Functional Characterization of Procaryotic
NCX by Solid Supported Membrane Technology
Maria Barthmes1, Jun Liao2, Youxing Jiang3, Andrea Brüggemann1
1Nanion Technologies GmbH, Gabrielenstr. 9, 80636 Munich, Germany, 2 Shanghai Tech University, 3 University of Texas, UT Southwestern Medical Center, Dallas, USA

MethodAbstract
Sodium/calcium exchangers (NCXs) are membrane transporters
which play an important role in Ca2+ homeostasis and Ca2+
signaling. These proteins have been implicated in various Ca2+
dependent physiological processes such as neurotransmission,
skeletal and smooth muscle contraction, cardiac contractility andapoptosis. Here, we use solid supported membrane (SSM)
technology to performed functional analysis of NCX_Mj, anarchaeal NCX isoform which has recently been crystalized. Using
this approach we characterized the substrate affinity, ion specificity
and inhibition by divalent cations of this archaeal exchanger
protein. In conclusion we found a high functional similarity of
NCX_Mj and eukaryotic NCX isoforms, although the prokaryotic
protein lacks a large regulatory domain. Furthermore our results
demonstrate that SSM based technology is well suited for state-ofthe-art functional characterization of transporter proteins.

SSM Technique
SSM based biosensors
A gold electrode of 3 mm diameteris functionalized by a thiol- and alipid layer. This allows to immobilize
membrane fragments or liposomes
containing the target transporter on
the sensor.

Accumulated transport current
The protein on the sensor issynchronously activated by a fast
perfusion system. The transport ofcharged substrates over the membrane generates an electricalcurrent. It’s measured as a sum
signal of all the protein on one
sensor, thereby enabling a highamplification.

Platform
The experiments were performed on the SURFE2R
N1, a system for SSM based transporter recordings.
It’s including a specialized perfusion system andperforms amplification and data recording.

Inhibition
Inhibition of NCX_Mj bydivalent cations.
(A) Na+ and Ca2+activated
NCX_Mjcurrents are inhibitedby 100 µM Cd2+.
(B) Concentration response curves of Cd2+,
Mg2+ and Mn2+.
(C) Reversibility of Cd2+
inhibition. After activation of INa/Ca by
Ca2+ and subsequent
block by Cd2+ only
partialrecoveryofINa/Ca upon washout ofCd2+ was observed.
(D) Ca2+ affinity of NCX
in the absence andpresence of 100 µM or
4 mM Mg2+. In thepresence of 4 mM Mg2+, Ca2+ induced NCX_Mj activity is shifted towards
higher Ca2+ concentrations indicating competition between Mg2+ and Ca2+.

The NCX_Mj protein wasreconstituted
inliposomes, which wereimmobilized on the SSM
sensor. The NCX current isgeneratedbytheexchange of Na+ versus
Ca2+, usually with anelectrogenic
stoichiometry of 3 Na+ to 1 Ca2+,
leading to a net cation
flux.
Toinduce
anoutward flowing INa/Ca,
lipid vesicles were loadedwith 140 mM Na+ andINa/Ca was activated byapplication of calciumcontaining buffer (100 µM
free Ca2+). Alternatively
liposomes were loadedwith 20 mM Ca2+ andtransport activated with
10 mM Na+.

Apparent Affinity
Apparent Ca2+ and Na+affinity of NCX_Mj (blackcircles) and human NCX1(greycircles).
Control
experiments (triangles) wereperformedwith
empty
liposomes/parental HEK cell
membranes (A) Apparent
Ca2+ affinity was measured
using vesicles loaded with
140 mM Na+. Outward INa/Ca
wasactivatedbyapplicationofsolution
containing differing freeCa2+concentration
asindicated(n=5).
(B)
Apparent Na+ affinity wasdetermined using vesicles
loaded with 20 mM Ca2+. Inward INa/Ca was activated by application ofvarying Na+ concentrations as indicated. (C+D) Representative current
traces of outward/inward NCX_Mj INa/Ca, evoked by different Ca2+/ Na+concentrations, respectively.

Selectivity