Supplementary Text (Methods):
Electrophysiology & Statistical Analysis: HERG currents were measured using the whole-cell configuration of the patch-clamp technique as described previously.15 The Axopatch-200 patch clamp amplifier (Axon Instruments Inc., Union City, CA, USA) was used to record currents and to measure cellular capacitance. The pCLAMP 8.0 software (Axon Instruments) and Origin 7.5 (Origin-Lab Corp., Northampton, MA, USA) were used for generation of voltage-clamp protocols and data analysis. Pipettes were pulled from borosilicate glass capillaries using a micropipette puller (model P-57, Sutter Instruments, Novato, CA, USA). Open tip resistances of pipettes filled with intracellular fluid for current recordings were 2–3 MΩ. The extracellular bath solution contained 137mM NaCl, 4mM KCl, 1.8mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES (pH 7.4 with NaOH). The intracellular pipette solution contained 130 mM KCl, 1 mM MgCl2, 5 mM EGTA, 5 mM MgATP, and 10 mM HEPES (pH 7.2 with KOH). After obtaining intracellular access, only cells with seal resistances >1 GΩ were used. Series resistance was compensated between 75 and 85%. Currents were filtered at 5 kHz and digitized at 10 kHz. The holding potential in all experiments was -80 mV. Current density was calculated by dividing current amplitude by cell capacitance (Cm). Cm was estimated by dividing the decay time constant of the capacitive transient in response to 5-mV hyperpolarizing voltage-clamp steps from -40 mV by the series resistance (Rs). All experiments were performed at room temperature within 2 hours of removing the cells from their culture conditions. Voltage-clamp protocols: details of each voltage-clamp protocol are given schematically in the figures. HERG tail currents (Itail) were measured by depolarizing cells to 50 mV for 5 s followed by a test pulse to -120 mV for 3 seconds to elicit Itail. Activation characteristics of WT and Q1070X channels were determined by depolarizing cells were from -80 to 60 mV in 10 mV increments for 5 s, followed by a test pulse to -50 mV for 5 s. Current-voltage (I-V) relations were generated by normalizing Itail to maximum value, measured during test pulse, and plotting as a function of the prepulse voltage. The data were fitted to a Boltzmann distribution curve to obtain V1/2 (membrane potential for half maximal activation) and slope factor k values. To measure channel inactivation, cells were depolarized from a holding potential of -80 to 50 mV for 1.5 s, hyperpolarized to -100 mV for 2.5 ms, and next stepped to test voltages between 0 and 100 mV in 10 mV decrements for 1.5 s. The decay of the generated HERG currents during the test pulses was fitted to a single exponential function to obtain a time constant for channel inactivation. To measure recovery from inactivation cells were depolarized from -80 mV to 50 mV for 1.5 s, followed by hyperpolarizing test voltages between -120 and -30 mV in 10 mV increments for 3 s. The initial phase of Itail (indicated by arrow in Fig. 1D) reflects recovery from inactivation and was fitted to a single exponential function. The same protocol as recovery from inactivation was used to measure deactivation time constants. To obtain the fast and slow time constants of inactivation the decay of Itail (indicated by arrow in Fig. 1E) was fitted to a double-exponential function. The time constants of inactivation, recovery from inactivation and deactivation were plotted as a function of the test voltage.
Statistics: Values are expressed as mean ± SEM. Unpaired t-test was used for statistical analysis of current densities, V1/2 and slope factors. Values were considered significantly different if P<0.05. Two-way repeated measure of analysis of variance followed by pair-wise comparison using the Student-Newman-Keuls test was used for statistical analysis of time constants of inactivation, recovery from inactivation and deactivation.