Electrochemical studies of human nAChR a7 subunit phosphorylation by kinases PKA, PKC and Src

Analytical Biochemistry 574 (2019) 46–56

Contents lists available at ScienceDirect

Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio

Electrochemical studies of human nAChR a7 subunit phosphorylation by kinases PKA, PKC and Src

T

Syed Ahmada,b, Zhe Shea,b, Heinz-Bernhard Kraatza,b,* a b

Department of Physical and Environmental Sciences, University of Toronto Scarborough, 1265 Military Trail, Toronto, M1C 1A4, Canada Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, M5S 3H6, Canada

A R T I C LE I N FO

A B S T R A C T

Keywords: Electrochemistry Phosphorylation XPS Nicotinic acetylcholine receptors Enzyme Kinetics

Nicotinic acetylcholine receptors (nAChR) are ion channels which regulate a numerous of neurotransmitters, including acetylcholine, norepinephrine, dopamine, serotonin and glutamate. These receptors are important targets for the study of a plethora of diseases such as Alzheimer's disease, schizophrenia, Parkinson's Disease, cancer, inflammation, etc. The α7 subunits are especially interesting in that they are commonly occurring and are critical sites of regulation. Herein we report the phosphorylation of the human nAChR α7 subunits, by the kinases PKA, PKC and Src, by both biochemical and electrochemical techniques along with the kinetics of each phosphorylation reaction. Phosphorylation was investigated through changes in current density as well as impedance and X-ray photo electron spectroscopy (XPS) and the kinetics were determined electrochemically using the surface Michaelis–Menten model. Our results clearly demonstrate the phosphorylation of the nAChR α7 and the invaluable strength of surface electrochemical techniques in the investigation of protein phosphorylation.

1. Introduction Nicotinic acetylcholine receptors (nAChR) are pentameric ligandgated ion channels composed of 5 subunits (α, β, γ, δ, and ε), and responsible for mediating the cellular response to neurotransmitters [1,2]. nAChRs are important targets of study for Alzheimer's disease [3–5], memory loss [5], schizophrenia [4,6,7], Parkinson's Disease [8,9], melancholic and bipolar depression [4], inflammation (and the damage resulting thereof) [4,10–12], chronic pains resulting from posttraumatic stress disorder [13,14], hyperalgesia [13,14], lung and epithelial cancers [8,9,15,16], HIV-caused brain damage (though the HIV1 glycoprotein120 virotoxin), as well as methamphetamine and nicotine induced nervous system injuries [17]. The nAChRs are also involved in pre-and postsynaptic transmission where nAChR activation regulate acetylcholine, norepinephrine, dopamine, serotonin, gammaaminobutyric acid and glutamate, also making them desirable targets for anesthetic drugs [2]. The α subunits are unique in that they protrude the cell and contain a “cysteine (Cys) loop” and contain the ACh binding site (two adjacent Cys amino acids) [18]. Among the α subunits, the α7 is unique in that it is the most involved in nervous system disorders, cancers, inflammation, and is unaffected by common nAChR α inhibitors such as isoflurane and propofol [2,4,5,10–14,16,17,19]. The current understanding of nAChR regulation is through *

phosphorylation-induced desensitization by cAMP Dependent Protein Kinase A (PKA) and receptor stabilization by Protein Kinase C (PKC) and Sarcoma Related Kinase (Src). PKA is able to phosphorylate the γ and δ subunits of both membrane bound and free nAChRs and causes, two distinct states of desensitization on chicken and rat nAChR α7 homologs. The first is easily reversible, requiring only a few seconds to recover (minor desensitization) and the second requires minutes (severe desensitization) [18,20,21]. It has been shown that PKA phosphorylation prevents the severe desensitization phase [18]. Thus, phosphorylation is at the heart of understanding the nAChR α7 receptor and acetylcholine regulation. However, it has never been shown empirically that the human nAChR α7 subunit undergoes PKA phosphorylation. Research into the nAChR regulation has been conducted in rat and chicks, but is lacking in human cells. In fact, to our knowledge, despite the overwhelming involvement of the α7 subunit in a plethora of diseases and healthy states, there has been no empirical evidence of the phosphorylation of human nAChR α7. Furthermore, although biochemical techniques such as gel electrophoresis and Western blots or ELISA are reliable, they require multiple steps, reagents and specialized equipment for each step. For example, gel electrophoresis can establish phosphorylation, but cannot provide real-time enzyme kinetics information. In contrast, electrochemical techniques are well established for protein phosphorylation detection and provide reliable, efficient data

Corresponding author. Department of Physical and Environmental Sciences, University of Toronto Scarborough, 1265 Military Trail, Toronto, M1C 1A4, Canada. E-mail address: [email protected] (H.-B. Kraatz).

https://doi.org/10.1016/j.ab.2019.03.012 Received 9 January 2019; Received in revised form 19 March 2019; Accepted 19 March 2019 Available online 23 March 2019 0003-2697/ © 2019 Elsevier Inc. All rights reserved.

Analytical Biochemistry 574 (2019) 46–56

S. Ahmad, et al.

along with 0.1% Coomassie Brilliant Blue G-250, pH 8.5 (0.1% SDS was present for the SDS PAGE). The anode buffer was identical to the cathode buffer except that it lacked the Coomassie dye. Electrophoresis was started at 75 V for 30 min to allow the proteins to enter the stacking gel. Thereafter the gel was run at 100 V for 2 h, after which the gel was swiftly moved into the staining solution.

about peptide/protein phosphorylation along with kinetics with reduced reagents and equipment. Previously it has been shown that phosphorylation of surface bound proteins leads to decreased charge transfer from the electrode surface [22,23]. Since then electrochemistry has been used for drug IC50 and inhibition studies [24–27], electrochemical enzyme studies and sensor development [28–40], and much more. Our group has studied protein phosphorylation extensively and have shown that electrochemical techniques of phosphorylation detection can be on par with standard biochemical immunoassays [33]. Furthermore, using our electrochemical techniques, we have demonstrated that the tau protein undergoes significant conformational changes when going from a tyrosine kinase phosphorylated state to a GSK-3β hyperphosphorylated state [34]. Moreover, we have also demonstrated that the enzyme phosphorylation kinetics of peptides and Tau can be determined electrochemically [25,28]. More recently our group has demonstrated, through the use of electrochemistry, that tau dimerization is a result of iron binding [40]. In the present contribution we utilize the previously established electrochemical techniques, supplemented with Blue-Native and SDSPAGE along with X-ray photon spectroscopy (XPS) surface studies, to investigate the phosphorylation of the human nAChR α7 protein subunit. The electrochemical techniques used are square wave voltammetry (SWV), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) and each of these methods is also utilized to determine the surface enzyme kinetics of the nAChR α7 and PKA/PKC/Src phosphorylation reactions. The results of this contribution empirically establish that the human nAChR α7 is phosphorylated as well as enzyme kinetics and provides insights into future explorations of nAChR α7 phosphorylation studies.

2.2.3. Protein staining and destaining of gels The gels were incubated for 60 min under constant shaking at 90 rev min−1 in a protein staining solution consisting of a 50/40/10 mixture of distilled water/methanol/glacial acetic acid and 0.1% Coomassie Brilliant Blue G-250. After 1 h, the gel was transferred into a destaining solution, which was identical to the protein staining solution except that it lacked the Coomassie stain, for 8 h. 2.3. Protein transfer and Western blot analysis Following SDS-PAGE electrophoresis the proteins were transferred onto a nitrocellulose membrane using electroelution for 30 min. Subsequently, the membrane was washed with millipore water and then stained with Ponceau Red solution (59.85% Millipore water. 0.2% Ponceau solution, 3% trichloroacetic acid and 3% sulfosalicylic acid). The protein ladder was marked using a pencil. Subsequently, the membrane was socked in a blocking solution of 5% BSA* in 100 mM PBS for 60 min under gentle agitation. BSA was chosen over dried milk because, although dried milk is inexpensive, milk contains casein, a protein which itself is a phosphoprotein and could potentially cross react. After washing, the membrane was incubated in a solution of primary antibody (1:10,000 dilution of Ab in blocking buffer) for 2 h under gentle agitation. Successively, the membrane was washed once with 100 mM PBS containing 0.25% Tween-20 and then thrice with water. Lastly, the membrane was incubated in a solution of secondary rabbit anti-mouse antibody (rAB) (1:10,000 dilution of rAb in 100 mM PBS buffer) for 1 h at room temperature.

2. Materials and methods 2.1. Materials and reagents Nicotinic acetylcholine receptor α7 subunit (nAChR α7) (ab114193), PKC (ab55672) and Src (ab60884) was purchased from Abcam whereas the monoclonal anti-phosphoserine antibody (Ab) (P3430), PKA catalytic subunit α (PKA), ethanolamine (EA), mercaptohexanol (MCH) and adenosine triphosphate (ATP) were purchased from Sigma-Aldrich. Lipoic acid N-hydroxysuccinimide ester (LPANHS) was purchased from Synchem UG & Co. All other chemicals were purchased from Sigma-Aldrich unless otherwise specified.

