Planar patch clamp approach to characterize ionic currents from intact lysosomes.
Since its launch in the early 1980s, the patch clamp method has been extensively used to study ion channels in the plasma membrane, but its application to the study of intracellular ion channels has been limited. Unlike the plasma membrane, intracellular membranes are usually not stable enough to withstand mechanical manipulation by glass electrodes during seal formation and rupturing of the membrane. To circumvent these problems, we developed a method involving the immobilization of isolated organelles on a solid matrix planar glass chip. This glass chip contains a microstructured hole that supports the formation of gigaseals and subsequent electrophysiological recordings despite the high fragility of intracellular membranes. Here, we report the experimental details of this method using lysosomes, which are the smallest cellular organelles, as a model system. We demonstrate that we can record endogenous ionic currents from wild-type lysosomes, as well as from lysosomes overexpressing ion channels, and expect that this method will provide electrophysiological access to a broad range of intracellular ion channels.
Center for Integrated Protein Science CIPS-M and Zentrum für Pharmaforschung-Department Pharmazie, Ludwig-Maximilians-Universität München, Butenandtstrasse 5 -13, D-81377 München, Germany.
Michael
M
Schieder
Katrin
K
Rötzer
Andrea
A
Brüggemann
Martin
M
Biel
Christian
C
Wahl-Schott
D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology*; D020013:Calcium Signaling / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D007425:Intracellular Membranes / Q000502:physiology; D008247:Lysosomes / Q000378:metabolism*; D018408:Patch-Clamp Techniques / Q000295:instrumentation / Q000379:methods; D015203:Reproducibility of Results; D012984:Software
D015220:Calcium Channels; D002118:Calcium