M335A/E373A and M335A/E373Q mutants show similar single-channel characteristics with the WT rSlack channel. A, The sample traces of M335A mutant in the presence of different cytosolic sodium ions at −100 mV membrane potential. B, The Hill equation fitted the Slack channel open probability versus sodium concentration data. The EC50 value is 46.5 ± 2.3 mm. C, The sample single-channel traces of M335A/E373Q mutant at indicated voltages in intracellular solution with 0 mm cytosolic Na+. D, The plotted traces show M335A single-channel conductance is 170 ± 5 pS while M335A/E373Q and M335A/E373A single-channel conductance is 163 ± 10 and 167 ± 4 pS at 0 mm cytosolic sodium, respectively. E, The sample single-channel traces of the M335A mutant were blocked by bepridil at indicated concentrations. F, The Hill equation fitted the dose–response curve of the bepridil blocking on M335A, M335A/E373A, and M335A/E373Q mutants. The blocking EC50 values of bepridil on these mutants are 1.55 ± 0.17, 1.2 ± 0.03, and 1.18 ± 0.04 μm, respectively.