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UP896787

Unit U22206 Cellular & Molecular Drug Targets Two-Electrode Voltage Clamp of Xenopus laevis Oocytes Workshop Overview In this laboratory session you will observe ion channel current recordings being made directly from post-injected Xenopus laevis oocytes using the two-electrode voltage clamp technique. You will gain an appreciation of how glass microelectrodes are fabricated for both injection and recording purposes, gain hands-on experience of microinjection of oocytes, and learn about the equipment used for making electrophysiological recordings. You will be provided with real data from recordings of an “unknown” ion channel and will be required to analyse the data and answer basic biophysical questions.

Your write up should be less than two pages of written text, plus the figures requested. Please submit your write-up via Turnitin on the CMDT Moodle site by 5 pm on Tuesday 16th March 2021.

Basics of voltage clamp The voltage clamp technique is used to measure ionic currents in response to precisely controlled changes in the transmembrane potential of an isolated cell. Large cells (e.g., frog oocytes) can be studied using two-electrode voltage clamp (Figure 1). The oocyte is impaled by two glass microelectrodes, one for voltage sensing and one for current injection. The transmembrane potential is measured by the voltage-sensing electrode (V1) connected to a high input impedance amplifier (Amp 1). This signal is compared to a command voltage generated by a computer at the input of Amp 2. The output of the high gain feedback Amp 2 is a current delivered to the cell interior by the second micropipette. This current is sufficient to force the transmembrane potential to equal the command voltage. The current delivered by micropipette 2 is monitored as “I2” via a current-to-voltage converter. In this lab exercise you will observe recordings of whole cell ionic currents conducted by an “unknown” ion channel heterologously expressed in Xenopus oocytes using the two-electrode voltage clamp technique. A Warner 0C-725C amplifier (Figure 2) will be used to record currents in response to changes in membrane voltage in oocytes that were injected 24 hours previously with cRNA encoding the “unknown” ion channel. Data acquisition will be performed using a personal computer and an Axon low-noise 1440A analog-to-digital (A/D) interface.

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UP896787 Fig. 2: Warner OC-725C oocyte clamp

Microelectrodes Microelectrodes are pulled from 1.0 mm (external diameter) borosilicate glass tubing using a Narishighe PC-10 vertical micropipette puller. The method used by this puller is to stretch the glass capillary vertically using the gravitational force of its own and added weights, thereby thinning the glass tubing until it breaks resulting in two glass microelectrodes with a tip diameter of ~1 µm and a tip resistance of 0.2 - 1 MΩ. Microelectrodes are backfilled with a 3M KCl solution using a special syringe needle and one electrode secured into each of the two electrode holders, making sure that Ag/AgCl wire makes contact and therefore electrical connection with KCl solution in the pipette.

Preparation of oocytes Ovaries are obtained from Xenopus laevis (Figure 3) killed humanely in accordance with regulations issued by the Home Office of the United Kingdom under the Animals (Scientific Procedures) Act, 1986. Oocytes are manually dissected into clumps of ~10 oocytes with forceps and washed in sterile, calcium-free, OR-2 solution containing (in mM) 82.5 NaCl, 2.5 KCl, 1 MgCl 2, 10 HEPES (pH 7.4 with NaOH). Oocytes are then rotated in OR-2 solution containing 2 mg/ml collagenase type II at room temperature for ~60-90 minutes to dissociate oocytes and remove the follicular layer (Figure 4). Oocytes are then washed with calcium containing ND96 storage solution containing (in mM) 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, 10 HEPES, 1% penicillin/streptomycin solution, 0.1% gentamycin (pH 7.6 with NaOH) and stored at 16 °C in a refrigerated incubator.

Fig 3: Xenopus laevis

Fig 4: Dissociated Xenopus laevis oocytes

Preparation and microinjection of cRNA Plasmid DNA containing the “unknown” ion channel to be expressed is linearised with a single cut restriction enzyme (PacI) following the terminating stop codon of the gene. Freshly linearised DNA is in-vitro transcribed into cRNA using the mMESSAGE mMACHINE® T7 transcription kit (Ambion, Life Page 2 of 14

UP896787 Technologies) and diluted to 0.2 ng/23nl in RNase/DNase free molecular grade water ready for injection into oocytes. Stage IV and V (mature) oocytes (Figure 4) are selected and injected with 23nl (equivalent to 0.2 ng) of cRNA using a positive-displacement, hydraulic Nanoject II injector and incubated in ND96 storage solution at 16°C for at least 24 hours to allow for ion channel expression at the cell membrane.