2.4. Construction of the modified electrode surface Prior to surface modification, the electrodes were immersed in a piranha solution of 3:1 H2SO4/H2O2 v/v for 30 s and rinsed with Millipore water. The bare gold electrodes were polished using aqueous alumina slurries of 1 μm, 0.3 μm and 0.05 μm to obtain a mirror surface which was subsequently cleaned electrochemically using 0.5 M H2SO4 and 0.5 M KOH separately. The cleaned electrodes were immediately immersed in a 50 μL of 2 mM LPA-NHS ethanol solution at 4 °C for 16 h. The LPA-NHS monolayer here served as the linker between the nAChR α7 and Au electrodes in the following step. The modified electrodes were then thoroughly rinsed with ethanol and blown dry by N2 (g). The gold surface was subsequently immersed in 10 mM mercaptohexanol for 15 min at room temperature prior to incubation in 50 μL of nAChR α7 (10 μg mL−1) in 10 mM HEPES buffer (pH 7.4) at 4 °C for 18 h. The nAChR α7-modified electrodes were rinsed with an excess volume of washing buffer and Milli-Q water and blown dry followed by incubation in a 100 mM ethanolamine solution for 1 h at room temperature to block any unreacted LPA-NHS. Phosphorylation was obtained by incubating the modified electrodes in a solution of 100 mM ATP, 30 μg/mL PKA in a final volume of 50 μL of 10 mM HEPES buffer (pH 7.4) for 2 h at 37 °C followed by rinsing. Immunodetection was done by incubating the phosphorylated electrodes in 100 mM HEPES buffer (pH 7.4) containing a 1:100 dilution of anti-phospho-antibodies for 2 h at 37 °C followed by rigorous rinsing.

2.2. Determination of phosphorylation through gel electrophoresis Two TruPAGE™ precast gels (PCG2003-2 EA), 12% polyacrylamide, were obtained from Sigma-Aldrich. One gel was used for Blue-Native PAGE (BN-PAGE) electrophoresis and the other was used for SDS PAGE electrophoresis. The procedure was adopted from Coster, R. et al., 2001 [41]. 2.2.1. Preparation of the samples Each of the following four samples were prepared and incubated at 37 °C for 2 h: 1) PKA, Ab and ATP, 2) PKA/PKC/Src, Ab and the nAChR α7, 3) PKA/PKC/Src, nAChR α7 and ATP and 4) PKA/PKC/Src, Ab, nAChR α7 and ATP. The concentrations of kinase, Ab, nAChR α7 and ATP in all samples was 30 UN mL−1, 1:100 dilution, 10 μg/mL, and 100 mM respectively. The SDS gel contained an additional lane for the protein ladder. Immediately after incubation, 5 μL of 5% Coomassie Brilliant Blue G-250 and were loaded into the gel. A 2 h incubation/ phosphorylation time was selected due as it is a convenient time for monitoring phosphorylations electrochemically.

2.5. Electrochemical measurements

2.2.2. Conditions of electrophoresis The gels were stored at 4 °C until used and run at room temperature. The cathode buffer contained 100 mM Tris/HCl and 10 mM of Tricine

Cyclic 47

voltammograms

(CV),

electrochemical

impedance

Analytical Biochemistry 574 (2019) 46–56

S. Ahmad, et al.

beginning with the immobilization of lipoic acid N-hydroxysuccinimide ester (LPA-NHS) onto clean Au surfaces. Subsequently, the surface is submerged in 1 mM mercaptohexanol (MCH) in order to remove any non-specifically bound LPA-NHS and to backfill the surface. Next, the nAChR α7 is immobilized onto the surface through the LPA-NHS linker. Lastly, ethanolamine (EA) is introduced to the surface to block any unreacted ester sites and to backfill any potential defect sites.

spectroscopy (EIS), and square wave voltammetry (SWV) were recorded on a CHI-660C electrochemical station (CH Instruments, Austin, Texas) with an enclosed Faraday cage. All electrochemical measurements were performed in 100 mM HEPES buffer, pH 7.4, in the presence of 5 mM [Fe(CN)6]3-/4- In a typical electrochemical experimental set up the peptide-modified Au electrode was used as a working electrode with a platinum counter electrode and an Ag/AgCl reference electrode in 3 M KCl, all of which was connected via a KNO3 salt bridge. For SWV measurements, the potential was scanned from 0.2 to 0.6 V with a step potential of 4 mV, frequency at 15 Hz, quiet time at 2 s and a pulse amplitude of 25 mV in the same buffer. The kinetics of the kinase catalyzed phosphorylation was evaluated using the surface Michaelis–Menten equation with respect to the co-substrate, ATP. The parameters for the surface catalyzed reactions were evaluated based in Eq. (1) as previously reported for the surface enzyme catalyzed reactions [25,42,43]:

V=

Vmax [E ] KM + [E ]

3.1. Phosphorylation detection using gel electrophoresis Gel electrophoresis and Western Blot assays are among the most well established and reliable techniques for the determination of protein phosphorylation. In order to establish phosphorylation of the nAChR α7, an SDS-PAGE gel, followed by a Blue-Native polyacrylamide gel electrophoresis (BN-PAGE) gel were performed. The advantage of SDS-PAGE is that each interaction can be easily identified by its molecular weight. However, in order to prevent a decrease in selectivity, a very low concentration of SDS must be utilized. The benefit of a BNPAGE gel, as compared to an SDS-PAGE gel, is that it maintains the native charge, structure, function and specificity of the proteins in the gel, thus making it a good test for native interactions. For both gels, three different kinases, PKA, PKC and Src, were utilized for the phosphorylation of the nAChR α7, with an anti-phosphoserine antibody for detection of phosphorylation. As mentioned earlier, SDS-PAGE can easily identify the bands by their molecular weights. The SDS-PAGE gels were run as described in Section 2.2 with a 0.1% SDS-PAGE buffer. The reason for utilizing such a small amount of SDS was to prevent over denaturation of the Ab, which would impede binding or its specificity. Fig. 1 shows the resulting SDS-PAGE gel. In Fig. 1, lane L) is the column containing the protein ladder, ranging from 245 kDa to 6.5 kDa. Since the ATP/ADP were smaller than the smallest ladder marker (0.5 kDa), it was difficult to distinguish from the dye-front and so was disregarded. The molecular weights of each of the components are: PKA = 40 kDa, PKC = 115 kDa, Src = 105 kDa, Ab = 50 kDa, nAChR α7 = 61 kDa and ATP = 0.5 kDa. Lane 1) (PKA, Ab and ATP) shows two distinct bands located at 40 kDa (PKA) and 50 kDa (Ab). This establishes that there is no interaction between PKA and the Ab in the absence of the α7. Lanes 2, 5 and 8) (PKA/PKC/Src, Ab and nAChR α7) show 3 bands located at 40 kDa (PKA)/110 kDa (PKC and Src), 63 kDa (α7) and 50 kDa (Ab). The presence of the three distinct bands clearly establishes that there are no false positives due to Ab-α7 interactions. Lanes 3, 6 and 9 (kinase, α7 and ATP) also show three bands located at 40 kDa (PKA)/110 kDa (PKC and Src), 63 kDa (α7) and 68 kDa (P-

(1)

The V0 is the steady-state current density per minute, Vmax is the maximal reaction speed, [E] is the enzyme concentration and KM is the Michaelis–Menten constant. 2.6. X-ray photoelectron spectroscopy (XPS) The Thermo scientific Kα X-ray Photoelectron Spectrometer (XPS) System was utilized for XPS surface studies. The XPS equipped with a monochromated Al Kα (1486.7 eV) X-ray source and 180° double focusing hemispherical analyzer with two-dimensional parallel angle resolved XPS (PARXPS) detector. The typical operating pressure is less than 5 × 10−9 mbar. The binding energies were referenced to the Au 4f7/2 peak energy at 84.0 eV. The peak fitting procedure was performed using the Thermo Avantage Software. 3. Results and discussion The present work investigates the phosphorylation of the nAChR α7 and establish the effectiveness of electrochemical techniques in determining protein phosphorylation as well as enzyme kinetics. Surface based electrochemical techniques are utilized as they are highly sensitive, easy to use and cost-effective. Scheme 1 shows the principle of surface based electrochemical techniques for phosphorylation detection on a gold electrode surface. The modified surfaces were developed stepwise from clean Au surfaces using the active ester chemistry, developed previously [12,13]. The surfaces were prepared in a step-wise fashion

Scheme 1. A schematic illustration of the electrochemical measurement of the phosphorylation reactions. In the absence of the negatively charged phosphate groups, the [Fe(CN)6]3-/4- redox probe is capable of permeating the monolayer. Upon reaching the surface, the [Fe(CN)6]3-/4- can be either oxidized or reduced, thus transferring charge (current) to or from the surface. However, upon phosphorylation the phosphate groups restrict diffusion of the redox probe which causes a decrease in the electrochemical response. 48

