Two microelectrode voltage clamp (TEVC) of Xenopus oocytes Introduction 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-microelectrode voltage clamp (Fig. 1). The oocyte is impaled by two glass micropipettes, 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 (amp1). 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 record whole cell ionic currents conducted by K+ channels heterologously expressed in Xenopus oocytes (Fig. 3) using the two-microelectrode voltage clamp technique. A Geneclamp 500 amplifier will be used to record currents in oocytes that were injected 3 days ago with cRNA encoding Kv11.1 (hERG) channels. Data acquisition is performed using a personal computer and an analog-to-digital (A/D) interface. Electrophysiological measurements Fabrication and testing resistance of glass micropipettes Microelectrodes are pulled from 1.0 mm o.d. borosilicate glass tubing using a Flaming/Brown micropipette puller. Backfill pipettes with a 3M KCl solution using the special syringe needle and place pipettes in storage jar until ready for use. Place one electrode into each of the two holders, making sure that Ag/AgCl wire makes contact with KCl solution in the pipette. Insert holders into the two headstages of the Geneclamp 500 amplifier (Fig. 2). Fig. 2: GENECLAMP 500 amplifier: Zero1, Zero2 offsets R1 R2 headstages Virtual ground headstage (bath clamp amplifier)Using the micropositioners, immerse tips of both electrodes into the KCM211 extracellular solution within the oocyte chamber. Look through the compound microscope to visualize the tips of the electrodes. Break the tip off the pipettes with fine forceps until a tip resistance of ~1 MΩ is achieved. To determine resistance of pipette #1, depress “R1” on DC METER; for pipette #2, depress “R2” on DC METER. Press “Zero 1” and “Zero 2” buttons on amplifier; this will remove voltage offset; panel meter should now read “0 mV” for each electrode. Measurement of resting membrane potential Initial settings of amplifier: MODE: “SETUP” DC METER: “V1” and “I2/V2” SCALED OUTPUT: “I2” FREQ (low-pass filter): “500”; GAIN: “x1” Impale oocyte with both microelectrodes. DC meter will read resting membrane potential (in mV) for electrode #1 (V1) and #2 (V2). The numbers should be about the same and will vary from -40 to -70 mV for healthy oocytes. Voltage clamp (whole cell currents) Now switch MODE to “VOLTAGE CLAMP” VOLTAGE CLAMP controls should be as follows: GAIN (controls loop gain of voltage clamp): “9k” STABILITY (introduces phase lag into feedback loop): “200 µs” HOLDING POTENTIAL (dial): off – full counter clockwise The DC meter will now display the holding potential in mV (V1) and the holding current in µA (I2). The reading for I2 is the current required to “clamp” the membrane potential to V1. A virtual ground headstage amplifier is used to actively clamp the bath potential to zero. Data acquisition and analysis software PCLAMP software will be used; CLAMPEX for data acquisition and CLAMPFIT for analysis. In addition, you will use ORIGIN software to plot and analyze current-voltage relationships. Lab Instructors will walk you through the basics of using these programs. ………………………………………………………………………………………………………………… Fig. 3: Xenopus frog and oocytesLab Exercise: biophysical properties of wild-type and mutant hERG K+ channels You will be supplied with oocytes injected with cRNA encoding wild-type and two mutant forms of hERG. The mutant channels contain a single point mutation (introduced by site-directed mutagenesis) that alters one or more biophysical property of the channel. Include the following data in the RESULTS of your LAB REPORT: 1. Under current clamp (SETUP MODE), record resting membrane potential of the oocyte._______ mV 2. Under VOLTAGE CLAMP MODE, record currents elicited in response to test voltages that range from -130 mV to +40 mV, applied in 10-mV increments from a holding potential of -80 mV. First use a pulse duration of 0.3 sec; next use a pulse duration of 5 sec. Measure currents at the end of each pulse and plot the “NORMAL” current-voltage (I-V) relationships for both sets of data. Pulse protocols: “hERG 300ms IV” and “hERG 5s IV” 3. Measure peak “tail” currents (Itail) and plot as a function of test voltage (Vt) for both sets of data. These plots define the voltage dependence of channel activation. Fit the relationship with a Boltzmann function to determine the half-point for activation (V1/2) and the slope factor (k, a measure of steepness of the relationship; k = RT/zF): Itail /Itail-max = 1/{1 + exp[(V1/2 – Vt)/k]} For 0.3 s pulses V0.5: ________mV; k: ______mV For 5.0 s pulses V0.5: ________mV; k: ______mV 4. Under VOLTAGE CLAMP MODE, record currents using a “FULLY-ACTIVATED I-V” protocol. From a holding potential of -80 mV, apply a prepulse to +40 mV for 1 sec, followed by test pulses that range in voltage from -130 mV to -10 mV, applied in 10-mV increments. Measure peak tail currents and plot as a function of test potential. Measure the reversal potential (Erev) of the I-V relationship. Pulse protocol: “hERG fully activated IV” 5. Estimate the time- and voltage-dependent contribution of hERG current to a cardiac action potential. Pulse protocol: hERG_AP” 6. Repeat steps 1-4 using oocytes injected with cRNA encoding the two different mutant hERG channels. Issues to consider when writing Discussion section of LAB REPORT: a) At what potential does hERG current first appear to activate, and how does it compare to the resting membrane potential of the oocyte when first impaled by an