By Iris Yang, M.S., Cathy Smith-Maxwell, Ph.D., Naibo Yang, Ph.D. and David Yamane, Molecular Devices, 1311 Orleans Drive, Sunnyvale, CA 94089.
Ligand-gated ion channels
Ligand-gated ion channels (LGICs) are multisubunit transmembrane proteins involved in neurotransmission. LGICs, as the name implies, are ion channels that open upon the binding of a ligand. Examples include the amino acid neurotransmitters, γ-aminobutyric acid (GABA) receptors and acetylcholine receptors, which mediate synaptic transmission at the neuromuscular junction. The opening and closing of ligand-gated ion channels are characterized by a fast activation phase and a slower desensitization phase. Activation and desensitization are commonly reported as time constants, τact and τdes, and are in the range of milliseconds to seconds. Therefore, a rapid solution exchange rate is an important consideration when studying ligand-gated ion channels.
Two ligand-gated ion channels, γ-aminobutyric acid type A (GABAA) receptor and acid-sensing ion channels (ASICs), were used to evaluate the feasibility of using the PatchXpress 7000A system to study ligand-gated ion channels. GABAA receptors are the major neuronal ion channel mediating fast synaptic inhibitory transmission in the mammalian brain. GABAA receptors are composed of five subunits surrounding a chloride-selective pore with the binding sites for GABAA, benzodiazepines and several other modulators. ASICs are ligand-gated cation channels activated by extracellular protons. ASICs have been suggested to play an important role in the physiology and pathology of sensory transmission, behavioral memory, retinal function, epileptogenesis and ischemia.
A new feature in PatchXpress Commander 1.6 software which runs the PatchXpress 7000A System is the Lock Compound Robot command. This function locks the drug addition pipettor to a single chamber for a defined series of procedural steps.
By locking the compound addition robot to a single chamber, timing for compound additions will be the same for all cells since the pipettor is not available to add compounds to the other chambers during the “locked” steps. When studying ligand-gated ion channels and/or when incubating cells with antagonist or modulator compounds, locking the compound addition robot ensures compound addition and incubation times are exactly the same for each cell for more consistent results. In this application note, PatchXpress 7000A and the Lock Compound Robot function are evaluated for conducting ligand-gated ion channel studies.
- Cells: fibroblast L-tk cells expressing the α1β3γ2 GABAA receptors; Human Embryonic Kidney (HEK293) cells endogenously expressing ASIC receptors; RBL-1 cells endogenously expressing inward rectifier potassium channel (Kir)
- Reagents and Buffers: γ-aminobutyric acid (Sigma Cat. #A-5835), bicuculline (TOCRIS Cat. #0130), CL 218872 (TOCRIS Cat. #1709), Amiloride (Sigma Cat. #A-7410), DMSO (Sigma Cat. #105879)
For L-tk cells expressing GABAA receptors The internal buffer contained (in mM): 130 CsCl (Sigma Cat. #C-3309), 10 Hepes (Sigma Cat. #H-4034), 10 Cs-EGTA (Sigma Cat. #E-0396), 2 MgCl2 (Fisher Cat. #M33- 500), 53 sucrose (Sigma Cat. #S-7903), pH 7.3 with CsOH; the external buffer contained (in mM): 149 NaCl (Sigma, Cat. #S-5886), 3.25 KCl (Fisher Cat. #P217-500), 2 CaCl2 (Fisher Cat. #C79-500), 2 MgCl2 (Fisher Cat. #M33-500), 10 Hepes (Sigma Cat. #H- 4034), 11 glucose (Sigma Cat. #G-7528), 22 sucrose (Sigma, Cat. #S-7903), pH 7.4 with NaOH
For HEK293 cells expressing ASIC receptors The internal buffer contained (in mM): 20 KCl (Fisher Cat. #P217-500), 8 NaCl (Sigma Cat. #S-5886), 110 K-Glu (Sigma Cat. #G-1501), 1 MgCl2 (Fisher Cat. #M33-500), 10 EGTA (Sigma Cat. #E-0396), 4 Mg ATP (Sigma Cat. #A-9187), 10 Hepes (Sigma Cat. #H-4034), pH 7.25 with KOH; the external buffer contained (in mM): 140.8 NaCl (Sigma Cat. #S-5886), 4 KCl (Fisher Cat. #P217-500), 1.2 MgCl2 (Fisher Cat. #M33-500), 1 CaCl2 (Fisher Cat. #C79- 500), 10 Hepes (Sigma Cat. #H-4034), 10 MES (Sigma Cat. #M-2933), pH 7.45 with NaOH or pH 4.4 with HCl
For RBL-1 cells The buffers are the same as used with HEK293 cells. The high potassium buffer exchanging solution was a modified external buffer containing 30 mM KCl and 115 mM NaCl. The rest of the compositions are the same.