Analytical Biochemistry 574 (2019) 46–56

S. Ahmad, et al.

Fig. 1. A Coomassie stained SDS-PAGE gel containing 0.1% SDS showing the interactions of the PKA catalytic subunit α (PKA; MW = 40 kDa), PKC (MW = 115) and Src (MW = 105) the anti-phosphoserine antibody (Ab; MW = 50 kDa), the nAChR α7 (α7; MW = 61 kDa) and ATP (MW = 0.5 kDa). Lane L) shows the protein ladder truncated at 35 kDa and lane 1) (PKA, Ab and ATP) shows two distinct bands located at 40 kDa (PKA) and 50 kDa (Ab). This shows that there is not interaction between PKA and the Ab in the absence of the α7. Lanes 2, 5 and 8) (kinase, Ab and α7) show three distinct bands located at 40 kDa (PKA)/110 kDa (PKC and Src), 63 kDa (α7) and 50 kDa (Ab). The presence of the three distinct bands clearly establishes that there are no false positives due to Ab-α7 interactions. Lanes 3, 6 and 9 (kinase, α7 and ATP) also show three bands located at 40 kDa (PKA)/110 kDa (PKC and Src), 63 kDa (α7) and 68 kDa (P- α7). Lastly, lanes 4, 7 and 10 (kinase, α7, ATP and Ab) show 4 bands located at 40 kDa (PKA)/110 kDa (PKC and Src), 63 kDa (α7), 68 kDa (P-α7) and 165 kDA (Ab-P-α7). This confirms the P-α7 band since it interacted with the Ab to form the Ab-P-α7 complex. Lanes 9 and 10 show an extra band located at 100 kDa. This implies the formation of an intermediary P-α7 complex not present with PKA or PKC.

α7). This establishes that the α7 protein is phosphorylated since the small increase in the molecular weight is as expected also other possible interactions have been ruled out based on the previous lanes. Lastly, lanes 4, 7 and 10) (kinase, α7, ATP and Ab) show 4 bands located at 40 kDa (PKA)/110 kDa (PKC and Src), 63 kDa (α7), 68 kDa (P-α7) and 165 kDA (Ab-P-α7). This confirms the P-α7 band since it interacted with the Ab to form the Ab-P-α7 complex. Notice that the Ab does not interact with the kinase or the unphosphorylated α7 (lanes 2, 5 and 8). Lanes 9 and 10 show an extra band located at 100 kDa as a result of Src phosphorylation. This indicates the formation of a P-α7 complex not present with PKA or PKC phosphorylation, suggesting that perhaps Src phosphorylates at different sites, which has implications for the role of the P-α7 under physiological Conditions. The details of this complex may be an interesting topic for future research endeavors. The results of the SDS-PAGE were confirmed by running a BN-PAGE gel, Fig. S1. The BN-PAGE gel shows an identical band pattern, except that the P-α7 band (in Lanes 3, 4, 6, 7, 9 and 10) are indistinguishable from the α7 bands and also the P-α7 complex (in Lanes 9 and 10) is indistinguishable from the α7 band. This is not unexpected since BN gels are sensitive to the charge, size and even conformations of proteins, including globularity [44–47]. Since phosphorylation is known to induce conformational changes of the nAChR α7, which may increase folding (degree of globularity), it may be that the increase in globularity counteracts the additional negative charges of the phosphate groups [48–50]. Moreover, upon changing confirmation, it is possible that the negatively charged phosphate groups are directed inwards of the globular protein, thus minimizing their charge effect on the overall mobility of the protein. Our group has shown that sequential phosphorylation of Tau protein can lead to conformational changes which can bury the phosphorylated groups within the structure of the protein, effectively insulting its electrochemical properties [51]. Thus, the lack of a distinct phosphorylated α7 band does not imply a lack of phosphorylation. However, both the SDS-PAGE gel and BN-PAGE gels are unable to exactly establish that the P-α7 is formed, they can only imply it thorough the expected molecular weights and the Ab interaction. Therefore, to ensure the correct interpretations of the bands, a Western Blot was also performed in which all of the P-α7 containing bands could be easily and unambiguously identified. The resulting membrane is shown in Fig. S2. As expected, the Western Blot only shows secondary antibody

binding in Lanes where phosphorylation occurred as expected. However, Fig. 1 also shows that a very low percentage of α7 is phosphorylated after 2 h (only approximately 10–20%). Therefore, to ensure that the α7 can be phosphorylated to a larger degree, the SDS-PAGE gel and subsequent western blot were preformed over 2 h (a repeat of Fig. 1.) 6 h, 24 h and 48 h. The results are shown in Fig. 2. As such the phosphorylation of the nAChR α7 by PKA, PKC and Src is clearly established through gel electrophoresis. However, electrophoresis is incapable of determining the phosphorylation kinetics, which will be done through electrochemistry. Thus, the next step is to electrochemically detect phosphorylation of the nAChR α7 on an electrode surface and subsequently measure the enzyme kinetics of the surface phosphorylation. This will also go to show that electrochemistry is an equally reliable technique for the phosphorylation detection, kinase discovery and enzyme kinetics. 3.2. Electrode development and surface characterization In order to detect the phosphorylation and enzyme kinetics electrochemically, the nAChR α7 must first be immobilized onto a conducting surface. Scheme 2 shows each of the developmental states of the nAChR α7 thin film on an Au surface. After each preparatory step, the surface was characterised both electrochemically, by SWV, CV and EIS, as well as with XPS. Each step in the surface modification, as depicted in Scheme 2, was characterised by SWV, CV and EIS measurements using the [Fe(CN)6]3-/ 4redox couple, shown in Fig. 4. Briefly, each Au surface was initially modified with LPA-NHS, as previously established [40,51] with the following modifications. The first modification the use of 10 mM HEPES buffer and the second was to add MCH before the protein (nAChR α7) solution. The electrochemical measurements were conducted in 10 mM HEPES solution, pH 7.4, with 5 mM [Fe(CN)6]3-/4-. As the electrode surface is modified, charge transfer decreases resulting in a decreased current in the CV and SWV and an increasing impedance by EIS. SWV and CV measurements are shown in Fig. 3A) and Fig. 3B), respectively and both show that there is a decrease in current density upon surface modification. The decrease in the current density is due to the formation and increasing thickness of the surface monolayer (the nAChR α7 thin film) and results in an increased separation between the oxidation and reduction peaks of the of the [Fe 49

Analytical Biochemistry 574 (2019) 46–56

S. Ahmad, et al.

Fig. 2. A side-by side comparison between the SDS-PAGE (A) and its corresponding Western blot (B) of the showing the identification of the phosphorylated nAChR α7 (P-α7) though its binding to the primary anti-phosphoserine antibody (Ab) and further identification using the secondary rabbit anti-mouse antibody (rAb). Fig. 2A shows the SDS-PAGE gel containing 0.1% SDS shows the results of α7 phosphorylation by the three kinases, PKA catalytic subunit α (PKA; MW = 40 kDa), PKC (MW = 115) and Src (MW = 105), containing anti-phosphoserine antibody (Ab; MW = 50 kDa), 10 μg mL−1 nAChR α7 (α7; MW = 61 kDa) and 100 mM ATP (MW = 0.5 kDa). Lane L) shows the protein ladder truncated at 35 kDa and the subsequent lanes show the results of phosphorylation with phosphorylation times of 2 h, 6 h, 24 h and 48 h for each of the kinases. For each kinase, there is a decrease in the concentration of α7 as phosphorylation time increases from 2 h to 6 h and by 24 h of phosphorylation, there is no detectable unphosphorylated α7 remaining in the sample. Fig. 2B shows the corresponding western blot which confirms that the concentration of unphosphorylated α7 decreases as the duration of time of phosphorylation increases, and the level of unphosphorylated α7 is undetectable at times above 24 h.

