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

  • Josefina del Mármol
  • Robert A. Rietmeijer
  • Stephen G. BrohawnEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1684)


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.

Key words

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


  1. 1.
    Ranade SS, Syeda R, Patapoutian A (2015) Mechanically activated ion channels. Neuron 87:1162–1179. doi: 10.1016/j.neuron.2015.08.032 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Delmas P, Coste B (2013) Mechano-gated ion channels in sensory systems. Cell 155:278–284. doi: 10.1016/j.cell.2013.09.026 CrossRefPubMedGoogle Scholar
  3. 3.
    Nilius B, Honore E (2012) Sensing pressure with ion channels. Trends Neurosci 35:477–486. doi: 10.1016/j.tins.2012.04.002 CrossRefPubMedGoogle Scholar
  4. 4.
    Anishkin A, Loukin SH, Teng J, Kung C (2014) Feeling the hidden mechanical forces in lipid bilayer is an original sense. Proc Natl Acad Sci U S A 111:7898–7905. doi: 10.1073/pnas.1313364111 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Wu J, Lewis AH, Grandl J (2017) Touch, tension, and transduction – the function and regulation of piezo ion channels. Trends Biochem Sci 42:57–71. doi: 10.1016/j.tibs.2016.09.004 CrossRefPubMedGoogle Scholar
  6. 6.
    Hao J, Padilla F, Dandonneau M et al (2013) Kv1.1 channels act as mechanical brake in the senses of touch and pain. Neuron 77:899–914. doi: 10.1016/j.neuron.2012.12.035 CrossRefPubMedGoogle Scholar
  7. 7.
    Schmidt D, del Mármol J, Mackinnon R (2012) Mechanistic basis for low threshold mechanosensitivity in voltage-dependent K+ channels. Proc Natl Acad Sci U S A 109:10352–10357. doi: 10.1073/pnas.1204700109 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Enyedi P, Czirják G (2010) Molecular background of leak K+ currents: two-pore domain potassium channels. Physiol Rev 90:559–605. doi: 10.1152/physrev.00029.2009 CrossRefPubMedGoogle Scholar
  9. 9.
    Honore E (2007) The neuronal background K2P channels: focus on TREK1. Nat Rev Neurosci 8:251–261. doi: 10.1038/nrn2117 CrossRefPubMedGoogle Scholar
  10. 10.
    Brohawn SG (2015) How ion channels sense mechanical force: insights from mechanosensitive K2P channels TRAAK, TREK1, and TREK2. Ann N Y Acad Sci 1352:20–32. doi: 10.1111/nyas.12874 CrossRefPubMedGoogle Scholar
  11. 11.
    Brohawn SG, Campbell EB, Mackinnon R (2014) Physical mechanism for gating and mechanosensitivity of the human TRAAK K+ channel. Nature 516:126–130. doi: 10.1038/nature14013 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Hao J, Delmas P (2011) Recording of mechanosensitive currents using piezoelectrically driven mechanostimulator. Nat Protoc 6:979–989. doi: 10.1038/nprot.2011.343 CrossRefPubMedGoogle Scholar
  13. 13.
    Coste B, Mathur J, Schmidt M et al (2010) Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels. Science 330:55–60. doi: 10.1126/science.1193270 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Besch S, Suchyna T, Sachs F (2002) High-speed pressure clamp. Pflugers Arch 445:161–166. doi: 10.1007/s00424-002-0903-0 CrossRefPubMedGoogle Scholar
  15. 15.
    Sokabe M, Sachs F, Jing ZQ (1991) Quantitative video microscopy of patch clamped membranes stress, strain, capacitance, and stretch channel activation. Biophys J 59:722–728. doi: 10.1016/S0006-3495(91)82285-8 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Sukharev SI, Sachs F (2012) Molecular force transduction by ion channels - diversity and unifying principles. J Cell Sci 125:3075–3083. doi: 10.1242/jcs.092353 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Opsahl LR, Webb WW (1994) Lipid-glass adhesion in giga-sealed patch-clamped membranes. Biophys J 66:75–79. doi: 10.1016/S0006-3495(94)80752-0 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Moe P, Blount P (2005) Assessment of potential stimuli for mechano-dependent gating of MscL: effects of pressure, tension, and lipid headgroups †. Biochemistry 44:12239–12244. doi: 10.1021/bi0509649 CrossRefPubMedGoogle Scholar
  19. 19.
    Lewis AH, Grandl J (2015) Mechanical sensitivity of Piezo1 ion channels can be tuned by cellular membrane tension. eLife 4:143. doi: 10.7554/eLife.12088 Google Scholar
  20. 20.
    Sukharev SI (2002) Purification of the small mechanosensitive channel of Escherichia coli (MscS): the subunit structure, conduction, and gating characteristics in liposomes. Biophys J 83:290–298. doi: 10.1016/S0006-3495(02)75169-2 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Brohawn SG, Su Z, Mackinnon R (2014) Mechanosensitivity is mediated directly by the lipid membrane in TRAAK and TREK1 K+ channels. Proc Natl Acad Sci U S A 111:3614–3619. doi: 10.1073/pnas.1320768111 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Berrier C, Pozza A, de Lacroix de Lavalette A et al (2013) The purified mechanosensitive channel TREK-1 is directly sensitive to membrane tension. J Biol Chem 288(38):27307. doi: 10.1074/jbc.M113.478321 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Martinac B, Rohde PR, Battle AR et al (2009) Studying mechanosensitive ion channels using liposomes. In: Lipid-protein interactions. Humana Press, Totowa, NJ, pp 31–53Google Scholar
  24. 24.
    Teng J, Loukin S, Anishkin A, Kung C (2015) The force-from-lipid (FFL) principle of mechanosensitivity, at large and in elements. Pflugers Arch 467:27. doi: 10.1007/s00424-014-1530-2 CrossRefPubMedGoogle Scholar
  25. 25.
    Dubin AE, Murthy S, Lewis AH et al (2017) Endogenous Piezo1 can confound mechanically activated channel identification and characterization. Neuron 94:266–270.e3. doi: 10.1016/j.neuron.2017.03.039 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Josefina del Mármol
    • 1
    • 2
  • Robert A. Rietmeijer
    • 3
  • Stephen G. Brohawn
    • 1
    • 2
    Email author
  1. 1.Department of Molecular and Cell BiologyUniversity of California – BerkeleyBerkeleyUSA
  2. 2.The Helen Wills Neuroscience InstituteUniversity of California – BerkeleyBerkeleyUSA
  3. 3.Biophysics Graduate GroupUniversity of CaliforniaBerkeleyUSA

Personalised recommendations