Practical course
Functional characterization of voltage-dependent potassium
channels in Xenopus oocytes: Voltage clamp and Patch clamp
Preparation and supervision of the course:
Dr. Vitya Vardanyan, Institute of Molecular Biology NAS RA ([email protected])
Prof. Dr. Jürgen R. Schwarz, Institute für Molekulare Neurogenetik, ZMNH, Hamburg,
Germany ([email protected])
Event duration and location:
The internship takes place in the Institute of Molecular Biology, at the laboratory of
Molecular Neuroscience (3th flour, room 34). Hasratyan 7, Yerevan, Armenia
From 01.06 - 05.06 2015,
Duration: 9:30 - 17:30
Course overview
The aim of the practical course is to provide knowledge in molecular and cellular
aspects of modern neuroscience research with emphasis on practical work with
electrophysiological methods. For this purpose, the function of the voltage-dependent
potassium channel KCNQ1 involved in human Long QT syndrome will be investigated in
the Xenopus laevis expression system. In this year the patch clamp method will be
additionally introduced to the participants of the course.
KCNQ1 channels plays a crucial role in the repolarization of cardiomyocyte action
potential. In non-excitable tissues KCNQ1 channels are responsible for distribution of
potassium ions, control of endocytosis and exocytosis of specialized cells. Therefore, it is
not surprising that many mutations have been found in KCNQ1 channel genes that are
linked to a number of inherited diseases (such as Long QT syndrome, Jervell LangeNielson syndrome, Diabetes mellitus type two etc.).
During the practical course of this year KCNQ1 potassium channels will be
expressed in Xenopus laevis oocytes via microinjection of corresponding mRNA into
cytoplasm of the cells. Subsequently, the biophysical properties of the heterologously
expressed channels will be investigated via Two-Electrode Voltage-Clamp (TEVC) and
patch clamp techniques.
Seminars: In the morning of each day theoretical backgrownd of the techniques, function
of the ion channels and related pathologies will be introduced in the form of seminars by
Prof. Dr. J.R. Schwarz. To make the course more effective it is desirable that participants
to read corresponding literature beforehand in order to get involved in active discussion
during the course.
Practical part
The practical part begins with the surgical removal of oocytes from the African
clawed frog Xenopus laevis (performed by trained personnel). Subsequently, the selected
oocytes will be injected with 20-50 nl of mRNA encoding KCNQ1 channels. After incubation
at appropriate conditions corresponding channels will be expressed after 2-3 days.
Functional properties of expressed channels will be investigated with two-electrode
voltage-clamp technique and patch-clamp techniques and corresponding analysis will be
performed.
Each participant will have the opportunity to record from oocytes by himself or
herself during the course.
Computer programs
The following table lists acquisition and analysis software used during the course.
Program
Application
Clampex9.2 (axon instruments)
Pulse (HEKA)
Instrument control and data acquisition
Clampfit
Data analysis (amplitude measurement,
kinetic analysis, curve fitting)
Pulse, Pulsefit (HEKA)
Igor pro (wavemetrics)
Kaleidograph (Synergy Software)
Data analysis (amplitude measurement,
kinetic analysis, curve fitting)
Schedule
The course will start at 9:30 in the laboratory of Molecular Neurosciences at IMB NAS RA
(room 34, 3th flour). There will be a break at around noon. The practical part will start in the
afternoon. Depending on the progress of experiments, certain course days may last longer.
Day 1. Monday, 01.06.2015
In the morning
- Overview seminar: Ion channels – Basic properties of excitable membranes (Prof.
Schwarz)
In the afternoon
- Operation of frog and preparation of oocytes (Dr. Vardanyan)
- Enzymatic defolliculisation of oocytes
- General introduction to working conditions and equipment in the electrophysiological
laboratory
Day 2. Tuesday, 02.06.2015
in the morning
- Lecture-seminar: Sodium and potassium channels (Prof. J.R. Schwarz)
in the afternoon
- Injection of mRNA into oocytes
- Measurements of oocytes already injected with mRNA (prepared beforhand).
