Abstract
In multi-cellular organisms, cells communicate with each other utilizing chemical messengers. For many of these messenger molecules, the membrane is an insurmountable barrier. Yet, they act by binding to surface proteins often triggering a cascade of reactions inside the cell. Accordingly, studying ligand-receptor interactions at the cellular surface is key to understanding important aspects of membrane biology. However, despite a multitude of approaches to study membrane features, there is a need for developing techniques that can measure ligand binding with high temporal resolution and on a single cellular level. We recently developed a label-free approach to study ligand binding in real time. This methodology capitalizes on changes of the membrane's surface potential induced by the adsorption of a charged ligand. The resulting apparent alteration of membrane capacitance is measurable by capacitance recordings. Herein, we describe the implementation of the same using recordings obtained from HEK293 cells stably expressing the human serotonin transporter (SERT), which were challenged with the inhibitor cocaine.
Highlights
[Background] Ligand binding to surface proteins is a key event in cell-to-cell signaling
Most methods to measure ligand binding rely on the use of radiolabeled or fluorescence-labeled surrogates
These include (i) a small sampling rate (i.e., 0.2 s-1) (ii) distorted binding properties of the ligand by virtue of the necessary label (iii) non-physiological experimental conditions, implemented to ensure success of the method and (iv) the difficulty of tracking ligand binding on single cellular level
Summary
Note: PDL-coating is required for the cells to stay attached during rapid solution exchange in the experiment. Plate cells at low density (2,000-4,000 cells ml-1) onto PDL-coated 35 mm dishes containing 2 ml maintenance medium supplied with 1 μg∙ml-1 tetracycline. 2. Coat the tip of a patch pipette with Sylgard to reduce the stray capacitance (CS) of the pipette and fluctuations thereof (Figure 2). (sweep duration: 20 ms, sweep number: 4,096, sampling frequency: 100 kHz, Figure 3). Protocol to measure the stray capacitance of the pipette and the membrane patch beneath it: 2.5 ms step from +20 mV to -20 mV (sweep duration: 5 ms, sweep number: 4,000, sampling frequency: 100 kHz, Figure 4). Protocol specifications for recording of the residual stray capacitance: UStim: 20 mV TS: 5 ms Number of sweeps: 4,000 Sampling frequency: 100 kHz
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
