A label-free approach to detect ligand binding to cell surface proteins in real time

Burtscher, Verena; Hoťka, Matej; Li, Yang; Freissmuth, Michael; Sandtner, Walter

doi:10.7554/elife.34944

“…The fit revealed that the residual, which had originated from cocaine binding to SERT is comprised of a battery (red trace) and a capacitive component (blue trace) ( Figure 6B-upper panel). This was unexpected and disproved our original claim that the cocaine-induced change in CM was fully attributable to changes in surface potential (8). However, in partial exoneration of this claim, we found that the battery residual accounted in average for a slightly larger part of the change.…”

Section: Analysis Of the Current Responses Recorded From Mammalian Cecontrasting

confidence: 50%

“…Ligand induced capacitance changes are apparent changes. We previously showed that it is possible to monitor ligand binding to membrane proteins in real time by utilizing measurements of the cell membrane capacitance (CM) (8). We investigated the ligand-induced capacitance change in HEK293 cells stably expressing hSERT.…”

Section: Resultsmentioning

confidence: 99%

“…The difference of 0.0016 C/m 2 is accounted for by the number of SERT moieties (i.e. 2*10 7 ) expressed on the surface of our stable cell line (8), to which cocaine binds in a 1:1 stoichiometry at saturating concentrations. The Gouy-Chapman model predicts that ΔVS in the presence of cocaine is 7.9 mV, which differs by 1.5 mV from that in the absence of cocaine.…”

Section: Resultsmentioning

confidence: 99%

“…we measured the membrane capacitance with an approach, which relied on the analysis of current responses to voltage square pulses (8). More often, however, membrane capacitance measurements are conducted utilizing a sinusoidal voltage stimuli in combination with a hardwired (16) or a software-based lock-in amplifier (17).…”

Section: Ligand-induced Capacitance Changes Assessed With Sinusoidal mentioning

confidence: 99%

“…We hypothesized that the change evoked by cocaine originated from a change in the outer surface potential caused by binding of the charged ligand to SERT rather than from a true capacitance change. Accordingly, we referred to this alteration as an apparent change in capacitance (8).…”

Section: Introductionmentioning

confidence: 99%

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An electrophysiological approach to measure changes in the membrane surface potential in real time

Burtscher

¹

,

Hoťka

²

,

Freissmuth

³

et al. 2019

Preprint

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Biological membranes carry fixed charges at their surfaces. These arise primarily from phospholipid head groups. In addition, membrane proteins contribute to the surface potential with their charged residues. Membrane lipids are asymmetrically distributed. Because of this asymmetry the net negative charge at the inner leaflet exceeds that at the outer leaflet. Changes in surface potential are predicted to shape the capacitive properties of the membrane (i.e. the ability of the membrane to store electrical charges). Here, we show that it is possible to detect changes in surface potential by an electrophysiological approach: the analysis of cellular currents relies on assuming that the electrical properties of a cell are faithfully described by a three-element circuit -i.e. the minimal equivalent circuit -comprised of two resistors and one capacitor. However, to account for changes in surface potential it is necessary to add a battery to this circuit connected in series with the capacitor. This extended circuit model predicts that the current response to a square-wave voltage pulse harbors information, which allows for separating the changes in surface potential from a true capacitance change. We interrogated our model by investigating changes in capacitance induced by ligand binding to the serotonin transporter (SERT) and to the glycine transporters (GlyT1 and GlyT2). The experimental observations were consistent with the predictions of the extended circuit. We conclude that ligand-induced changes in surface potential (reflecting the binding event) and in true membrane capacitance (reflecting the concomitant conformational change) can be detected in real time even in instances where they occur simultaneously. Statement of SignificanceThe plasma membrane of a cell possesses fixed charges on both surfaces. Surface charges play an important role in many biological processes. However, the mechanisms, which regulate the surface charge densities at the plasma membrane, are poorly understood. This is in part due to lack of experimental approaches that allow for detecting changes in surface charges in real time. Here, we show that it is possible to track alterations in the electric potential at the membrane surface with high temporal resolution by an electrophysiological approach. Importantly, the described method allows for discriminating between a change in surface potential and a change in true membrane capacitance (e.g. a change in membrane area), even if these occur in parallel.

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An Electrophysiological Approach to Measure Changes in the Membrane Surface Potential in Real Time

Burtscher

¹

,

Hoťka

²

,

Freissmuth

³

et al. 2020

Biophysical Journal

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Biological membranes carry fixed charges at their surfaces. These arise primarily from phospholipid headgroups. In addition, membrane proteins contribute to the surface potential with their charged residues. Membrane lipids are asymmetrically distributed. Because of this asymmetry, the net-negative charge at the inner leaflet exceeds that at the outer leaflet. Changes in surface potential are predicted to give rise to apparent changes in membrane capacitance. Here, we show that it is possible to detect changes in surface potential by an electrophysiological approach; the analysis of cellular currents relies on assuming that the electrical properties of a cell are faithfully described by a three-element circuit (i.e., the minimal equivalent circuit) comprised of two resistors and one capacitor. However, to account for changes in surface potential, it is necessary to add a battery to this circuit connected in series with the capacitor. This extended circuit model predicts that the current response to a square-wave voltage pulse harbors information, which allows for separating the changes in surface potential from a true capacitance change. We interrogated our model by investigating changes in the capacitance induced by ligand binding to the serotonin transporter and to the glycine transporters (GlyT1 and GlyT2). The experimental observations were consistent with the predictions of the extended circuit. We conclude that ligand-induced changes in surface potential (reflecting the binding event) and in true membrane capacitance (reflecting the concomitant conformational change) can be detected in real time even in instances in which they occur simultaneously.

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Extracellular loops of the serotonin transporter act as a selectivity filter for drug binding

Esendir

¹

,

Burtscher

²

,

Coleman

³

et al. 2021

Journal of Biological Chemistry

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A label-free approach to detect ligand binding to cell surface proteins in real time

Cited by 17 publications

References 51 publications

An electrophysiological approach to measure changes in the membrane surface potential in real time

An electrophysiological approach to measure changes in the membrane surface potential in real time

An Electrophysiological Approach to Measure Changes in the Membrane Surface Potential in Real Time

Extracellular loops of the serotonin transporter act as a selectivity filter for drug binding

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