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Methods Mol Biol. 2018;1684:129-150. doi: 10.1007/978-1-4939-7362-0_11.

Studying Mechanosensitivity of Two-Pore Domain K+ Channels in Cellular and Reconstituted Proteoliposome Membranes.

Author information

1
Department of Molecular and Cell Biology, University of California - Berkeley, 289 Life Science Addition, Berkeley, CA, 94720, USA.
2
The Helen Wills Neuroscience Institute, University of California - Berkeley, 289 Life Science Addition, Berkeley, CA, 94720, USA.
3
Biophysics Graduate Group, University of California, Berkeley, CA, 94720, USA.
4
Department of Molecular and Cell Biology, University of California - Berkeley, 289 Life Science Addition, Berkeley, CA, 94720, USA. brohawn@berkeley.edu.
5
The Helen Wills Neuroscience Institute, University of California - Berkeley, 289 Life Science Addition, Berkeley, CA, 94720, USA. brohawn@berkeley.edu.

Abstract

Mechanical force sensation is fundamental to a wide breadth of biology from the classic senses of touch, pain, hearing, and balance to less conspicuous sensations of proprioception, blood pressure, and osmolarity and basic aspects of cell growth, differentiation, and development. These diverse and essential systems use force-gated (or mechanosensitive) ion channels that convert mechanical stimuli into cellular electrical signals. TRAAK, TREK1, and TREK2 are K+-selective ion channels of the two-pore domain K+ (K2P) family that are mechanosensitive: they are gated open by increasing membrane tension. TRAAK and TREK channels are thought to play roles in somatosensory and other mechanosensory processes in neuronal and non-neuronal tissues. Here, we present protocols for three assays to study mechanical activation of these channels in cell membranes: (1) cell swelling, (2) cell poking, and (3) patched membrane stretching. Patched membrane stretching is also applicable to the study of mechanosensitive K2P channel activity in a cell-free system and a procedure for proteoliposome reconstitution and patching is also presented. These approaches are also readily applicable to the study of other mechanosensitive ion channels.

KEYWORDS:

Cell poking; Cell swelling; K2P ion channel; Mechanosensitive ion channel; Membrane tension gating; Patch clamp; Patch inflation; Proteoliposome reconstitution; TRAAK; TREK1; TREK2

PMID:
29058189
PMCID:
PMC6202064
DOI:
10.1007/978-1-4939-7362-0_11
[Indexed for MEDLINE]
Free PMC Article

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