Preparation of Microsomes to Study Ca²⁺ Channels

Abstract

Native cerebellar microsomes are used to study the properties of native cerebellar inositol(1,4,5)-trisphosphate receptor 1 (InsP₃R1) and ryanodine receptor 1 (RyanR1) Ca²⁺ release channels. Additionally, microsomes prepared from Sf9 cells infected with InsP₃R- or RyanR-expressing recombinant baculoviruses can be used to compare properties of different InsP₃R and RyanR isoforms and to perform structure–function studies of both types of receptors. This protocol describes how to prepare ER microsomes from native cerebellar tissue or Sf9 cells infected with InsP₃R- or RyanR-expressing recombinant baculoviruses for planar lipid bilayer (also called black lipid membranes or BLM) experiments. Prepared material is aliquoted and can be stored at −80°C freezer for many months before BLM experiments.