Unlocking the (Reversal) Potentialof SSM Electrophysiology:
Transporter Stoichiometry with the SURFE2R N1Nathan ThomasHenzler-Wildman Lab
University of Wisconsin – Madison

Stoichiometry is a crucial determinant oftransporter function
LacY

PepTSa1 proton per lactose

5 protons per dipeptide(West and Mitchell, 1972)(Parker, Mindell, and Newstead, 2014)-160mV PMF can drive:-160mV PMF can drive:

400-fold lactose gradient*

10 trillion-fold dipeptide gradient**Assumes tight couplingMDR efflux pump EmrE is not tightly coupled

Multiple pathways – multiple stoichiometries?

How to measure transport stoichiometry:
Traditional Reversal Potential assay with GdX

How to measure transport stoichiometry:
Traditional Reversal Potential assay with GdX++++n𝐻𝑜𝑢𝑡 + m𝐺𝑑𝑚𝑖𝑛 ⇌ n𝐻𝑖𝑛 + m𝐺𝑑𝑚𝑜𝑢𝑡

How to measure transport stoichiometry:
Traditional Reversal Potential assay with GdX++++n𝐻𝑜𝑢𝑡 + m𝐺𝑑𝑚𝑖𝑛 ⇌ n𝐻𝑖𝑛 + m𝐺𝑑𝑚𝑜𝑢𝑡

Kermani, et al. 2018

How to measure transport stoichiometry:
Traditional Reversal Potential assay with GdX++++n𝐻𝑜𝑢𝑡 + m𝐺𝑑𝑚𝑖𝑛 ⇌ n𝐻𝑖𝑛 + m𝐺𝑑𝑚𝑜𝑢𝑡

𝑛𝜇𝑖 + 𝑚𝜇𝑠𝐸𝑟𝑒𝑣 =−𝐹(𝑛𝑧𝑖 + 𝑚𝑧𝑠 ሻKermani, et al. 2018

How to measure transport stoichiometry:
Traditional Reversal Potential assay with GdX++++2𝐻𝑜𝑢𝑡 + 1𝐺𝑑𝑚𝑖𝑛 ⇌ 2𝐻𝑖𝑛 + 1𝐺𝑑𝑚𝑜𝑢𝑡

𝑛𝜇𝑖 + 𝑚𝜇𝑠
𝐸𝑟𝑒𝑣 =−𝐹(𝑛𝑧𝑖 + 𝑚𝑧𝑠 ሻ
Kermani, et al. 2018

Issues with reversal potential assays• Technically difficult and time consuming• Need to ensure internal solutions are accurate• Different internal conditions require separate reconstitutions• Need fluorophores or radioactivity to monitor transport• Relatively large amounts of sample requiredCan SSM-electrophysiology be
used to measure stoichiometry?

Overview of an SSM-electrophysiology
experiment

2
SURFE R N1

Reversal Potential AssayAssay Scheme – Proton gradient drives
guanidinium transport

Assay Scheme – Guanidinium gradient drives
proton transportTransport current can be reversed

Transport current can be reversedPeak current does not reverse at publishedreversal potential
WT GdXPeak current does not reverse at publishedreversal potential

Integrate on-signal

Integrated signal agrees with publishedstoichiometry
WT GdXSignal is independent of protein concentration
WT GdX, LPR 150
WT GdX, LPR 400

No significant currents for empty liposomes ortransport-dead mutants
WT GdX, LPR 150
WT GdX, LPR 400
E13Q GdX, LPR 150
E13Q GdX, LPR 400“Empty” Liposomes

Transported Charge is a linear function ofchemical potential

Transported Charge is a linear function ofchemical potential
R2 = 0.998
x-intercept = 1.98 ± 0.06

Sensor internal solution can be changed• Reconstitute and prepare sensors at pH 7, 1 mM Gdm+
• Flow pH 6.7, 1 mM Gdm+ buffer over sensor• Monitor solution exchange until no current is observed – about 3 mL

• Repeat reversal potential assay• Internal buffer: 200 nM H+, 1 mM Gdm+
• External buffer: 100 nM H+, varied Gdm+

Sensor internal solution can be changed
Internal pH 6.7

R2 = 0.999
x-intercept = 2.02 ± 0.06

EmrE has variable stoichiometry

EmrE has variable stoichiometry

35 experimental conditions tested <3 hours, using <50 picomol protein

Advantages of SURFE2R Reversal Potential• No need for fluorescent probes or radioactive isotopes• Much higher throughput – hundreds of measurements in a day• Requires very little sample• 10-10 mol protein per sensor• Up to 100 measurements per sensor• Can measure multiple conditions on each sensor

Acknowledgements
Henzler-Wildman LabGrant Hisao
Vilius Karauskas
Adam Lewis
Peyton Spreacker
Andrea Killian

Nanion Technologies
Dr. Maria Barthmes
Dr. Andre Bazzone

Funding
NIH 1R01GM095839 and T32 GM007215
Nanion SURFE2R N1 Grant 2018

SURFE2R Reversal PotentialDerivation of reversal potential equation

Assay Scheme – Guanidinium gradient drives
proton transport

Thermodynamics of proton coupled drug efflux
H+H+H+H+H+H+H+H+H+H+Antiport++𝑛𝐻𝑜𝑢𝑡+ 𝑚𝐷𝑟𝑢𝑔𝑖𝑛 ⇌ 𝑛𝐻𝑖𝑛
+ 𝑚𝐷𝑟𝑢𝑔𝑜𝑢𝑡𝑛+
𝐻𝑜𝑢𝑡=+𝐻𝑖𝑛𝐷𝑟𝑢𝑔𝑜𝑢𝑡
𝐷𝑟𝑢𝑔𝑖𝑛E. coli EmrE is a model drug/proton antiporter𝑚SSM Reversal Assay is quantitative
WT GdX, LPR 150
WT GdX, LPR 400“Empty” Liposomes