Day 3. Wednesday, 03.06.2015
in the morning
- Lecture-seminar: Ion channels and diseases (Prof. J.R. Schwarz)
- Measurements on RNA - injected oocytes
- Injection of RNA into oocytes
in the afternoon
- Measurements of control and mRNA - injected oocytes
- Patch-clamp of oocytes
Day 4. Thursday, 04.06.2015
full-time
- patch-clamp measurements of mRNA - injected oocytes
- preliminary data analysis
Day 5. Friday, 05.06.2015
- concluding remarks
- end of the course
Methods
Frog operation and harvest of oocytes
1. Dissolve the anesthetic MS222 in dechlorinated water in an induction tank at
a dose of 500mg/L.
2. Place frog in tank shallow enough to ensure that the frog will not drown. Leave in tank
for approximately 15 minutes.
3. A clean lab coat and sterile gloves should be used during frog operation.
4. Once the animal is anesthetized, remove it from the solution and place the frog on the
non-absorbent side of a clean/unused pad and keep the skin moist.
5. Make a small, paramedian, coelomic incision (0.5-2cm) through the skin and muscle
layers on either the right or left side of the abdomen.
6. Exteriorize a portion of the corresponding ovary with sterile forceps and remove
necessary amount of oocytes by cutting with scissors and place it on 10 cm dish on ice
containing OR2 solution.
7. Replace the remaining ovarian tissue into the coelomic cavity and check for
hemorrhage.
8. Both tissue layers must be first closed using a absorbable suture (e.g. vicryl). The skin
must be closed separately.
9. After surgery, the frog should be gently placed in dechlorinated water in a shallow
recovery container with a level of water that does not cover the nostrils of the frog (to
reduce risk of drowning).
10. Once the frog is active and mobile, it can be placed in a tank by itself or with a small
number of post-operative frogs.
Defoliculation of oocytes
1. After mechanical trituration of ovary tissue by Drummond Nr. 5 forceps transfer the small
pieces of ovary into a 50 ml Falcon tube containing 15-30 ml OR-2 solution with addtion of
collagenase Type A (Roche) at concentrations 2-3µg/µl.
2. Shake gently at room temperature for 1.5 to 4 hours till more than 50% of oocytes are
clean.
3. Pour off most of the collagenase solution and wash the oocytes 4 to 6 times with fresh
OR-2 to ensure complete removal of the collagenase A.
4. Do this by adding some OR-2 solution into the wells of sterile 10cm dish.
5. Select stage V-VI oocytes in necessary amount and incubate these oocytes overnight at
17°C in 10cm petri dish with incubation solution containing gentamicin and carbone source.
Injection of oocytes
1. Next day check the oocytes visually and remove those that are broken and have no
distinct boundary between the dark brown animal pole and the light green/yellow vegetal
pole. If the animal pole has a mottled appearance, the oocyte is unlikely to survive for long
and so should not be used.
2. Make pipettes for oocyte injection use 7" long glass capillaries from Drummond made
specially for manual microinjector.
3. Adjust the capillary on Narishige PC-10 puller and pull injection needles using 2-step
pulling program.
4. Using a fine forceps, break off the end of pipettes to have a tip diameter between 10 to
20 µm.
5. Bake the pipettes in metallic box for around 4h at 200°C to render them RNAse free.
Thereafter, they must always be handled only with gloves!
6. Holding the baked needle point up, fill it partially with RNase-free mineral oil and adjust
to the microinjector by squeezing out additional oil. They must be completely free of air
bubbles.
7. In order to load the pipette with RNA, insert the tip into the drop or RNA pipetted on
paraphin film or on 35 mm dish cover.
8. Inject the oocytes in special chamber filled with OR2 solution with 20-50nl RNA and
place them into 35 mm dish half-filled with incubation solution with gentamicin.
9. Incubate oocytes at 17oC up to 1 week and record when appropriate expression level is
reached.
