and also to the expression of the other receptor subtype.
Dependence of Ca2+ of the ACh-evoked nicotinic current increment
The increase of the ACh peak currents shown under control conditions in Figure 1A is reduced in the absence of Ca2+ (Fig. 4A). The r Pearson's correlation coefficient achieved was 0.15 (R2 = 0.0212, p = 0.6878; n = 7–10), reflecting that there is not an increase of currents with successive ACh pulses. Neither the τactivation decreased with repetitive pulses (r = −0.65; R2 = 0.4299, p = 0.1098), indicating that the lack of new activatable α3β4 receptors in the absence of Ca2+ (see below) also affects the τactivation of the whole currents (Fig. 4B). We wanted to investigate how Ca2+ flux through either α7 and α3β4 receptors could be affecting this response. To achieve that purpose, the “potentiation protocol” was performed using an external solution free of Ca2+. Representative recordings of the currents achieved under both conditions are shown in Figure 4C. The peak current elicited by choline was largely increased from 220.4 ± 58.6 to 783.4 ± 169.1 pA (W(14) = 105, p = 0.0001, n = 14). However, the ACh increase of peak currents did not occur in a Ca2+-free solution, changing from 1223 ± 208.9 to 1387 ± 217.6 pA (W(21) = 146, p = 0.5213, n = 21). The average values of the peak currents elicited by ACh and choline are shown in Figure 4D. Also, the percentages of peak current increments elicited by both agonists are drawn in Figure 4E. They amounted to 33.3 ± 5.6% and 4.2 ± 5% for ACh in the presence and absence of Ca2+, respectively (t(41) = 2.577, p = 0.0136, n = 21–23), and to 59.61 ± 6.3% and 65.5 ± 7% for choline in the presence and absence of Ca2+, respectively (t(42) = 0.581, p = 0.5643, n = 14–30). These experiments prove that Ca2+ is a requirement for α3β4 receptors to increase their flux of current but not for α7 receptors.
α7 and α3β4 receptors are physically interacting
The functional interaction above described for α7 and α3β4 receptors might be derived from a possible physical interaction. This was investigated in the present study in human chromaffin cells by FRET. The scheme of this protocol is shown in Figure 5A. We used BuIA-Cy3 as an acceptor and BgTx-488 as a donor to perform these experiments. In Figure 5B, the fluorescence images obtained under prebleaching and postbleaching conditions for the donor and the acceptor, the FRET efficiency, and the correlation index values measured as normalized mean deviation product (nMDP) are shown. Dot plots of the percentage of the FRET efficiency, calculated as explained in Materials and Methods, and the nMDP, are drawn in Figure 5C,D, respectively. Without any treatment, FRET efficiency was high, amounting to 23.3 ± 1.3% (n = 25), which suggests that, in human chromaffin cells, α7 and α3β4 are physically interacting. The FRET efficiency was the same with the different treatments after seven successive pulses of ACh, desensitizing previously the α7 receptor, or treating previously the cells with ArIB, TxID, or both toxins together (22.6–26.4%, n = 5–23; Table 1). Accordingly, the nMDP values obtained for the colocalization between α7 and α3β4 receptors varied between 0.76 and 0.85 under these conditions (n = 6–43), showing a high degree of colocalization (Table 1).
Also, the efficiency of the interaction did not vary when the agonists ACh or choline were applied in the absence of Ca2+, showing that Ca2+ does not modulate the physical interaction. Values were 30.1 ± 4.6%, 36.6 ± 4.4%, 33.1 ± 3%, and 33.6 ± 7.5% (n = 6) for ACh in 2 mm and 0 Ca2+, and choline 2 mm and 0 Ca2+. Representative fluorescence images obtained under prebleaching and postbleaching conditions for the donor and the acceptor, the FRET efficiency, and the correlation index values are shown in Figure 6A. Dot plots of the percentage of the FRET efficiency and the nMDP are drawn in Figure 6B,C, respectively.
Tyrosine and serine/threonine phosphatases regulate α7 and α3β4 receptor expression and interaction
Phosphorylation is a common regulatory mechanism of various processes. Regulation of native nonhuman α7 nicotinic receptors by tyrosine kinases and phosphatases has been already reported (Charpantier et al., 2005; Cho et al., 2005). Here, we investigated whether the expression and interaction of α7 and α3β4 receptors are regulated by tyrosine and serine/threonine-mediated phosphorylation/dephosphorylation processes. BgTx-488 and BuIA-Cy3 were again used to record α7 and α3β4 receptors expression.
