2.4 Single Channel Analysis

Single channel measurements with wild type and mutant RyRs were performed in planar lipid bilayers (12). Membrane fractions expressing RyRs were pretreated with myosin light chain kinase CaM binding domain peptide (CaMBP) for 30 min at room temperature to remove endogenous CaM (10). Membranes were added to the cis cytosolic chamber of the bilayer apparatus. A strong dependence of single channel activities on cytosolic Ca²⁺ concentration indicated that the large cytosolic “foot” region faced the cis chamber of the bilayers. The trans sarcoplasmic reticulum luminal side of the bilayer was defined as ground. Measurements were made in symmetrical buffer containing 0.25 M KCl, 20 mM K-Hepes, pH 7.4. Activating and inhibitory effects of exogenous cis cytosolic CaM were determined at the indicated cytosolic Ca²⁺ concentrations. Electrical signals were filtered at 2 kHz, digitized at 10 kHz and analyzed (12). Open probability (P_o) was obtained from recordings lasting a minimum of 2 min. P_o values in multichannel recordings were calculated using the equation P_o = Σ iP_o,i/N, where N is the total number of channels and P_o,i is channel open probability of the i^th channel. To obtain the number of channels incorporated into the bilayers, the number of channel current levels were determined with 2 µM cis cytosolic Ca²⁺.