10. Inject a sufficient number of oocytes for the required set of experiments. On the
following days of injection dead oocytes should be identified by careful inspection and
removed. One must assume that up to 20% of oocytes will not survive the injection. If
necessary, a fresh gentamicin containing solution should be replaced.
Recordings
The voltage clamp technique is a method that allows the measurement of ionic currents
through the membrane by fixation of the membrane potential to a certain desired value.
This is achieved by a feedback amplifier. The membrane of the oocyte is penetrated by two
microelectrodes, one for voltage sensing and one for current injection. The membrane
potential as measured by the voltage-sensing electrode and current is injected through
current electrode to reach a certain given command value of voltage. The electric signals
are amplified, digitized and controlled by computer via interfaces.
1. Switch on the set-up and leave 10-30 min, so that electrical circuits rich the stationary
thermal noise level.
2. Meanwhile prepare the recording pipettes by pulling capillary glass on Narishige PC-10
puller with appropriate program.
3. Brake the tips 10-15 µm diameter leaving preferable sharp endings to facilitate the
penetration.
4. Prefill the tips with the tip-agar-solution (3M KCl solution containing 1% agar) by melting
the agar via the suction using the syringe. Keeping the solution on 60oC water bath for
repeated use in the same day.
5. Backfill pipettes with 3M KCl solution.
6. Prepare the bathclamp circuit by placing the 3M KCl reservoirs and corresponding agarbridges.
7. Put electrodes into the holders and immerse them into the recording chamber filled with
recording solution (ND96). Measure the electrode resistance.
8. Place oocyte into the recording chamber, switch the amplifier mode on CC (current
clamp) mode and insert the electrodes into the cell. The display of the amplifier now should
read the membrane potential of the cell.
9. Switch the amplifier mode to voltage-clamp and give the holding potential from
appropriate icon of the software.
10. Run protocols that correspond to characteristic properties of the particular channel.
11. When the protocols are finished switch the amplifier mode to current-clamp mode, after
which you can safely remove electrodes from oocyte. Otherwise, it is possible to damage
the amplifier if it remains in voltage-clamp mode.
12. Save files in appropriate directory of your computer.
13. At the end of experiments carefully remove electrodes from the holders, clean
recording chamber from salt, put the agar-bridges into corresponding container, wash the
perfusion system with distilled water, bring the set-up to its start-up condition.
Solutions
Oocyte Ringer 2 (OR2)
82.5 mM NaCl
2.0 mM KCl
1.0 mM MgCl2
5.0 mM HEPES
adjusted pH to 7.5 with NaOH, autoclave and store at 4° C.
For collagenase solutions add collagenase A to 2-4μg/μl (final volume 15-30ml)
Incubation (gentamicin) solution
75.0 mM NaCl
2.0 mM KCl
2.0 mM CaCl2
1.0 mM MgCl2
5.0 mM Na-Pyruvate
5.0 mM HEPES
adjusted pH to 7.5 with NaOH, autoclave or steril filter
add 50 μg/ml gentamicin and store 4° C.
Measuring solution (modified ND96)
59.5 mM NaCl
20.0 mM KCl
2.0 mM CaCl2
1.0 mM MgCl2
5.0 mM HEPES
adjusted pH to 7.5 with NaOH, autoclave or sterile filter and store at 4° C.
Tricain-solution: 0.6 g in 500 ml tab-water
Tip-agar-solution for electrodes: add 0.5g Agar to 50 ml 3 M KCl solution and boil.
Patch clamp solutions
Bath-solution for patches
70 mM K-citrate
20 mM KCl
2 mM EGTA
4 mM MgCl2
10 mM HEPES
adjusted pH to 7.4 with KOH, autoclave or sterile filter and store at 4° C.
Patch pipette solution
90 mM NaCl
2 mM KCl
1.8 mM CaCl2
10 mM HEPES
adjusted pH to 7.4 with NaOH, autoclave or sterile filter and store at 4° C.