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 single-channel permeation and gating properties of these Ca²⁺-release channels using the powerful patch-clamp 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.