(CN)6]3-/4- [28,52]. A decrease in the current density indicates an increase in the film resistance, which was measured using EIS, shown in Fig. 2C). The Nyquist plots were analyzed by the Randles’ equivalent circuit model using the ZSimpWin 2.0 software. From the analysis, it was determined that the solution resistance (Rs) was constantly recorded at 24.1 ± 0.3 Ω. The Nyquist plots in Fig. 2C) clearly demonstrate a significant increase in the Rct from 24.1 ± 0.3 Ω (bare gold) to 3267 ± 342 Ω (nAChR α7 modified surfaces). A comprehensive table for the equivalent circuit elements is provided in Table S1. The increase from 24 Ω to 3243 Ω is due to the formation of the nAChR α7 thin film which prevents charge transfer to the electrode surface. To confirm the correct preparation of the nAChR α7 surface, XPS was utilized. XPS analysis reveals the atomic identity at each stage of development and so is a useful technique to unequivocally determine the atomic composition of the molecules present on the surface. In the case of phosphorylation (before and after step e) in Scheme 2.), it is a powerful analytical technique since none of the components of then thin film contain a phosphorus prior to phosphorylation. Thus, XPS measurements were performed to confirm surface phosphorylation through the emergence of the phosphate signal, P(2p), before and after step e) in Scheme 2. Fig. 4 shows a selective XPS spectrum confirming the presence of the phosphate groups on the thin film following phosphorylation. It is to be noted that in Fig. 4, the presence of the P(2p) peak (with a binding energy of 134 eV) is not observed prior to the phosphorylation of the α7 film (Fig. 4A), but is present post-phosphorylation (Fig. 4B), indicating the presence of a phosphate group. In order to investigate the surface preparation in greater detail, XPS was also used to characterize similarly modified gold surfaces at key

stages and to estimate the thickness of the monolayer at the various stages (Fig. 5). The atomic characterization allows us to accurately determine which compounds are present on the surface and the film thickness confirms subsequent addition of layer components. Au surfaces were prepared in the same manner as the gold electrodes, as described in Section 2.3. The four key stages are: 1) a clean gold surface, 2) a gold surface modified with MCH and LPA-NHS, 3) a gold surface modified with MCH, LPA, nAChR α7 and EA, and 4) a gold surface modified with MCH, LPA and nAChR α7, EA and phosphorylation by PKA. The bare Au surface shows 2 peaks; the first peak is at 79.95 eV and represents the Au(4f) peak, and the second peak is at 116 eV and represents the AueS bound (a result of cleaning the electrodes with H2SO4). The C(1s) spectral peaks in Fig. 5B were fitted with three carbon species: 1) carboxyl and amides (binding energies of 288.3 eV), 2) aromatic carbons (binding energy of 286.3 eV), and aliphatic carbons (binding energy of 285.1 eV). The N(1s) spectra in Fig. 5C were fitted according to amide groups (the most common N species in biomolecules) with binding energy of 400.3 eV. Between the clean surface and the MCH and LPA-NHS incubation, there is a significant increase in C(1s) and N(1s) peaks along with a S peak, corresponding to the immobilization of MCH and LPA-NHS. The ratio of the peaks is 20:1 C:N which corresponds to the ratio of C(1s) atoms to N(1s) (ideally 18:1 for a perfectly clean gold surface immobilized with only MCH and LPANHS). Between MCH and LPA-NHS immobilization and protein and EA incubation, there is a significant increase in C(1s), N(1s) and O(2p) peaks, corresponding to the binding of the nAChR α7 protein (and ethanolamine). Lastly, before and after phosphorylation, there is the emergence of the P(2p) peak at 134 eV, corresponding to the presence of the phosphate group (Fig. 5.). These results agree with the work of

Scheme 2. Step-wise formation of the thin film formation on a gold surface for the study of nAChR α7 phosphorylation. The clean gold electrode was initially modified by a 2 mM lipoic acid N-hydroxysuccinimide ester (LPA-NHS) incubation for 18 h at 4 °C (a) followed by incubation in 10 mM mercaptohexonal solution at room temperature for 15 min (b). The electrode was subsequently immersed in the 10 μg/mL nAChR α7 solution (in 10 mM HEPES, pH 7.4) for 8 h at 4 °C (c). Lastly, the gold electrode was incubated in 100 mM ethanolamine solution (d) for 1 h at room temperature before phosphorylation with 100 mM ATP solution and 30 μg/mL kinase solution in 10 mM HEPES, pH 7.4, at 37 °C for 2 h (e). 50

Analytical Biochemistry 574 (2019) 46–56

S. Ahmad, et al.

Fig. 3. Characterizations of the gold surface for the study of nAChR α7 phosphorylation with Square Wave Voltammetry (SWV), Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS). The measurements were carried out using a three-electrode system consisting of a gold disc working electrode, a Pt wire counter electrode and an Ag/AgCl reference electrode. All measurements were started at the open circuit potential (OCP) and were conducted in 10 mM HEPES solution, pH 7.4, with 5 mM [Fe(CN)6]3-/4-. SWV was conducted at the potential window −0.1 V to 0.6 V with an amplitude of 0.025 V and increments of 0.002 V and a 2 s quiet time. CV started with a 30 s quiet time followed by a positive scan polarity which swept between the potential window of −0.1 V to 0.6 V at a scan rate of 0.1 Vs–1. EIS measurements were also started with a 30 s quiet time and were performed between the frequency range of 0.1 Hz–100 kHz with a 5 mV amplitude. The step-wise electrode modification was: bare Au electrode (blue), after binding of LPA-NHS (red), MCH (orange), nAChR α7 (purple), and finally EA (black). (A) SWV showing the voltammograms of modifications from LPA-NHS onwards and (inlet) showing the relative peak height of the bare Au as compared to modified surfaces, (B) CV and (C) EIS Nyquist plots with (inlet) a depiction of the Randles' circuit used to analyze the data.

In order to confirm the formation of the thin film, its thickness must be measured. An increase in the C(1s) and N(1s) peak area also suggests an increase of the electrode film thickness. The thickness of the film was determined by measuring the attenuation of the Au(4f5/2) signal (Is/IO) as previously reported and calculated [40,55,56]. Due to the inverse relationship between film thickness (and inelastic mean free path) and the probability of electron escape, the Au signal diminishes as the film thickness increases. The equation of the signal attenuation is given by:

I t = (−λ cos θ) ln ⎛ S ⎞ I ⎝ O⎠ ⎜

⎟

(2)

where t is the film thickness, λ is the Au(4f5/2) electron effective attenuation length (NIST Standard Reference Database), ϴ is the takeoff angle (0°), Is is the substrate Au(4f5/2) signal intensity after modification, and IO is the substrate Au(4f5/2) signal intensity of clean gold. Using Eq (2), the film thickness was determined to be 0.5 nm for the MCH-LPA film. Chemical attachment of the nAChR α7 increases the film thickness to 0.9 nm. No change in film thickness is observed upon protein phosphorylation. 3.3. Electrochemical detection of the phosphorylation of the nAChR α7 After explicitly demonstrating phosphorylation of the nAChR α7 biochemically and through XPS, electrochemical techniques were utilized. Phosphorylation of the nAChR α7 was performed by incubation of the thin film surface with PKA, PKC or Src in 10 mM HEPES buffer with 100 mM ATP followed by incubation in 1:100 dilution of Ab for 2 h each. Example SWV, CV and EIS measurements of PKA phosphorylation are shown in Fig. 6 and the electrochemical results of each all three kinases together are shown in Fig. S3. CV and SWV are voltammetry techniques which measure the current in solution in response to a change in the potential. From Fig. 5 it can be shown that phosphorylation results in a decrease in charge transfer, a result of the addition of the negatively charged phosphate groups to the electrode surface. The negatively charged phosphate groups repel the negatively charged [Fe(CN)6]3-/4- redox probe, preventing charge transfer. Conversely, EIS measures resistance, which increases with decreasing charge transfer. The equivalent circuit which best models the EIS spectra, shown in Fig. 5C, is the Randles’ circuit and a table of the electrochemical parameters is provided in Table S1. The EIS shows an increase in the

Fig. 4. X-ray photoelectron spectroscopy (XPS) using a monochromatic Al Kα (1486.7 eV) X-ray source at the P(2p) peak (134 eV), on nAChR α7 modified thin film gold surfaces both prior to phosphorylation (A) and after 2 h incubating in 100 mM ATP and 30 μg/mL PKA solution (B).

Amaral et al. 2005, Rains et al. 2013, Ghanadpour et al. 2015 and Ahmadi et al. 2017 [40,51,53,54]. The strong agreement between the electrochemical measurements and the XPS measurements establish the occurrence of phosphorylation. 51

Analytical Biochemistry 574 (2019) 46–56

S. Ahmad, et al.

Fig. 5. X-ray photoelectron spectroscopy (XPS) using a monochromatic Al Kα (1486.7 eV) X-ray source on four key stages of gold surface modification: clean gold surface (blue), MCH-LPA-NHS modified surface (red), MCH-LPA-nAChR α7 modified surface (orange) and finally MCH-LPA-nAChR α7-EA-phosphorylated gold surface (purple). A) shows the attenuation of the Au(4f5/2) and Au(4f7/2) peaks as the surfaces are modified. The increasing intensities of C(1s) and N(1s) as the surfaces are modified are shown in B) and C) respectively.

