Application Note

Organelles:
Tools:

Mitochondria
Port-a-Patch

Recordings from mitochondria and mitoplasts on Nanion’s Port-a-Patch
The electrophysiology team at Nanion Technologies GmbH, Munich.
Mitoplasts courtesy of Dr. Shin-Young Ryu and Professor Shey-Shing Sheu at the University of Rochester, USA.
Mitochondria courtesy of Dr. Valentin Gribkoff, Dr. Armando Signore and Dr. Steven
Dworetzky, Knopp Neurosciences Inc., Pittsburgh, PA, USA.
Summary

Results

Mitochondria are often referred to as the “power house”

Mitochondria play a central role within eukaryotic cellsof the cell since they are responsible for making mostproviding the cell with energy. They are generally rod-of the cell’s energy supply in the form of adenosine

shaped and have an outer and inner membrane. Figure

triphosphate (ATP). In addition to providing the cell with

1 shows a schematic representation of a mitochondrion.

energy, mitochondria are thought to have roles in cellsignalling, cellular differentiation and apoptosis1. They
have also been implicated in the pathophysiology ofneurodegenerative disorders such as Parkinson’s Disease2, and may also play a role in diabetes3 and in theageing process4.
Mitochondria are usually rod shaped and range insize from approximately 1 - 10 µm. They have an outermembrane and a highly folded inner membrane. The
outer membrane is highly permeable and contains oneof the most well studied mitochondrial proteins, thevoltage-dependent anion channel (VDAC). The inner membrane contains many ion channels, including
the Ca2+ uniporter, a KATP channel, the Ca2+-activated

K+ channel (KCa) and the inner membrane anion chan-nel (IMAC)(for brief review see ref. 5). To study themitochondrial inner membrane using the patch clamp
technique, mitoplasts were formed. This is a process
whereby the mitochondria are swelled, thus rupturing theouter membrane and exposing the inner membrane.

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Figure 1: Schematic representation of a mitochondrion showing outer andinner membranes. (Adapted from http://micro.magnet.fsu.edu/cells/
mitochondria/mitochondria.html)

Application Note
We were able to successfully capture and record from
intact mitochondria using the Port-a-Patch. Figure 2 shows
the current response to a voltage ramp from -120 mV to+100 mV over 500 ms from a mitochondrion.
20 pA
100 msFigure 3: Single channel events recorded from mitoplasts on the Port-aPatch. Currents were elicited using a voltage ramp protocol, the portion
here shows from 0 to -80 mV over 1 s. Single channel events are seen as
small downward deflections at voltages more negative than -20 mV0Besides intact mitochondria we were also able to record
from mitoplasts. The recording in Figure 3 shows single
channel events from a mitoplast. A voltage ramp protocolwas used in the cell attached configuration from 0 mV to50pA-80 mV over 1 s. Inward currents can be seen at voltages

100ms

more negative than approximately -20 mV.

Figure 2: The current response of a mitochondrion to a voltage ramp
from -120 mV to +100 mV over 500 ms approximately 30 secs after
application of 100 µM Ca2+ on the external surface. There is a small “bump”
that appeared on the ramp at approximately -55 mV. It is not known
whether the mitochondrion, however, was in the “on-cell” or “wholecell” configuration. It was not determined whether this is an inward Na+ orCa2+ current at negative potentials, or an outward K+ (or other positivelycharged ion) at positive potentials.

In conclusion, we successfully captured and recorded

References

Methods

1. Duchan, M.R., 1999. J. Physiol. 516.1: 1-17

from mitochondria and mitoplasts using the Port-a-Patch.
Both macroscopic and single channel events could beelicited using a voltage ramp.

Organelles

2. Shapira, A.H.V., et al., 1998. Ann Neurol. 44(Suppl. 1):

Intact mitochondria were acutely isolated from liver at

S89-S98

Knopp Neurosciences. Mitoplasts prepared from mito-

3. Sivitz, W.I., & Yorek, M.A., 2010. Antioxid. Redox Signal.

chondria isolated from rat heart were prepared by Dr.

12: 537-577.

Shin-Young Ryu at the University of Rochester, USA.

4. Bratic, A., & Larsson, N-G., 2013. J. Clin. Invest. 123(3):Electrophysiology

951-957
5. O’Rourke, B. 2000. J. Physiol. 529.1: 23-36

Patch clamp recordings were conducted on the Port-aPatch using NPC-1 chips with high resistance either usingan Axon Axopatch 200B or a Multiclamp 700B amplifier.

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