Nature Methods
- 5, 87 - 93 (2008)
Published online: 16 December 2007; | doi:10.1038/nmeth1144
Tracking transmitter-gated P2X cation channel activation in vitro and in vivoEsther Richler1, 4, Severine Chaumont1, 4, Eiji Shigetomi1, 4, Alvaro Sagasti3 & Baljit S Khakh1, 21
Department of Physiology, University of California Los Angeles, 10833 LeConte Avenue, Los Angeles, California 90095, USA. 2
Department of Neurobiology, David Geffen School of Medicine, University of California Los Angeles, 10833 LeConte Avenue, Los Angeles, California 90095, USA. 3
Department of Molecular, Cellular and Developmental Biology, University of California Los Angeles, 10833 LeConte Avenue, Los Angeles, California 90095, USA. 4
These authors contributed equally to this work.
Correspondence should be addressed to Baljit S Khakh bkhakh@mednet.ucla.edu We present a noninvasive approach to track activation of ATP-gated P2X receptors and potentially other transmitter-gated cation channels that show calcium fluxes. We genetically engineered rat P2X receptors to carry calcium sensors near the channel pore and tested this as a reporter for P2X2 receptor opening. The method has several advantages over previous attempts to image P2X channel activation by fluorescence resonance energy transfer (FRET): notably, it reports channel opening rather than a conformation change in the receptor protein. Our FRET-based imaging approach can be used as a general method to track, in real time, the location, regional expression variation, mobility and activation of transmitter-gated P2X channels in living neurons in vitro and in vivo. This approach should help to determine when, where and how different receptors are activated during physiological processes.
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