Patch-Clamp Study of Single Ryanodine Receptor Channels in the Outer Nuclear Membrane

Abstract

Modulation of cytoplasmic free calcium (Ca2+) concentration is a universal signaling pathway that regulates numerous cellular processes. Ubiquitous intracellular Ca2+ release channels – inositol 1,4,5-trisphosphate receptor (InsP3R) and ryanodine receptor (RyR) channels – localized in the sarco/endoplasmic reticulum (ER) play a central role in this pathway in all animal cells. Electrophysiological study of the single-channel conductance and gating properties of these Ca2+ release channels with conventional patch-clamp approach has been hindered by their intracellular localization. To overcome this limitation, patch-clamp electrophysiology has been applied on isolated nuclei where these Ca2+ release channels are found abundantly in the outer nuclear envelope. We have successfully uterlized this nuclear membrane electrophysiology to study the gating properties of single InsP3R channels in several cellular systems. Whereas, all the current single channel data, including channel conductance, permeation properties, and ligand regulation, of the RyR channels were done exclusively by reconstituting the channels into artificial planar lipid bilayers. To gain insights into the single channel properties of the RyR in its native membrane milieu, we applied nuclear membrane electrophysiological study on isolated nuclei from stable-inducible mouse RyR2 HEK-293 cells. Using potassium as charge carrier, caffeine activated single channel current with conductance of 750 pS in isolated nuclei. This caffeine activated current showed a linear current/voltage relationship under symmetrical ionic conditions and was sensitive to non-specific RyR inhibitor, ruthenium red. Furthermore, the single RyR channels recorded from the outer nuclear membrane exhibited bi-phasic Ca2+ regulation. In conclusion, we demonstrated, for the first time, that single RyR channels recordings from isolated nuclei and our results suggested that the nuclear membrane electrophysiology could be a sensitive and robust technique to study the gating properties of intracellular channels, including the InsP3R and RyR.