The results of Fig. 7 agree with the results shown in Fig. 2. In Fig. 7 it can be seen that, for each kinase, after 24 h, the phosphorylation reaction has reached completion. Furthermore, it can be seen that at 6 h, the α7 exists in an approximate 50:50 ratio of α7:P-α7. Table 1 shows the electrochemical response which results from the mixing of α7:P-α7 in ratios of 9:1, 1:1 and 1:9 along with the equivalent value of percent phosphorylated obtained from Fig. 7. For example, a 1:9 ratio of α7:P-α7 of PKA corresponds to 10% P-α7 on the electrode surface, which should have an Rct of 3225 Ω according to Fig. 7. However, it should be noted that the average Rct obtained from mixing ratios (Fig. S6) was slightly higher than the average for continuous measurement (Fig. 7.). This is to be expected since the mixture of α7:P-α7 also contained some kinase and ATP from the P-α7 solution. Although the electrodes were stored at 4 °C, the surface phosphorylation would be greater than the desired ratio due to the presence of the kinase and ATP. Overall, the Rct values are extremely similar, follow the same trends and are within the standard deviation of the values obtained from Fig. 7. Thus, it can be concluded that the change in electrochemical properties is due to the increase in surface protein phosphorylation.

impedance, and conversely a decrease in charge transfer, with surface phosphorylation and subsequent anti-phosphoserine antibody (Ab) binding. Phosphorylation of the nAChR α7 leads to the minor increase in the Rct (ΔRct) by 200 Ω cm2. The Rct is a result of two factors on the electrode surface, the first being pinhole formation due to surface reorientation and the second being the charge on the electrode surface [28,57]. Subsequent modification with the anti-phosphoserine antibody resulted in a ΔRct of 4118 Ω cm2. The large increase the Rct implies the formation of the phosphor-immunocomplex sandwiches which not only clearly indicates phosphorylation of the nAChR α7, but also establishes that a serine residue is phosphorylated. However, based on the results of Figs. 1 and 2, the film is presumably not homogeneous. To ensure that the electrochemical response is a result of protein phosphorylation, rather than two subsequent experiments were carried out. The first is to measure the change in electrochemical properties of the α7 modified Au electrodes over the course of 2 h, 6 h, 24 h and 48 h. The results are shown in Fig. 7. The second experiment involves preparing electrode surfaces modified with different α7:P-α7 ratios (9:1, 1:1 and 1:9). The Rct values of the modified electrodes (EIS spectra shown in Fig. S6) are compared in Table 1 below.

Fig. 6. (A) Square wave voltammetry (SWV), (B) cyclic voltammetry (CV) and (C) electrochemical impedance spectroscopy (EIS) Nyquist plots showing the electrochemical responses for the modified Au surface (black), the phosphorylated surface (green) and after incubation of anti-phospho-serine antibodies (yellow). Phosphorylation was performed 100 μM ATP, 0.1 M HEPES buffer (pH 7.4) for 2 h at 37 °C and subsequently measured with Ag/AgCl as the reference electrode and a Pt wire counter electrode. All measurements were started at the open circuit potential (OCP). SWV was conducted at the potential window −0.1 V to 0.6 V. CV started with a positive scan polarity which swept between the potential window of −0.1 V to 0.6 V at a scan rate of 0.1 V s-1. EIS measurements were performed between the frequency range of 0.1 Hz–100 kHz with a 5 mV amplitude. 52

Analytical Biochemistry 574 (2019) 46–56

S. Ahmad, et al.

Fig. 7. Charge transfer resistance (Rct) values of α7 modified Au electrodes vs phosphorylation time for A) PKA, B) PKC and C) Src. Each kinase solution contained 30 UN mL−1 of kinase, 10 μg mL−1 of α7 and 100 mM ATP in 10 mM HEPES buffer, pH = 7.5 and kept at 37 °C. For each kinase, 3 modified Au electrodes were prepared and subsequently phosphorylated for 2, 6, 24 or 48 h, in triplicate, in the kinase solutions. For each kinase, as the phosphorylation time increases, so does the Rct. Furthermore, each phosphorylation reaction is completed within 24 h. An Ag/AgCl reference electrode was used with a platinum wire. All experiments were performed at the OCP.

technique for their work. Herein, using PKA as an example kinase, we present our method for obtaining kinetic information from all three techniques and finally compare and analyze the kinetics obtained from EIS. The kinetics of the PKA were analyzed from the SWV and modelled by the surface Michaelis–Menten equation, Eq (1). Briefly, nAChR α7 modified surfaces were incubated with 100 mM ATP and various concentrations of PKA (0, 0.06, 0.125, 0.5, 2.5, 5, 10, 20 and 30 μg/mL) followed by a 2-h incubation in 1:100 diluted solution of Ab. Current densities were obtained by dividing the SWV peaks by the area of the electrode, 0.03 cm2 (calculation shown in the Supplementary Information). It can be seen from the normalized current density plots that the current density decreases with increasing enzyme concentration (Fig. 8B). This is consistent with the data obtained in Fig. 6, as higher enzyme concentrations will increase the amount of substrate phosphorylated resulting in decreased in charge transfer. From Fig. 8B a saturation in current response is seen at concentrations larger than 5 μg/mL. However, it is difficult to apply the surface Michaelis–Menten equation to a decreasing graph, such as the normalized current densities. In order to obtain a traditional Michaelis–Menten curve, the current densities were inverted and the reaction speeds were subsequently obtained by dividing by inverted current by time (120 min, which is 2 h). The resulting graph was then fitted with Eq (1), as shown in Fig. S3, and the values of KM and Vmax were determined to be 0.53 ± 0.04 μg/mL and 8.273 ± 0.147 cm2 A−1 min−1 respectively. Enzyme kinetics obtained via CV were analyzed in the same manner as that of SWV. The resulting curves are shown in Fig. S4. The KM and Vmax for the CV analysis were determined to be 0.52 ± 0.04 μg/mL and 1.344 ± 0.130 cm2 A−1 min−1. For EIS, the surface Michaelis–Menten equation, Eq. (1), can be applied directly to the resistance without inverting the current. Therefore, the change in resistance, ΔRct, between the unphosphorylated and phosphorylated states (after antibody incubation) were normalized by dividing by the electrode area and the time, as done for SWV and CV. From the resulting curves, shown in Fig. 8, the KM and Vmax were determined to be 0.53 ± 0.04 μg/mL and 551.200 ± 19.7 Ω cm−2 min−1. Following the same techniques, the kinetic data for PKC and Src were also obtained. The resulting EIS graphs for all three kinases are presented in Fig. 9 and the data is summarized in Table 2. It is interesting to note that the KM of PKC and Src are both lower than that of PKA and also that their Vmax values are higher than that of

Table 1 A comparison of the chare transfer resistance, Rct, (Ω) of different ratios of α7 and P-α7 immobilized onto gold electrode surfaces by PKA, PKC and Src. A comparison of the Rct is provided using the Rct of each kinase as it phosphorylates the electrode surface (see Figs. 8 and 9). In triplicates, each LPA modified electrode was incubated in either 9:1, 1:1 or 1:9 of α7:P- α7. Additionally the Pα7 solution was prepared by mixing prepared by mixing 30 UN mL−1 of kinase, 100 mM ATP and 10 μg mL−1 of α7 in 10 mM HEPES buffer at pH = 7.5 and left to incubate at 37 °C for 24 h. The phosphorylation time of the P- α7 was 24 h to ensure that the P-α7 solution was homogeneously phosphorylated. The solution was then subsequently mixed with a solution of α7 to the desired ratios without prior purification. Kinase

Ratios of P-α7

Rct (Ω)

PKA

9:1 α7:P-α7 10% Phos 1:1 α7:P-α7 50% Phos 1:9 α7:P- α7 90% Phos 9:1 α7:P-α7 10% Phos 1:1 α7:P-α7 50% Phos 1:9 α7:P- α7 90% Phos 9:1 α7:P-α7 10% Phos 1:1 α7:P-α7 50% Phos 1:9 α7:P- α7 90% Phos

3335 ± 154 3225 ± 242 7463 ± 165 7303 ± 304 10,183 ± 276 10,067 ± 196 5225 ± 273 4932 ± 156 8825 ± 293 8653 ± 206 12,825 ± 283 12,662 ± 136 4687 ± 129 4647 ± 407 8042 ± 203 7956 ± 441 12,136 ± 174 11,749 ± 203

PKC

Src

3.4. Enzyme kinetics The enzyme kinetics were performed using all three PKA, PKC and Src with SWV, CV and EIS as described in the literature [25] with a slight modification for the SWV and CV. The literature discusses how to analyze kinetics with signals that increase as enzyme concentration increases, and thus, modelled by Michaelis-Menton [25,29]. In this section a method is presented on how to handle the opposite case, such seen here with SWV and CV, where the signal decreases as enzyme concentration increases. It should be noted here that each of the electrochemical techniques provides identical kinetic information albeit providing different units of Vmax. Thus, a one may use the following protocol to obtain kinetic information employing the most convenient 53

Analytical Biochemistry 574 (2019) 46–56

S. Ahmad, et al.

Fig. 8. (A) Electrochemical Impedance Spectra (EIS) for PKA concentrations: 0 (blue), 0.06 (red), 0.125 (orange), 0.5 (purple), 2.5 (black), 5 (green), 10 (yellow), 20 (aquamarine) and 30 (brown) μg/mL. (B) The speed of the reaction obtained from the inverted current densities divided by time. The concentrations of PKA are 0, 0.06, 0.125, 0.5, 2.5, 5, 10, 20 and 30 μg/mL. The light green fitted line in (B) is the Michaelis–Menten curve based on Eq (1). The parameters for the experiments were: 100 μM ATP, 10 mM HEPES buffer (pH 7.5), scan rate at 0.1 V s−1, Ag/AgCl as the reference electrode and a Pt wire working electrode. The data points show the average of three measurements with the error bars indicating the relative standard deviation.

