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Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy
08:27

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Published on: January 7, 2019

Native TRPC7 channel activation by an inositol trisphosphate receptor-dependent mechanism

Guillermo Vazquez1, Gary St J Bird, Yasuo Mori

  • 1NIEHS, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709, USA. vazquez1@niehs.nih.gov

The Journal of Biological Chemistry
|July 11, 2006
PubMed

Insights

Canonical Transient Receptor Potential 7 (TRPC7) channels in DT40 B lymphocytes require inositol trisphosphate receptors (IP3R) for diacylglycerol-activated currents at low expression. At higher levels, TRPC7 functions independently of IP3R.

Area of Science:

  • Ion channel physiology
  • Cell signaling in lymphocytes

Background:

  • Canonical Transient Receptor Potential 7 (TRPC7) is a diacylglycerol-activated cation channel in DT40 B lymphocytes.
  • Previous studies suggested non-store-operated calcium (Ca2+) entry relies on inositol trisphosphate receptors (IP3R).

Purpose of the Study:

  • To investigate the role of TRPC7 and IP3R in diacylglycerol-activated cation currents in DT40 B lymphocytes.
  • To clarify the relationship between TRPC7 channel activity and IP3R dependence under varying expression levels.

Main Methods:

  • Utilized DT40 B lymphocyte cell lines with genetic knockouts for TRPC7 and IP3R.
  • Employed cell-attached patch-clamp electrophysiology to measure single channel activity.
  • Assessed channel activity induced by oleyl-acetyl-glycerol (OAG) and rescued by TRPC7 or IP3R re-expression.

Main Results:

  • Oleyl-acetyl-glycerol (OAG)-induced 75 pS single channel activity was absent in TRPC7 knockout cells but rescued by TRPC7 re-expression.
  • OAG-induced activity was also absent in IP3R knockout cells but rescued by IP3R re-expression.
  • Single channel properties were consistent across wild-type, rescued, and knockout cell lines, indicating TRPC7 forms the channel, but its activity is IP3R-dependent at low expression.

Conclusions:

  • TRPC7 forms or is part of the endogenous diacylglycerol-activated cation channel in DT40 B lymphocytes.
  • Native TRPC7 channel activity is dependent on IP3R at low expression levels.
  • TRPC7 functions independently of IP3R at higher expression levels, potentially resolving conflicting prior research.

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