Nicotinic acetylcholine receptors (nAChRs) are a class of pentameric ligand-gated ion channels involved in a wide variety of complex brain functions (memory, cognition, etc.) and neuromuscular transmission and plays a role in pathological conditions such as Alzheimer’s and Parkinson’s diseases, schizophrenia, and nicotine addiction. Additionally, nicotinic receptor agonists have potential as analgesics, anxiolytics and cognitive enhancers. The search for modulators of nAChR channels has been hampered by the lack of suitable high-throughput electrophysiology platforms with the ability to interrogate fast desensitizing ligand-gated channels.
Here, we present assay data, EC50, and recovery time scale measurements for the nACh receptor channel (α1) using the IonFlux™ Automated Patch Clamp System (Figure 1). The IonFlux system is a desktop instrument that utilizes microfluidic compound delivery on 100 ms timescales, facilitating the recording of fast-activating, ligand-gated ion channels (Fig. 1). A large number of cells (20 per ensemble) under voltage clamp can be exposed to a compound within a short time scale in parallel across the plate. Continuous recording, coupled with fast solution exchange, enables high-throughput screening against ligand-gated ion channel targets.
Materials and methods
Cells expressing hnAChR (Millipore PrecisION™ hnAChR α1/β1/γ/ε -HEK 293 Recombinant Cell Line, Cat # CYL3052) were cultured in medium containing DMEM/F12 glutamax, Fetal Bovine Serum, Non-essential amino acid, Geneticin, and Hygromycin B at 37°C and 5% CO2. For cell isolation, flasks were first washed with 10 mL of Ca- and Mg-free PBS, followed by 2 mL of Detachin™ solution (Genlantis), after which cells were treated with Detachin™ solution. After release, the cell suspension was spun for 90 seconds (1000 rpm) and re-suspended in extracellular solution (5x106 cell/mL). The extracellular solution contained (mM): 138 NaCl, 4 KCl, 1 MgCl2, 1.8 CaCl2, 10 HEPES, 5.6 glucose, pH 7.45 with NaOH. The intracellular solution for the whole cell voltage clamp contained (mM): 15 NaCl, 60 KCl, 70 KF, 5 HEPES, 5 EGTA, pH 7.25 with KOH. Cell suspension in extracellular solution was dispensed into an IonFlux plate.
The IonFlux plate layout consists of units of twelve wells: two wells contain intracellular solution, one contains ECS plus cells, eight contain ECS plus compounds of interest, and one well is for waste collection. Cells are captured from suspension by applying suction to microscopic channels in ensemble recording arrays. Once the array is fully occupied, the applied suction breaks the cell membranes of captured cells, establishing whole-cell voltage clamp. For compound applications, pressure is applied to the appropriate compound wells, introducing the compound into the extracellular solution rapidly flowing over the cells. For recording nAChR currents, cell arrays were voltage clamped at a holding potential of -80 mV.
Acetylcholine (ACh), the nAChR agonist, was purchased from Sigma-Aldrich (Cat# A6625). ACh was dissolved in deionized water to make a 10 mM stock solutions, and diluted serially into extracellular buffer. Data analysis and graphical presentation were performed using a combination of IonFlux software and Origin Lab. A threshold was set at -2nA for responses to 10 µM ACh. Ensemble responses below this value were excluded from the analysis.
To start, an experiment was run in order to determine the ACh EC50, measured at 6 µM (Fig. 2). The time required for recovery from desensitization was determined using repeated agonist applications. A first agonist application (10 µM ACh, 1 s duration) was followed by a variable wash interval of 1 to 10 s and a repeat of the 10 µM application (Fig. 3 A, B). For clarity, both applications were done within the same recording sweep. When the recovery period is only 1 s long, we measure roughly a 40% current amplitude for the second application (Fig. 3A); at the other extreme, a 10 s recovery period results in a 95% current amplitude for the second application (Fig. 3B).
Recovery was measured by comparing the negative current amplitude for the second application as a percentage to the first, with the results shown in Fig. 3C. The IonFlux system has the advantage of being a continuous flow system with very small dead volumes, so agonist washout is very efficient (the volume in the recording region gets exchanged roughly 40 times over a 10 s recovery period). As a result, we observe recovery rates that are very fast: approx. 3 s time constant, 15 s for full recovery.
Other important parameters are success rates and consistency of current intensity. An experiment was designed to measure the above parameters as well as the EC50 of ACh response across IonFlux HT plates (Fig. 4). Each pattern was loaded with identical solutions as follows: C1 (compound well 1) – 0 µM control, C2 – 1 µM, C3 – 3 µM, C4 – 10 µM, C5 – 30 µM, C6 – 100 µM. The consistency of the 100 µM response is shown in an overlay of 16 such curves obtained during simultaneous compound applicaitons (left inset Fig. 4). During HT experiments, current trace charts show changes in the peak amplitude in response to compound applications (Fig. 4). Each quadrant of the plate contains 16 recording channels, which are displayed in the same viewing window. A tab control lets the user change views between the quadrants. For this particular experiment, 62/64 channels were successful, 16 of which are shown in Fig. 4. That means that the system had a 97% success rate for this plate. A typical current distribution standard error for 3, 10, 30 and 100 µM points are shown in Fig. 2.
The consistency and high success rates for recoding of nAChR responses on the IonFlux platform shows the potential for conducting robust pharmacology studies of nACh receptors.