PKC. Given that PKA is known to cause desensitization through phosphorylation and PKC is known to cause recovery of desensitization, this implies, that recovery from desensitization, via PKC phosphorylation, may occur faster than desensitization in vivo. Furthermore, the low KM and relatively high Vmax of Src implies its significant role in the regulation of the nAChR α7. A comparison of the nAChR α7 with well-known substrates reveals the strength of importance and the likelihood of relevance to diseases. It is noteworthy that the KM of PKA for the nAChR α7 is the same as for Tau films [58]. Furthermore, these results indicate that PKC has a

Table 2 Summary of the Surface Michaelis-Menten kinetic Parameters of PKA, PKC and Src in the phosphorylation of the nAChR α7 analyzed using EIS. Kinase

KM (μM)

Vmax (Ω cm−2 min−1)

PKA PKC Src

13.1 ± 0.5 3.9 ± 0.5 7.0 ± 0.5

551 ± 19 979 ± 22 784 ± 14

Fig. 9. A comparison of the kinetic data collected by EIS (top) and the corresponding surface Michaelis–Menten kinetics (bottom). EIS spectra were obtained for kinase concentrations of 0 (blue), 0.06 (red), 0.125 (orange), 0.5 (purple), 2.5 (black), 5 (green), 10 (yellow), 20 (aquamarine) and 30 (brown) μg/mL. The corresponding surface Michaelis-Menten curve (light green) is shown below the EIS spectra with the standard deviation of three measurements (blue). The MichaelisMenten curves show the KM and the Vmax of the enzymatic reaction between PKA, PKC and Src kinases. The KM values of each kinase is 0.52, 0.45 and 0.73 μg/mL while the Vmax values are 551, 979, 784 Ω cm−2 min−1 for PKA, PKC and Src, respectively. 54

Analytical Biochemistry 574 (2019) 46–56

S. Ahmad, et al.

Table 3 A Comparison of various kinetic parameters for PKA, PKC and Src using electrochemical and non-electrochemical methods. Kinase PKA PKA PKC PKC PKC PKC Src Src Src Src a

Substrate Tau Films nAChR α7 Peptide (KRAKRKTAKKR) Renal mesangial cell homogenates sterol carrier protein 2 nAChR α7 Peptide (EGIYDVP) Peptide (RRRRRLEELLeNHe(CH2)2OH) Tau Films nAChR α7

KM (μM) 13.0 ± 0.5 13.1 ± 0.5 0.49 ± 0.13 40 6 3.9 ± 0.5 200 83 ± 32 6.6 ± 0.5 7.0 ± 0.5

Vmax

Ref −2

−1

0.015 ± 005 μA cm min 551 ± 19 Ω cm−2 min−1 10.0 ± 0.5 nM mL−1 mg−1 300 pM min−1 undetermined 979 ± 22 Ω cm−2 min−1 115 μA cm−2 min−1 38 ± 4 nmol mL−1 mg−1 0.012 ± 0.005 μA cm−2 min−1 784 ± 14 Ω cm−2 min−1

[29] a

[59] [60] [61] a

[25] [62] [29] a

Current work.

higher affinity for the nAChR α7 than for sterol carrier protein 2 (a major player in steroidogenesis) and renal mesangial cell homogenates. Lastly, a comparison of the nAChR α7 with Tau films reveals that there is a very similar affinity for Src with both substrates.

References [1] B. Zhang, P. Madden, J. Gu, X. Xing, S. Sankar, J. Flynn, K. Kroll, T. Wang, Uncovering the transcriptomic and epigenomic landscape of nicotinic receptor genes in non-neuronal tissues, BMC Genomics 18 (2017) 439–451, https://doi.org/ 10.1186/s12864-017-3813-4. [2] P. Flood, J. Ramirez-Latorre, L. Role, a4b2 neuronal nicotinic acetylcholine receptors in the central nervous system are inhibited by isoflurane and propofol, but a7-type nicotinic acetylcholine receptors are unaffected, Lab. Invest. 86 (1997) 859–865. [3] D. Puzzo, W. Gulisano, O. Arancio, A. Palmeri, The keystone of Alzheimer pathogenesis might be sought in Aβ physiology, Neuroscience 307 (2015) 26–36, https:// doi.org/10.1016/j.neuroscience.2015.08.039. [4] H.O. Kalkman, D. Feuerbach, Modulatory effects of a 7 nAChRs on the immune system and its relevance for CNS disorders, Cell. Mol. Life Sci. 73 (2016) 2511–2530, https://doi.org/10.1007/s00018-016-2175-4. [5] N. Cells, S.V. Maurer, C.L. Williams, The cholinergic system modulates memory and hippocampal plasticity via its interactions with, Front. Immunol. 8 (2017) 1489–1493, https://doi.org/10.3389/fimmu.2017.01489. [6] H. Stefansson, D. Rujescu, S. Cichon, O.P.H. Pietiläinen, A. Ingason, S. Steinberg, R. Fossdal, E. Sigurdsson, T. Sigmundsson, J.E. Buizer-Voskamp, T. Hansen, K.D. Jakobsen, P. Muglia, C. Francks, P.M. Matthews, A. Gylfason, B.V. Halldorsson, D. Gudbjartsson, T.E. Thorgeirsson, A. Sigurdsson, A. Jonasdottir, A. Jonasdottir, A. Bjornsson, S. Mattiasdottir, T. Blondal, M. Haraldsson, B.B. Magnusdottir, I. Giegling, H.-J. Möller, A. Hartmann, K.V. Shianna, D. Ge, A.C. Need, C. Crombie, G. Fraser, N. Walker, J. Lonnqvist, J. Suvisaari, A. Tuulio-Henriksson, T. Paunio, T. Toulopoulou, E. Bramon, M. Di Forti, R. Murray, M. Ruggeri, E. Vassos, S. Tosato, M. Walshe, T. Li, C. Vasilescu, T.W. Mühleisen, A.G. Wang, H. Ullum, S. Djurovic, I. Melle, J. Olesen, L.A. Kiemeney, B. Franke, C. Sabatti, N.B. Freimer, J.R. Gulcher, U. Thorsteinsdottir, A. Kong, O.A. Andreassen, R.A. Ophoff, A. Georgi, M. Rietschel, T. Werge, H. Petursson, D.B. Goldstein, M.M. Nöthen, L. Peltonen, D.A. Collier, D. St Clair, K. Stefansson, R.S. Kahn, D.H. Linszen, J. van Os, D. Wiersma, R. Bruggeman, W. Cahn, L. de Haan, L. Krabbendam, I. Myin-Germeys, Large recurrent microdeletions associated with schizophrenia, Nature 455 (2008) 232–236, https://doi. org/10.1038/nature07229. [7] J.W. Young, M.A. Geyer, Evaluating the role of the alpha-7 nicotinic acetylcholine receptor in the pathophysiology and treatment of schizophrenia, Biochem. Pharmacol. 86 (2013) 1122–1132, https://doi.org/10.1016/j.bcp.2013.06.031. [8] R.J. Hung, J.D. McKay, V. Gaborieau, P. Boffetta, M. Hashibe, D. Zaridze, A. Mukeria, N. Szeszenia-Dabrowska, J. Lissowska, P. Rudnai, E. Fabianova, D. Mates, V. Bencko, L. Foretova, V. Janout, C. Chen, G. Goodman, J.K. Field, T. Liloglou, G. Xinarianos, A. Cassidy, J. McLaughlin, G. Liu, S. Narod, H.E. Krokan, F. Skorpen, M.B. Elvestad, K. Hveem, L. Vatten, J. Linseisen, F. Clavel-Chapelon, P. Vineis, H.B. Bueno-De-Mesquita, E. Lund, C. Martinez, S. Bingham, T. Rasmuson, P. Hainaut, E. Riboli, W. Ahrens, S. Benhamou, P. Lagiou, D. Trichopoulos, I. Holcátová, F. Merletti, K. Kjaerheim, A. Agudo, G. MacFarlane, R. Talamini, L. Simonato, R. Lowry, D.I. Conway, A. Znaor, C. Healy, D. Zelenika, A. Boland, M. Delepine, M. Foglio, D. Lechner, F. Matsuda, H. Blanche, I. Gut, S. Heath, M. Lathrop, P. Brennan, A susceptibility locus for lung cancer maps to nicotinic acetylcholine receptor subunit genes on 15q25, Nature 452 (2008) 633–637, https://doi.org/10.1038/nature06885. [9] S. Trombino, A. Cesario, S. Margaritora, P. Granone, G. Motta, C. Falugi, P. Russo, α7-Nicotinic acetylcholine receptors affect growth regulation of human mesothelioma cells, Cancer Res. 64 (2004) 135–145, https://doi.org/10.1158/0008-5472. CAN-03-1672. [10] J. Egea, I. Buendia, E. Parada, E. Navarro, R. Lean, M. Lopez, Anti-inflammatory role of microglial alpha7 nAChRs and its role in neuroprotection, Biochem. Pharmacol. 97 (2015) 463–472, https://doi.org/10.1016/j.bcp.2015.07.032. [11] M. Noda, A.I. Kobayashi, Nicotine inhibits activation of microglial proton currents via interactions with a 7 acetylcholine receptors, J. Physiol. Sci. 67 (2017) 235–245, https://doi.org/10.1007/s12576-016-0460-5. [12] L.C. Gahring, E.J. Myers, D.M. Dunn, R.B. Weiss, W. Rogers, Nicotinic Alpha 7 Receptor Expression and Modulation of the Lung Epithelial Response to Lipopolysaccharide, (2017). [13] R. Sun, W. Zhang, J. Bo, Z. Zhang, Y. Lei, W. Huo, Y. Liu, Z. Ma, X. Gu, Spinal activation of a7 nicotinic acetylcholine receptor attenuates posttraumatic stress

