TY - JOUR
T1 - Sub-millisecond ligand probing of cell receptors with multiple solution exchange
AU - Sylantyev, Sergiy
AU - Rusakov, Dmitri A.
N1 - Acknowledgements
This work was supported by the Wellcome Trust, Medical Research Council (UK), the European Research Council (Advanced Grant), the European Commission COST Action BM1001 ECMNet, and the Biology and Biotechnology Research Council (UK).
PY - 2013/7
Y1 - 2013/7
N2 - The accurate knowledge of receptor kinetics is crucial to our understanding of cell signal transduction in general and neural function in particular. The classical technique of probing membrane receptors on a millisecond scale involves placing a recording micropipette with a membrane patch in front of a double-barrel (θ-glass) application pipette mounted on a piezo actuator. Driven by electric pulses, the actuator can rapidly shift the θ-glass pipette tip, thus exposing the target receptors to alternating ligand solutions. However, membrane patches survive for only a few minutes, thus normally restricting such experiments to a single-application protocol. In order to overcome this deficiency, we have introduced pressurized supply microcircuits in the θ-glass channels, thus enabling repeated replacement of application solutions within 10–15 s. This protocol, which has been validated in our recent studies and takes 20–60 min to implement, allows the characterization of ligand-receptor interactions with high sensitivity, thereby also enabling a powerful paired-sample statistical design.
AB - The accurate knowledge of receptor kinetics is crucial to our understanding of cell signal transduction in general and neural function in particular. The classical technique of probing membrane receptors on a millisecond scale involves placing a recording micropipette with a membrane patch in front of a double-barrel (θ-glass) application pipette mounted on a piezo actuator. Driven by electric pulses, the actuator can rapidly shift the θ-glass pipette tip, thus exposing the target receptors to alternating ligand solutions. However, membrane patches survive for only a few minutes, thus normally restricting such experiments to a single-application protocol. In order to overcome this deficiency, we have introduced pressurized supply microcircuits in the θ-glass channels, thus enabling repeated replacement of application solutions within 10–15 s. This protocol, which has been validated in our recent studies and takes 20–60 min to implement, allows the characterization of ligand-receptor interactions with high sensitivity, thereby also enabling a powerful paired-sample statistical design.
KW - Molecular neuroscience
KW - Patch clamp
KW - Receptor pharmacology
UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-84879326593&partnerID=MN8TOARS
U2 - 10.1038/nprot.2013.075
DO - 10.1038/nprot.2013.075
M3 - Article
VL - 8
SP - 1299
EP - 1306
JO - Nature Protocols
JF - Nature Protocols
SN - 1750-2799
IS - 7
ER -