Bilayer Measurement of Endoplasmic Reticulum Ca2+ Channels

Abstract

Reconstitution of ion channels into planar lipid bilayers (also called black lipid membranes or BLM) is the most widely used method to conduct physiological studies of intracellular ion channels, including endoplasmic reticulum (ER) calcium (Ca2+) channels. The two main types of Ca2+ release channels in the ER membrane are ryanodine receptors (RyanRs) and inositol(1,4,5)-trisphosphate receptors (InsP3Rs). Use of the BLM reconstitution technique enabled the initial description of the functional properties of InsP3R and RyanR at the single-channel level more than 20 years ago. Since then, BLM reconstitution methods have been used to study physiological modulation and to perform structure–function analysis of these channels, and to study pathological changes in the function of InsP3R and RyanR in various disease states. The BLM technique has also been useful for studies of other intracellular Ca2+ channels, such as ER Ca2+ leak presenilin channels and NAADP-gated lysosomal Ca2+ channels encoded by TPC2. In this article, basic protocols used for BLM studies of ER Ca2+ channels are introduced.