4. Conclusion We have demonstrated phosphorylation of human nAChR α7 contains serine/threonine phosphorylation sites of PKA and PKC as well as tyrosine phosphorylation via Src. Furthermore, this work establishes that electrochemistry is a viable and trustworthy technique for the analysis of phosphorylation states of neuronal membrane peptides and proteins. PKA and PKC are especially relevant to the study of neuronal membrane proteins such as nAChRs because of their role in regulation. PKA has been shown to cause desensitization of nAChRs in both major and minor states whereas PKC has been shown to facilitate recovery from the desensitized states. The results of this work imply that perhaps Src is also involved in the regulation of the nAChR because of its strong affinity and high kinetics. Thus, the study of the phosphorylation of the nAChR and acetylcholine regulation. We have shown through CV, SWV, EIS and XPS that the nAChR phosphorylated surface can be prepared. Furthermore, through EIS we have shown that the phosphorylation reaction can be easily monitored through an increase in the Rct and the charge transfer resistance can be further enhanced by the addition of monoclonal anti-serine antibodies. Lastly, we have demonstrated that enzyme kinetics calculations can be conducted electrochemically and provide a technique to handle kinetics with an inverse relationship between the electrochemical signal and enzyme concentration. Herein we have reported the Vmax of each kinase as obtained by EIS to be 551 ± 19, 979 ± 22, 784 ± 14 Ω cm2 min−1 with KM values of 13.1 ± 0.5, 3.9 ± 0.5 and 7.0 ± 0.5. Future work may be done to determine the exact sites of phosphorylation, the role of Src in nAChR α7 regulation and finding additional kinases. Furthermore, another research direction can be to determine if there are any kinetic differences between the different subtypes of the nAChR or measure the kinetics of these enzymes phosphorylating the full receptor. Declaration of interest The authors declare that there is no conflict of interest. Acknowledgements We gratefully acknowledge funding from University of Toronto Scarborough and from the Natural Sciences and Engineering Research Council of Canada (NSERC – RGPIN-2016-06122). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ab.2019.03.012. 55

Analytical Biochemistry 574 (2019) 46–56

S. Ahmad, et al.

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37] N. Wang, Z. She, Z. Ingar, S. Martic, H.B. Kraatz, A bioorganometallic approach to study histidine kinase autophosphorylations, Chem. Eur J. 23 (2017) 3152–3158, https://doi.org/10.1002/chem.201605253. [38] L. Liu, Probing of protein kinase activity by electrochemistry, Int. J. Electrochem. Sci. 11 (2016) 8405–8417, https://doi.org/10.20964/2016.10.59. [39] S. Martic, S. Ahmadi, Z. She, H.-B. Kraatz, Electrochemical detection of protein kinase-catalyzed phosphorylations, Kinomics Approaches Appl. (2015) 169–192, https://doi.org/10.1002/9783527683031.ch7. [40] S. Ahmadi, I.I. Ebralidze, Z. She, H.-B. Kraatz, Electrochemical studies of tau protein-iron interactions—potential implications for Alzheimer's Disease, Electrochim. Acta 236 (2017) 384–393, https://doi.org/10.1016/j.electacta.2017.03.175. [41] R.V.A.N. Coster, J. Smet, E. George, L.D.E. Meirleir, S. Seneca, J.V.A.N. Hove, G. Sebire, H. Verhelst, J.A.N.D.E. Bleecker, B.V.A.N. Vlem, P. Verloo, J. Leroy, Blue native polyacrylamide gel Electrophoresis : a powerful tool in diagnosis of oxidative phosphorylation defects, Pediatr. Res. 50 (2001) 658–665. [42] P.A. Neff, A. Serr, B.K. Wunderlich, A.R. Bausch, Label-free electrical determination of trypsin activity by a silicon-on-insulator based thin film resistor, ChemPhysChem 8 (2007) 2133–2137, https://doi.org/10.1002/cphc.200700279. [43] H.J. Lee, A.W. Wark, T.T. Goodrich, S. Fang, R.M. Corn, Surface enzyme kinetics for biopolymer microarrays: a combination of Langmuir and michaelis-men ten concepts, Langmuir 21 (2005) 4050–4057, https://doi.org/10.1021/la046822h. [44] I. Wittig, H. Schägger, Features and applications of blue-native and clear-native electrophoresis, Proteomics 8 (2008) 3974–3990, https://doi.org/10.1002/pmic. 200800017. [45] I. Wittig, H. Braun, H. Scha, Blue native PAGE, Nat. Protoc. 1 (2006) 418–428, https://doi.org/10.1038/nprot.2006.62. [46] H. Schagger, G. Jagow, Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form, Anal. Biochem. 231 (1991) 223–231. [47] H. Schagger, W. Cramer, G. Jagow, Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis, Anal. Biochem. 217 (1994) 220–230. [48] C. Biology, S. Talwar, J.W. Lynch, Phosphorylation mediated structural and functional changes in pentameric ligand-gated ion channels : implications for drug discovery, Int. J. Biochem. Cell Biol. 53 (2014) 218–223, https://doi.org/10.1016/ j.biocel.2014.05.028. [49] S. Swope, S. Moss, L. Raymond, R. Huganir, Regulation of ligand-gated ion channels by protein phosphorylation, Adv. Second Messenger Phosphoprotein Res. 33 (1999) 49–78. [50] V.V. Pollock, T. Pastoor, C. Katnik, J. Cuevas, L. Wecker, Cyclic AMP-dependent Protein Kinase A and Protein Kinase C Phosphorylate alpha4-beta2 nicotinic receptor subunits at distinct stages of receptor formation and maturation, Neuroscience 158 (2009) 1311–1325, https://doi.org/10.1016/j.neuroscience. 2008.11.032. [51] M.K. Rains, S. Martić, D. Freeman, H.B. Kraatz, Electrochemical investigations into kinase-catalyzed transformations of tau protein, ACS Chem. Neurosci. 4 (2013) 1194–1203, https://doi.org/10.1021/cn400021d. [52] M.K. Rains, S. Martić, D. Freeman, H.B. Kraatz, Electrochemical investigations into kinase-catalyzed transformations of tau protein, ACS Chem. Neurosci. 4 (2013) 1194–1203, https://doi.org/10.1021/cn400021d. [53] I.F. Amaral, P.L. Granja, M.A. Barbosa, Chemical modification of chitosan by phosphorylation: an XPS, FT-IR and SEM study, J. Biomater. Sci. Polym. Ed. 16 (2005) 1575–1593, https://doi.org/10.1163/156856205774576736. [54] M. Ghanadpour, F. Carosio, P.T. Larsson, L. Wågberg, Phosphorylated cellulose nanofibrils: a renewable nanomaterial for the preparation of intrinsically flameretardant materials, Biomacromolecules 16 (2015) 3399–3410, https://doi.org/10. 1021/acs.biomac.5b01117. [55] A.A. Azmi, I.I. Ebralidze, S.E. Dickson, J.H. Horton, Journal of Colloid and Interface Science Characterization of hydroxyphenol-terminated alkanethiol self-assembled monolayers : interactions with phosphates by chemical force spectrometry, J. Colloid Interface Sci. 393 (2013) 352–360, https://doi.org/10.1016/j.jcis.2012.10. 026. [56] M. Shamsipur, S. Habib, A. Alizadeh, Self-assembled monolayers of a hydroquinone-terminated alkanethiol onto gold surface. Interfacial electrochemistry and Michael-addition reaction with glutathione, 610 (2007) 218–226, https://doi.org/ 10.1016/j.jelechem.2007.07.014. [57] E. Snir, J. Joore, P. Timmerman, S. Yitzchaik, Monitoring selectivity in kinasepromoted phosphorylation of densely packed peptide monolayers using label-free electrochemical detection, Langmuir 27 (2011) 11212–11221, https://doi.org/10. 1021/la202247m. [58] B.C. Carlyle, A.C. Nairn, M. Wang, Y. Yang, L.E. Jin, A.A. Simen, B.P. Ramos, cAMPPKA phosphorylation of tau confers risk for degeneration in aging association cortex, Proc. Natl. Acad. Sci. U. S. A 111 (2014) 5036–5041, https://doi.org/10. 1073/pnas.1322360111. [59] P. Chemistry, B. Centre, A potent and highly selective peptide substrate for protein kinase C assay, Biochem. J. 460 (1997) 455–460. [60] J. Isbell, S. Christian, N. Mashburn, P. Bell, A non-Radioactive fluorescent method for measuring protein kinase C activity.pdf, Life Sci. 57 (1995) 1701–1707. [61] A. Steinschneider, M.P. McLean, J.T. Billheimer, S. Azhar, G. Gibori, Protein kinase C catalyzed phosphorylation of sterol carrier protein 2, Endocrinology 125 (1989) 569–571. [62] T.R. Lee, J.H. Till, D.S. Lawrence, W.T. Miller, Precision substrate targeting of protein kinases v-Abl and c-Src *, J. Biol. Chem. 270 (1995) 27022–27026.