- Tissue culture flasks: Cells grown in T-75 flasks (Corning Cat. #430641)
- Cell culture media: Dulbecco’s Modified Eagle Medium (Gibco Cat. #11965-126)
- 10 % Fetal Bovine Serum (Hyclone Cat. #SH30070.01), 1 % Penicillin-Streptomycin (Gibco Cat. #15140-122), Geneticin (G418) and Trypsin 0.25 %, 1 mM EDTA x 4Na (Gibco Cat. #252 00-056), DPBS without Ca2+ and Mg2+ (Sigma Cat. #8537)
- SealChip™ 16 planar electrodes (Molecular Devices)
- Compound plates: Costar® 96-well plate (Corning Cat. #3595)
For the solution exchange experiments, RBL-1 cells were cultured in suspension with DMEM/ F12 media in T75 flasks. Cells were passaged every 1–3 days at 1:4 to 1:40 dilutions. To prepare cells for use, cells were centrifuged at 1000 rpm and the supernatant removed. The pellet was resuspended in 4 mL of media at a density of 250,000 cells per mL. Immediately before each experiment, cells were centrifuged at 1000 rpm for 1 minute. The supernatant was decanted and the cells were resuspended in 150 µL of extracellular buffer for recording with PatchXpress 7000A.
For the ligand-gated ion channel studies on PatchXpress 7000A, a L-tk cell line expressing α1β3γ2 GABAA receptors and Human Embryonic Kidney (HEK293) cell line expressing endogenous ASIC receptors were used. Cells were cultured in T-75 flasks and passaged every 2–3 days at 1:4 or 1:10 dilutions. Cells were also maintained at a lower seeding density (1:50) and passaged every 3–5 days. To prepare cells for use, cells were grown to 70–80% confluence in a T-75 flask. Growth medium was aspirated from the culture flasks using 1 mL sterile aspirating pipette by vacuum. Cells were gently rinsed with 10 mL DPBS for approximately 1 minute before the solution was aspirated. The cells were immersed in 10 mL Trypsin solution for 2 minutes to dislodge the cells. DMEM growth medium in the amount of 10 mL was added to the flask. The medium was triturated to remove the cells from the flask surface and to dissociate cell clumps. The cell suspension was placed into a 50 mL sterile centrifuge tube. The 50 mL tube was centrifuged at 1000 rpm for 1.5 minutes. The cell supernatant was decanted and 5 mL of DMEM growth media was added to resuspend the cells. A cell suspension of 4 mL at a density of 250,000 cells per mL was put into a 15 mL centrifuge tube. The tube was placed in a 37°C, 5 % CO2 incubator with caps slightly closed for 15 minutes for cells to recover. For each experiment, one tube of cells was removed from the incubator and centrifuged at 1000 rpm for 1 minute. The supernatant was decanted. The cells were resuspended in 150 µL of extracellular buffer for recording with PatchXpress 7000A.