Typical Experiment Ionic currents will be recorded at room temperature with constant, gravity fed solution flow at 1-2 ml/min with a bath solution containing (in mM): 96 NaCl, 4 KCl, 1 MgCl2, 0.3 CaCl2, 10 HEPES (pH 7.6, tris-base). Data will be sampled at 5 kHz, filtered at 1 kHz and recorded using Clampex 10.1 software (Axon Instruments, Inc.). Currents will not be adjusted for leak or capacitance. Oocyte membrane potential will be held at -80 mV and step depolarised to potentials ranging from -120 mV to +60 mV in 10 mV steps for a duration of 1 s (Figure 5). This will be followed by a second voltage step to -30 mV for 0.5 s to visualise deactivating tail currents before returning to the holding potential. For selectivity measurements, NaCl will be isotonically substituted for KCl to give a final [K +]o of 4, 20 and 100 mM and a final [Na+]o of 96, 80, and 0.01 mM respectively. Liquid junction potentials have been calculated to be less than 4 mV and will therefore be ignored. Ionic currents will firstly be recorded using the above voltage protocol in bath solution containing 4 mM K+/96 mM Na+ (Figure 5). The solution will then be switched to one containing 20 mM K+/80 mM Na+ and allowed to equilibrate for 1-2 minutes before applying the voltage protocol again. This procedure will then be repeated after exchange of the bath solution again to one containing 100 mM K+/0.01 mM Na+.

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UP896787 Data Analysis and Results The responses of 6 oocytes will be recorded in each ionic condition and the steady state current measured for each voltage step. Mean steady state current ± SEM will be provided for each condition in the table below. Table 1. Mean steady-state current values (in µA) recorded in various extracellular conditions (in mM) at different voltage clamped potentials (in mV).

4 mM [K+]o/96 mM [Na+]o Voltage (mV) -120 -110 -100 -90 -80 -70 -60 -50 -40 -30 -20 -10 0 10 20 30 40 50 60

Mean (µA) -0.2 -0.18 -0.17 -0.12 -0.11 -0.04 0.05 0.15 0.27 0.46 0.67 1.04 1.4 1.9 2.46 3.18 3.99 4.91 6.01

SEM 0.09 0.07 0.09 0.09 0.11 0.14 0.12 0.17 0.18 0.31 0.42 0.55 0.76 0.85 0.96 0.98 1.11 1.29 1.32

20 mM [K+]o/80 mM [Na+]o Mean (µA) -0.95 -0.93 -0.88 -0.83 -0.76 -0.65 -0.42 -0.18 0.22 0.77 1.44 2.24 3.22 4.51 5.85 7.48 9.35 11.43 13.69

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SEM 0.22 0.29 0.28 0.29 0.22 0.28 0.36 0.39 0.32 0.48 0.54 0.71 0.84 0.95 0.99 1.11 1.26 1.48 1.52

100 mM [K+]o/0.01 mM [Na+]o Mean (µA) -4.85 -4.88 -4.77 -4.68 -4.42 -4.12 -3.64 -3.04 -2.23 -1.18 0.04 1.57 3.27 5.23 7.44 9.87 12.55 15.49 18.71

SEM 0.88 0.96 0.82 0.99 1.01 0.91 0.92 0.88 0.79 0.62 0.55 0.66 0.89 0.78 0.99 1.11 1.35 1.82 2.21

UP896787 Lab Report: Determining the properties of an ion channel current using basic biophysical concepts 1. Using the data in the table above plot the mean current-voltage (I-V) relationships for all extracellular K+ levels on the same graph using the SEM to generate error bars. Label the graph with axis labels, a key and provide a short figure legend underneath to accompany the graph. The figure and legend should be of journal quality. (8 marks)

Figure 1. Current-voltage relationships of extracellular K+ levels in oocytes. Steady-state current values of extracellular K+ ions increase at set voltage values (1s).

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UP896787 2. From the graph, determine the approximate reversal potential (Erev in mV) for the currents in each solution condition. You may need to adjust your scales to pinpoint the exact intersection. Note your answers (to 1 decimal place). (6 marks) 4 mM [K+]o/96 mM [Na+]o 20 mM [K+]o/80 mM [Na+]o 100 mM [K+]o/0.01 mM [Na+]o

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UP896787 3. Write a brief (...