Patch-clamp electrophysiology of intracellular Ca2+ channels

Abstract

The modulation of cytoplasmic free Ca2+ concentration ([Ca2+]i) is a universal intracellular signaling pathway that regulates numerous cellular physiological processes. Ubiquitous intracellular Ca2+-release channels localized to the endoplasmic/sarcoplasmic reticulum-inositol 1,4,5-trisphosphate receptor (InsP3R) and ryanodine receptor (RyR) channels-play a central role in [Ca2+]i signaling in all animal cells. Despite their intracellular localization, electrophysiological studies of the singlechannel permeation and gating properties of these Ca2+-release channels using the powerful patchclamp approach have been possible by application of this technique to isolated nuclei because the channels are present in membranes of the nuclear envelope. Here we provide a concise description of how nuclear patch-clamp experiments have been used to study single-channel properties of different InsP3R channels in the outer nuclear membrane. We compare this with other methods for studying intracellular Ca2+ release. We also briefly describe application of the technique to InsP3R channels in the inner nuclear membrane and to channels in the outer nuclear membrane of HEK293 cells expressing recombinant RyR. © 2013 Cold Spring Harbor Laboratory Press.