Measuring PatchXpress 7000A System solution exchange rates
RBL-1 cells endogenously expressing inward rectifying potassium channels were used to study the PatchXpress 7000A System solution exchange rates. To measure the solution exchange rate for compound addition, currents from RBL cells were recorded at -120 mV. A solution containing 30 mM potassium was applied to 15 cells to elicit a change in the holding current. The average time constant for the measured Kir current across fifteen cells was 19 ± 11 ms. (See Figure 1.) Following compound addition, cells were washed using the PatchXpress 7000A System dedicated wash station and the Kir currents again were recorded. Average time constant for solution wash out was 420 ± 281 ms for the fifteen cells tested. The rapid solution exchange rates allow the PatchXpress 7000A System to assay fast-acting ligand-gated ion channels.
Ligand-gated ion channel assay
GABA-elicited inward chloride currents at a holding potential of -60 mV in L-tk cells expressing α1β3γ2 GABAA receptors. Five concentrations, 0.1 µM, 0.3 µM, 1.0 µM, 3.0 µM, 10.0 µM and a saturating concentration of 30.0 µM of GABA were applied to the cells (Figure 2). Concentrationresponse curves for GABA were fitted to a Hill equation:
f(x) = Imax/[1+(EC50/x) n]
where x is the GABA concentration, Imax is the maximal current, EC50 is the concentration of GABA eliciting a half-maximal response and n is the Hill coefficient. The mean concentrationresponse relationship to GABA for cells expressing α1β3γ2 GABAA receptors is shown in Figure 3 and an average EC50 value of 3.41 ± 0.33 µM (mean ± S.D.; n=5) was calculated. To evaluate the effect of adding the competitive antagonist bicuculline, GABA currents were repeatedly induced by adding 3 µM of GABA to the cells. L-tk cells were then incubated with bicuculine for 15 seconds before co-application with 3 µM GABA. (See Figure 4.) By using the Lock Compound Robot function of PatchXpress Commander 1.6, the bicuculline incubation time was identical for each cell. GABA-induced currents returned to their baseline levels after washing out the bicuculline using the dedicated wash station of the PatchXpress 7000A System.
Benzodiazepines (BZD), one of the most commonly prescribed therapeutics in the treatment of panic disorder, insomnia and epilepsy, allosterically potentiate GABA currents by increasing the GABA-gated Cl- conductance. This decreases the GABA concentration needed to elicit half-maximal channel activity (EC50). GABA currents were repeatedly induced by adding 1 µM of GABA to the cells. L-tk cells were then incubated with benzodiazepine agonist CL218872 for 15 seconds before co-application with 1 µM GABA. CL218872 augmented the GABA-mediated currents (Figure 5) and the GABA-induced currents returned to baseline levels after washing out the CL218872.
HEK293 cells endogenously expressing ASIC receptors were another class of ligand-gated ion channels studied. Cells were voltage-clamped at a holding potential of -60 mV. Fast reduction of extracellular pH from 7.4 to 4.4 evoked rapidly desensitizing inward currents. The currents could be reversibly inhibited by adding 100 µM amiloride, a selective antagonist of ASIC. (See Figure 6.)
Ligand-gated ion channels represent a large, evolutionarily related group of intrinsic transmembrane proteins that open in response to binding a chemical ligand. These ion channels are characterized by a fast activation phase followed by a slower desensitization phase. To accurately record the activation phase, a system with a rapid solution exchange rate is necessary. The feasibility of using PatchXpress 7000A System for performing dose-response experiments with agonists and antagonists of ligand-gated ion channels was studied. The solution exchange and wash out time constants for PatchXpress 7000A System were of 19 ms and 420 ms, respectively.
In addition, the Lock Compound Robot function in PatchXpress Commander software allows the user to incubate cells with antagonist or modulator compounds. Locking the Cavro robot ensures each cell is treated the same, improves data consistency and enhances the applicability of the PatchXpress 7000A System to handle ligand-gated ion channels.
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