disorder-related related chronic pain via suppression of glial activation, Neuroscience 344 (2017) 243–254, https://doi.org/10.1016/j.neuroscience.2016. 12.029. A. Pacini, L. Di, C. Mannelli, L. Bonaccini, S. Ronzoni, A. Bartolini, C. Ghelardini, Protective effect of alpha7 nAChR : behavioural and morphological features on neuropathy, Pain 150 (2010) 542–549, https://doi.org/10.1016/j.pain.2010.06. 014. D.L. Carlisle, T.M. Hopkins, A. Gaither-davis, M.J. Silhanek, J.D. Luketich, N.A. Christie, J.M. Siegfried, Nicotine signals through muscle-type and neuronal nicotinic acetylcholine receptors in both human bronchial epithelial cells and airway fibroblasts, Respir. Res. 5 (2004) 27–43, https://doi.org/10.1186/14659921-5-27. K.C. Brown, H.E. Perry, J.K. Lau, D. V Jones, J.F. Pulliam, B.A. Thornhill, C.M. Crabtree, H. Luo, Y.C. Chen, P. Dasgupta, Nicotine increases alpha7-nAChR expression in human squamous cell lung cancer cells via the SP1/GATA pathway, J. Biol. Chem. 288 (2013) 33049–33059, https://doi.org/10.1074/jbc.M113.501601. L. Liu, J. Yu, L. Li, B. Zhang, L. Liu, C. Wu, A. Jong, Alpha7 nicotinic acetylcholine receptor is required for amyloid pathology in brain endothelial cells induced by Glycoprotein 120 , methamphetamine and nicotine, Sci. Rep. 7 (2017) 40467–40479, https://doi.org/10.1038/srep40467. K. Paradiso, P. Brehm, Long-term desensitization of nicotinic acetylcholine receptors is regulated via protein kinase A-mediated phosphorylation, J. Neurosci. 18 (1998) 9227–9237 http://www.jneurosci.org/cgi/content/abstract/18/22/9227. D. Zhao, X. Xu, L. Pan, W. Zhu, X. Fu, L. Guo, Q. Lu, J. Wang, Pharmacologic activation of cholinergic alpha7 nicotinic receptors mitigates depressive-like behavior in a mouse model of chronic stress, J. Neuroinflammation 14 (2017), https://doi.org/10.1186/s12974-017-1007-2 234–149. S.J. Moss, B.J. McDonald, Y. Rudhard, R. Schoepfer, Phosphorylation of the predicted major intracellular domains of the rat and chick neuronal nicotinic acetylcholine receptor aplha7 subunit by cAMP-dependent protein kinase, Neuropharmacology 35 (1996) 1023–1028, https://doi.org/10.1016/S00283908(96)00083-4. A.S. Nimnual, W. Chang, N.S. Chang, A.F. Ross, M.S. Gelman, J.M. Prives, Identification of phosphorylation sites on AChR δ-subunit associated with dispersal of AChR clusters on the surface of muscle cells, Biochemistry 37 (1998) 14823–14832, https://doi.org/10.1021/bi9802824. O.I. Wilner, C. Guidotti, A. Wieckowska, R. Gill, I. Willner, Probing kinase activities by electrochemistry, contact-angle measurements, and molecular-force interactions, Chem. Eur J. 14 (2008) 7774–7781, https://doi.org/10.1002/chem.200800765. O.I. Wilner, C. Guidotti, A. Wieckowska, R. Gill, I. Willner, Probing kinase activities by electrochemistry, contact-angle measurements, and molecular-force interactions, Chem. Eur J. 14 (2008) 7774–7781, https://doi.org/10.1002/chem.200800765. V.C. Diculescu, T.A. Enache, Electrochemical evaluation of Abelson tyrosine-protein kinase 1 activity and inhibition by imatinib mesylate and danusertib, Anal. Chim. Acta 845 (2014) 23–29, https://doi.org/10.1016/j.aca.2014.06.025. S. Martić, M. Labib, H.B. Kraatz, Electrochemical investigations of sarcoma-related protein kinase inhibition, Electrochim. Acta 56 (2011) 10676–10682, https://doi. org/10.1016/j.electacta.2011.05.013. S. Martić, S. Tackenburg, Y. Bilokin, A. Golub, V. Bdzhola, S. Yarmoluk, H.B. Kraatz, Electrochemical screening of the indole/quinolone derivatives as potential protein kinase CK2 inhibitors, Anal. Biochem. 421 (2012) 617–621, https:// doi.org/10.1016/j.ab.2011.11.017. K. Kerman, M. Chikae, S. Yamamura, E. Tamiya, Gold nanoparticle-based electrochemical detection of protein phosphorylation, Anal. Chim. Acta 588 (2007) 26–33, https://doi.org/10.1016/j.aca.2007.02.001. S. Martić, S. Beheshti, H.B. Kraatz, D.W. Litchfield, Electrochemical investigations of tau protein phosphorylations and interactions with pin1, Chem. Biodivers. 9 (2012) 1693–1702, https://doi.org/10.1002/cbdv.201100418. S. Martić, S. Beheshti, M.K. Rains, H.-B. Kraatz, Electrochemical investigations into Tau protein phosphorylations, Analyst 137 (2012) 2042, https://doi.org/10.1039/ c2an35097a. Z. Wang, Z. Yan, F. Wang, J. Cai, L. Guo, J. Su, Y. Liu, Highly sensitive photoelectrochemical biosensor for kinase activity detection and inhibition based on the surface defect recognition and multiple signal amplification of metal-organic frameworks, Biosens. Bioelectron. 97 (2017) 107–114, https://doi.org/10.1016/j.bios. 2017.05.011. H. Song, K. Kerman, H.-B. Kraatz, Electrochemical detection of kinase-catalyzed phosphorylation using ferrocene-conjugated ATP, Chem. Commun. 4 (2008) 502–504, https://doi.org/10.1039/b714383d. P. Miao, L. Ning, X. Li, P. Li, G. Li, Electrochemical strategy for sensing protein phosphorylation, Bioconjug. Chem. 23 (2012) 141–145, https://doi.org/10.1021/ bc200523p. S. Martić, M. Gabriel, J.P. Turowec, D.W. Litchfield, H.-B.B. Kraatz, Versatile strategy for biochemical, electrochemical and immunoarray detection of protein phosphorylations, J. Am. Chem. Soc. 134 (2012) 17036–17045, https://doi.org/10. 1021/ja302586q. M.K. Rains, S. Martić, D. Freeman, H.B. Kraatz, Electrochemical investigations into kinase-catalyzed transformations of tau protein, ACS Chem. Neurosci. 4 (2013) 1194–1203, https://doi.org/10.1021/cn400021d. Z. She, K. Topping, B. Dong, M.H. Shamsi, H.-B. Kraatz, An unexpected use of ferrocene. A scanning electrochemical microscopy study of a toll-like receptor array and its interaction with E. coli, Chem. Commun. 53 (2017) 2946–2949, https://doi. org/10.1039/C7CC00863E. J.O. Esteves-Villanueva, S. Martic-Milne, Electrochemical detection of anti-tau antibodies binding to tau protein and inhibition of GSK-3??-catalyzed phosphorylation, Anal. Biochem. 496 (2016) 55–62, https://doi.org/10.1016/j.ab.2015.12.002.

56