Application NoteCells:
Tools:

Mitoplasts
Port-a-Patch®

Mitoplasts from HEK cells on Nanion‘s Porta-Patch®
The electrophysiology team at Nanion Technologies GmbH, Munich.

Summary
Mitochondria play an important role in metabolism bynatant as good as possible. Mitochondria can now beproviding the cell with ATP due to oxidative phosphoryla-

stored for a day on ice. For the preparation of mitoplasts

tion. But they are also supposed to be involved in apop-

resuspend pellet in 100 µl of swelling buffer (mix 160 µl oftosis and cytoprotection.

hypotone buffer with 40 µl of hypertone buffer). Incubate

The mitochondrial membranes contain a large number offor 7 min at room temperature. Stop process with 20 µl ofion channels, transporters and pores. However the physio-

hypertone buffer. Mitoplasts can now be used for mea-

logical role of mitochondrial channels is largely unknown.

surements. Keep on ice.

The main channel of the outer membrane is VDAC, a wellmost prominent are the permeability transition pore (PTP)
and the inner membrane anion channel IMAC. Different
potassium channels are described as well (mitoBK, mitoKATP and Kv1.3) and a number of calcium conducting
channels and receptors (for example MCU).
To study the channels of the inner mitochondrial mem-Electrophysiology
Mitoplasts can be measured with high success rates byusing NPC1 chips with a resistance of 10-15 MOhm andNanion’s standard electrophysiological solutions. A mix ofdifferent conductances was observed under these conditions. Some examples are displayed in fig.1 and fig. 2

brane the outer membrane can be stripped of by os-600

motic swelling. This method was used for the examples of500electrophysiological measurement with the Port-a-Patch

400

shown below.

300

Preparation of mitoplasts
Use two 10 cm dishes or a T75 flask confluent with HEK
293. Harvest with 2 ml trypsin for 3 min. Add 2 ml of serum
containing media, and spin down cells at 1200 g for 5
min. From this point every step has to be done at 4°C.
Resuspend pellet in 800 µl of IBc buffer. Potter by hand 4050 times. Wash the potter with 800 µl. Spin down the cells
at 700 g for 10 minutes at 4 °C in 2 ml Eppendorf tubes.
Put the supernatant in two fresh 1.5 ml tubes. Centrifuge
at 3000 g for 15 min. Resuspend the pellet in 200 µl andcentrifuge again at 3000 g for 15 min. Remove the superDownload more Application Notes from www.nanion.de

200100mV-100-50-100

50100pA

described anion conductance. In the inner membrane

Figure 1:
Voltage ramp from -100 mV to 100 mV in standard solutions. Resulting
current is a mix of macroscopic and single channel currents. Different
conductances can be identified.

Application NoteThe Port-a-Patch allows exchanging the external solution as well as the internal solution. Such an experiment isshown in Figure 4.
10050mV-100-5050100-50-100

K internal
Cs internalpA

Figure 2:
Individual traces of mitoplast recordings at continuous voltage (-20 mV)in standard solutions are shown in this figure. Ion channels with different
single channels properties can be found.

Figure 4:
Mitoplast current with different internal solutions. Blue trace is with internal cesium, green with potassium.

A chloride conductance was identified by replacing external chloride by MSA, resulting in a reduced outward
current (see Figure 3).

To identify potassium channels, a chloride free external
solution was used in figure 5. The single channel on top of
the macroscopic current has a conductance of of about
300 pS, and therefore could be the mitoBK.

600
400
20050 msmV

100-5050-200100

80pA-10040 pA

12060pA

40

internal chloride
without internal chloride

Figure 3:
Chloride conductance. Black trace shows current with external chloride, red without.

Methods

20s

Figure 5:
Step to +60 mV with internal potassium and chloride free external solution.
0.5

0.6

0.7

0.8

0.9

Nanion Standard Solutions:Internal K Solution : 50 mM KCl, 10 mM NaCl, 60 mM KF, 20

Solutions and buffermM EGTA, 10 mM Hepes /KOH, pH 7.2

Buffer for cell mitochondria isolation (IBc): Prepare 100 mlof IB by adding 10 ml of 0.1 MTris–MOPS (ph7.4 with MOPS)
and 1 ml of 0.1 EGTA/Tris (pH7.4 with Tris) to 20 ml of 1 M
sucrose. Bring the volume to 100 ml with distilled water.
Adjust pH to 7.4. Add proteinase inhibitor before use 1:2025.
Hypothone buffer: 5 K-HEPES and 1 CaCl2 (pH 7.2).

Internal Cs Solution : 50 mM CsCl, 10 mM NaCl, 60 mM
CsF, 20 mM EGTA, 10 mM Hepes /CsOH, pH 7.2
External Solution : 140 mM NaCl 4 mM KCl, 1 mM MgCl2,2 mM CaCl2, 5 mM D-Glucose monohydrate, 10 mM
Hepes /NaOH pH 7.4

Hypertone buffer: 750 KCl, 80 K-HEPES, 1 CaCl2 (pH 7.2).

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80636 Munich, Germany

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