First, the regulation by tyrosine kinases was investigated. Representative images of the different treatments are shown in Figure 7A. Cells were incubated for 40 min with pervanadate, a tyrosine phosphatase inhibitor, at a concentration of 50 μm. Expression of α7 and of α3β4 receptor subtypes was inhibited by 30% and 34%, respectively (U = 207, p = 0.006; t(51) = 3.127, p = 0.0029, n = 26). This inhibition could be reversed by genistein (200 μm), an inhibitor of tyrosine kinases, incubated 10 min before and during cell treatment with pervanadate (U = 202, p = 0.0121 and U = 161, p = 0.0028 for Bgtx-488 and BuIA-Cy3, respectively, n = 26). Genistein alone did not modify the expression of α7 and α3β4 receptors (t(51) = 0.136, p = 0.8926; t(47) = 1.166, p = 0.2495, respectively, n = 25). Dot plots of the fold times of the integrated density of BgTx and BuIA with respect to controls are shown in Figure 7B,C.
The regulation of the efficiency of the interaction between α7 and α3β4 receptors by tyrosine phosphorylation/dephosphorylation was investigated by FRET. Efficiency was decreased by 20% in cells treated with pervanadate (t(56) = 2.124, p = 0.0381, n = 26), and recovered in cells previously incubated with genistein (t(49) = 2.858, p = 0.0062, n = 26). Genistein alone did not modify the efficiency of FRET achieved under basal conditions (t(54) = 0.2291, p = 0.8196, n = 26). Thus, the inhibition of tyrosine phosphatases has a higher impact on the interaction than the inhibition of tyrosine kinases (Fig. 7D).
Next, we investigated whether the expression and interaction of α7 and α3β4 receptors are regulated by serine/threonine-mediated phosphorylation/dephosphorylation processes. BgTx-488 and BuIA-Cy3 were again used to record α7 and α3β4 receptors' expression. Representative images of the different treatments are shown in Figure 7A. First, OA, an inhibitor of serine-threonine phosphatases, was incubated for 24 h at the concentration of 5 nm. Expression of α7 and α3β4 receptors was inhibited by 45% and 44.5%, respectively (t(48) = 4.369, p < 0.0001 and t(48) = 3.94, p = 0.0003, respectively, n = 22; Fig. 7B,C).
The efficiency of the interaction of α7 and α3β4 receptors was also investigated in cells incubated with OA, obtaining a significant decrease (t(52) = 2.333, p = 0.0236 n = 22; Fig. 7D). The values of the percentage of FRET efficiency are summarized in Table 2.
Sex dependence of the expression of α7 and α3β4 receptor subtypes
The degree of physical interaction between α7 and α3β4 receptor subtypes was investigated by comparing FRET efficiency in donors sorted by sex. BuIA-Cy3 and BgTx-488 were again used as acceptor and donor, respectively, in the FRET experiments. FRET efficiency did not vary between males (23.3 ± 1.6%, n = 18) and females (23.1 ± 2.4%, n = 18; t(34) = 0.0852, p = 0.9326). Representative images are shown in Figure 8A. However, expression of α7 and α3β4 receptor subtypes was larger in men when compared with women (Fig. 8B). Total BgTx fluorescence amounted to 323.5 ± 50.5 (n = 16) in males, and to 75 ± 12.6 (n = 27) in females (U = 87, p < 0.0001). Also, total BuIA fluorescence amounted to 194.7 ± 21.5 (n = 16) in males, and to 93.3 ± 13 (n = 27) in females (U = 95, p < 0.0001). Therefore, men express a significantly larger number of α7 (4.3-fold) and α3β4 (2.1-fold) nicotinic receptors than women in human chromaffin cells.
Discussion
Multiple nAChR subtypes are expressed in the various areas of the central and peripheral nervous systems. The finality of that coexpression is not fully understood, but it might happen that these receptors are cooperating to modulate their function, which is highly dependent on the kinetics of the receptor. nAChRs exhibit a very high rate of desensitization, mainly the α7 subtype. Therefore, the goal of the present study was to investigate the functional cross talk between human native α7 and non-α7 nAChR subtypes. In this study we have studied in particular the α7 and α3β4 subtypes in a peripheral neuroendocrine cell, the chromaffin cell of the adrenal gland. These two receptor subtypes are also expressed in different areas of the brain such as the pineal gland, but also the hippocampus, cerebellum, and medial habenula together with other nAChR subtypes. The advantage of using this neuroendocrine cell is mainly to work in a human cell, avoiding extrapolation from other species, which in addition expresses only two receptor subtypes (Pérez-Alvarez et al., 2012a,b; Hone et al., 2015, 2017), making easier to investigate the interaction between them. In this study, we have shown by FRET that these receptors physically interact. Interaction between native nAChR subtypes has not been previously reported. However, in Xenopus oocytes, Criado et al. (2012) showed that stable and unstable interactions between α7 with β4 or α3 subunits, respectively, were established among human and bovine receptor subunits.
