- Organellar Transporters and Ion Channels How to access their electrophysiology by using the2
SURFE R technology and Planar Patch Clamp

1Nanion Technologies GmbH,Gabrielenstrasse 9, Munich
Germany
Info@nanion.de

References
Schulz P., Dueck B., Mourot A., Hatahet L., Fendler K., Biophys. J. 2009 97, 388–396
Watzke N., Diekert K., and Obrdlik P., Biochemistry 2010 49 (48), 10308-10318
Barthmes M., 2015 Dissertation at Nanion Technologies GmbH
Kirichok, Y. ; Krapivinsky, G. ; Clapham, D. E., 2004 Nature 427, 6972, 360-364
Borecky, J. ; Jezek, P. ; Siemen, D., 1997 J. Biol. Chem. 272, 31, 19282-19289

Maria Barthmes1, Tom A. Goetze1, Stephan Holzhauser1, Melanie Giesler1, Christian Patzig1, Raphael Bondes1, Timo Stengel1, Marius Vogel1, Johannes Stiehler1, Andrea Brüggemann1,Michael George1, Niels Fertig1

Higher Trough-put SURFE2R N96

Abstract

Decreasing blocker concentration

Electrophysiological characterization of ion channels from organellar
membranes is vastly restricted due to limited accessibility. Even moreso the direct measurement of transporter activity in these membranes
is extremely challenging, because noise usually exceeds signal sizedue to comparably low transport rates.
Here we present electrophysiological data of the ADP-ATP exchanger(ANT) from purified inner mitochondrial membranes (IMM) acquiredwith the SURFE2R technology which is based on a solid supported

membrane approach. By measuring the total current of a large
number of transporter molecules triggered simultaneously, the signal
becomes available for pharmacological studies.
Also we show data from mitochondrial BKCa channel to demonstrate
how planar patch clamp technology facilitates the study of organellar
membrane ion channels.

The SURFE2R Technology

ANT and Respiratory Chain Complexes
from IMM preparations

Surface Electrogenic Event Reader (formerly IonGate)
Solid Supported Membrane (SSM) based biosensors A gold electrode of 3mm diameter is functionalized by a thiol- and a lipid layer. This allows toimmobilize membrane fragments or liposomes containing the target
transporter on the sensor.

Adenine Nucleotide Translocator (ANT)

Accumulated transport current
The protein on the sensor is synchronously activated by a fast perfusion
system. The transport of charged substrates over the membrane generates
an electrical current (like capacitor plates). It’s measured as a sum signal of
all the protein on one sensor, thereby enabling a high amplification.

RC Complex I

the future…
SURFE2R 96 SE

Planar Patch Clamp - from Port-A-Patch to SyncroPatch 384PE referenceelectrode

SURFE2R N1

preparation of agar
salt bridge (3M KCl)measureelectrodereferenceelectrodeSensor preparation
 3 mm gold surface
 30 min 0.5 M 1-octadecane-thiol
 1 µl 7.5 µg/µl DPHPC loading buffer added
 8 µl sonicated liposomes
 centrifugation 1 h, 2200 g

PepT1

measureelectrode

The Search of the Putative Mitochondrial BKCa channel

Two modes: ADP-ATP exchange andtransport separately. Preparations frompigheart
mitochondria
innermembrane.
Exampletraces:activation of ANT in the presence ofATP or ADP.

Does KCNMA1 gene not only code for KCa1.1 (plasma membrane) or also
mitochondrial BKCa?

Respiratory chain complexes (I, II+III, IV, V)

 Preparation of mitoplasts (2-5 µm) and mitochondria from HEK293
 mito-tracker red stained, OMM as „cap“ structure [Kirichok et al.] control for OMM, electroformation of liposomes + intact mitochondria
bilayer measurements, large activities, VDAC

A-C macroscopic current + single channel events largely attributed to Clchannel IMAC ~100pS (Boreckyes et al.)
 Propranolol: IMAC inhibitor, Cl- replaced by MSA- methane sulfonic acid
 NPC-chip: <1 µm aperture, R: 10-15MOhm

IIV

Oxidation of NADH leads to transport offour protons across the membrane. The
electrons
aretransferredtoubiquinone. The signal is generated byaddition of NADH.

Oxidation of succinate to fumarate.
The electrons are transferred tocytochrome c via complex III whilefour protons are transported to theintermembrane space. Signal isevoked by oxidized cytochrome c,succinate must be provided.

VII+III
A-B Four different categories of conductance: 60 pS, 120 pS, 190 pS, 370 pS
mitoplasts from wt BK+/+ and mutant BK-/- mice, ionic conditions allowed
potassium currents to flow only

Reduction of oxygen by consumptionof electrons from cytochrome c andtransport of two protons to theintermembrane space. Signal is evokedby reduced (ascorbate) cytochromec.

Complex V generates ATP from ADP
plus phosphate. Here it works reverse
asATPase. Therefore
signalisgenerated by addition of ATP.
Membranbes were treated with 5µM
BKA (Bongkrekic acid = specific ANTinhibitor)

Statistical analysis
A 4 channel appearances in wt BK+/+ and mutant BK-/mitoplasts
B Conductance of 190pS in different conditions, NS11021:
increased appearance by >10%, Paxillin reduced by >20%.
n-experiments: 27 (BK+/+), 26 (BK-/-)
Analysis of single channel characteristics
A current histogram at 80mVB linear IV at positive voltages, conductance of 190pS
C-D dwell time of closed and open state