Investigating lysosomal membrane proteins using SSMbased electrophysiology: Improving amplification andaccessibility
Andre Bazzone, Maria Barthmes, Rocco Zerlotti, Nina Brinkwirth, Cecilia George, Niels Fertig, Alison Obergrussberger
Nanion Technologies, Munich, Germany

1

www.nanion.de

Contact: ali@nanion.de3

Introduction

Electrophysiological properties oflysosomal TMEM175

TMEM175 is a novel, constitutively active ion channel involved inregulating lysosomal pH and autophagy. Mutations in this gene impair
normallysosomal

andmitochondrial

function,

therebyincreasingaggregation of insoluble proteins such as phosphorylated α-synuclein,leading to symptoms typical of Parkinson’s Disease (PD). Consequently,
TMEM175 demonstrates significant potential as a key player in thetreatment of PD. The lack of specific pharmacological tools has
hampered further investigation into the exact role of TMEM175 in normal
lysosomal function and pathological processes. Here, lysosomes from a
TMEM175 stable cell line were measured on instruments employing solid
supported membrane-based electrophysiology (SSME), the SURFE2R N1

Left: We measured different cations and found that TMEM175 conducts K+, Rb+, and Cs+, but not Li+, Na+or choline+. Center: A concentration sequence for K+ reveals two permeabilities, depending on the K+
concentration range. Right: H+ concentration jumps reveal that TMEM175 conducts H+ with highpermeability. We found an average permeability ratio between protons and potassium of PH/PK=48.500.

and SURFE2R 96SE.

2SSM-based electrophysiology – How it
works

SSME relies on the adsorption of any membrane,
native, cell culture-derived or organellar, to a lipidcoated electrode, i.e the solid supported membrane,
and the direct current read-out caused by thecapacitive charging of the membranes when thesubstrate is applied via fast solution exchange.

Left: K+ flux through TMEM175 is downregulated to 30% of Imax upon acidification on both sides of themembrane, with a pK of 7.0. Below pH 4.5, K+ flux is completely blocked, likely due to competition with
H+ fluxes. Right: In the presence of a pH gradient, keeping the cytosolic pH constant at pH=7.5 andonly acidifying the lysosomal pH, K+ flux through TMEM175 is stable over a broad pH range.
Consequently, downregulation with a pK of 7.0 is due to effects of the cytosolic pH.

The SSM itself consists of a lipid monolayer on top of athiolated gold coated sensor chip. One important
advantage compared to patch-clamp is the large
sensor size of up to 3 mm. This allows the measurementof about 109 transporters at the same time and a>1000-fold amplification of the currents compared withconventional patch clamp, allowing for themeasurements of low-conducting membrane proteins,
such as transporters.

4The fact that also intracellular membranes can beaccessed by SSME means that ion channels andtransporters located in these membranes can becharacterized using a more native preparation.

Pharmacology of lysosomal TMEM175

SURFE2R 96SE

SURFE2R N1

We tested four tool compounds on their ability to modulate K+ flux through TMEM175 in our SSME assay.
We employed the HTS platform SURFE2R 96SE, capable of recording up to 10,000 data points per day.
Zinc and 4-AP blocked TMEM175 whereas DCPIB and arachidonic acid enhanced TMEM175. With SSME,
also H+ conductivity assays are possible and the effects of native lysosomal pH gradients on compound
potency may be investigated.

5In SSM-based electrophysiology a substrate gradient established by a fast solution exchange is themain driving force. The transport of charged substrates or ions into the liposomes or vesicles generates amembrane potential. This potential can be detected via capacitive coupling between the sample
membrane and the SSM on the gold layer of the sensor. In short: The change in membrane potential
due to electrogenic transport is measured. At some point the membrane potential equals the chemical
driving force and the transport process comes to a halt. This is why any current measured with SSMbased electrophysiology is transient. The peak current amplitude reflects the transporter activity understeady-state conditions. Since the current decay is fast, one measurement takes only one second. Dueto the high stability of the SSM, multiple measurements can be performed using the same sensor anddifferent buffer conditions to determine kinetic parameters such as EC50, IC50 or even rate constants.

Cells kindly provided by SB Drug Discovery.

Conclusions•Functional and pharmacological investigations of TMEM175 were performed on lysosomes purified
from HEK293 cells overexpressing TMEM175, kindly provided by SB Drug Discovery.

•SSM-based electrophysiology enables the characterization of cation and H+ fluxes through TMEM175
and efficient compound screening, compatible with an HTS environment.

•This technology was developed for the characterization of intracellular target proteins which arebarely accessible via patch-clamp, but also for transporters, that are too slow for detection via
conventional electrophysiology.