Here, we have shown that after maximal efficacy of activation of α7 and α3β4 nAChRs subtypes with the physiological agonist ACh, or by a synthetic agonist such as DMPP (Hone et al., 2017; Jiménez-Pompa et al., 2021), a sequence of events occurs. There is an increase of nAChR subtypes expression at the plasma membrane that are physically interacting and cooperating, and that become activatable and do not desensitize because of their mutual cooperation. These statements are proved by the following evidences: (1) enhanced expression of α7 and α3β4 subtypes with repetitive pulses of ACh, which does not occur if α7 or α3β4 subtypes are desensitized or blocked; (2) identical FRET efficiency achieved under control conditions and after the potentiation protocol with ACh pulses; (3) activation of a population of receptors evidenced by the associated decreased desensitization and increase of currents, because of the reciprocal cooperation between nAChR subtypes to maintain the activity of the other subtype. The α3β4 subtype requires the α7 to increase their expression and activity, avoiding its desensitization, and the same occurs with the α7 subtype. This is a major finding of the present study that shows α3β4 and α7 subtypes do not desensitize after ACh stimulation.
However, activation of these receptors with partial efficacy provokes their desensitization. This is what happens with choline, a partial agonist of α3β4 nAChRs, and a full agonist at homomeric α7 receptors (Mandelzys et al., 1995; Papke et al., 1996). This agonist is obtained after degradation of ACh by the acetylcholinesterase and it is maintained at 10–20 μm in the plasma (Zeisel, 2000; Midttun et al., 2013; Cho et al., 2016; Mödinger et al., 2019). It is able to desensitize the α7 receptor because of its high affinity for this subtype and its low affinity for the α3β4 receptor subtype, controlling the unlimited increase of currents elicited by ACh. However, choline will act as a brake only in the absence of ACh, otherwise, it will increase notably α7 receptor currents, as shown in Figure 2F. Another limiting factor of nAChR current increase is Ca2+. This cation does not affect the physical interaction between nAChR subtypes. However, our functional data reveals that the absence of Ca2+ avoids α3β4, but not α7, nAChR currents enhancement.
The population of nAChRs that becomes activatable is not composed of new receptors, but most probably of receptors that have experienced a conformational change. This is proved by the following evidences: (1) expression of both subtypes is reversible, reduced by pervanadate, and recovered when genistein is perfused before application of the tyrosine phosphatase inhibitor; (2) increase of expression and currents occurs in a second-time scale. It has been reported that chronic exposure to a nAChR agonist such as nicotine induces an upregulation of nAChRs, not because of increased gene expression in adults. Numerous mechanisms have been proposed to be responsible for this phenomenon: increased nAChR subunit maturation and folding, increased receptor trafficking, decreased subunit degradation possibly through the pharmacological chaperoning of nAChRs by nicotine; and increased translation and second messenger signaling (Melroy-Greif et al., 2016). However, the upregulation of receptors in the present study observed is a very rapid process that occurs in a second-time scale after full activation of both receptor subtypes by the agonist.
Here, we show that physical interaction and expression of α7 and α3β4 subtypes were decreased by inhibition of tyrosine or serine/threonine phosphatases. Inhibition of FRET efficiency and expression by pervanadate were reversed by genistein, reflecting that phosphorylation decreases the efficiency of the interaction and expression of nAChR subtypes. In addition, the physical interaction is reduced by OA, an inhibitor of serine/threonine phosphatase, which might be relevant if translated to the pathophysiology of AD. In vivo models of AD exhibit increased levels and activity of endogenous PP2A inhibitors (Tanimukai et al., 2005). The reduced activity of this enzyme, which is responsible for 70% of tau dephosphorylation activity (Liu et al., 2005), might account for hyperphosphorylation of tau (Gong et al., 1993; Liu et al., 2005) and AD-like pathology (Gong et al., 2001; Sun et al., 2003). Furthermore, the inhibition of PP2A increases GSK-3β activity, enhancing hyperphosphorylation of tau (Wang et al., 2015). Future work will clarify whether a dysfunction of the physical and functional interactions between nAChR subtypes might be contributing to the physiopathology of AD.
In addition, our data show that expression, but not interaction of α7 and α3β4 receptors, varies with sex. Men exhibited a 2- to 3-fold increase of BgTx and BuIA receptors labeling with respect to women. It might be that the direct influence of adrenal gland steroids regulates the expression of nAChRs in the chromaffin cell. Indeed, recombinant human α3β4 receptors are noncompetitively inhibited by estrogens (Ke and Lukas, 1996; Nakazawa and Ohno, 2001). Our data constitute the first study in which human native α7 and α3β4 nicotinic receptors expression in males and females has been determined.
In summary, the present study shows that the cross talk between α7 and α3β4 nAChR subtypes avoids their desensitization when these receptors are activated with maximal efficacy. In this way, repetitive ACh stimulation leads to an increase of nicotinic currents, while successive choline pulses desensitize them in the absence of ACh, controlling the unlimited rise of currents by this agonist. These data also help to identify pharmacological targets to regulate the cholinergic function, opening new perspectives for the treatment of cholinergic-related diseases.
Footnotes
This work was supported by Spanish Ministerio de Ciencia y Tecnología Grants BFU2012-30997 and BFU2015-69092 (to A.A.) and National Institutes of Health Grants GM136430 and GM103801 (to J.M.M).
The authors declare no competing financial interests.
- Correspondence should be addressed to Almudena Albillos at almudena.albillos{at}uam.es