Figure 1.
(A) The hERG current in control (blue), after application of 10 µM celecoxib (green) and after wash-out (red). The currents were elicited by a 10 s pulse to −60 mV following a 10 s pre-pulse to 0 mV (HP = −80 mV). (B) Dose-response relations for the peak tail currents evoked by a pulse to −40 mV after a 2 s pre-pulse to +40 mV; here and in the following figures the dose-response relations have been fitted to the Hill equation; number of cells varied between 3 and 11.
Figure 2.
(A) Four 10 s pulses to 0 mV in control with 30 s inter-pulse intervals were followed by four pulses after 3 min exposure to 10 µM celecoxib. Data from 9 experiments were divided by maximum value of current in control in each cell and then averaged. Traces labeled ‘celecoxib’ overlap for pulses 5 (green) and 6 (red). (B) Celecoxib modulates closed hERG channels. Currents were elicited by 1 Hz pulse trains consisting of a 100 ms pre-pulse to +60 mV followed by a 150 ms pulse to −130 mV. After 20 pulses in control, the stimulation was ceased and 10 µM celecoxib was rapidly washed in (arrow). Stimulation was resumed after 3 min exposure to the drug while holding the cell at −80 mV; number of experiments, 4; inset shows an example of tail currents. (C) Inhibition of hERG was not use-dependent. Currents were evoked by 1 Hz pulse train consisting of a 400 ms pre-pulse to +20 mV followed by a 200 ms pulse to −40 mV and normalized to the magnitude of current during the first pulse in each sample; (n), number of experiments in this and the following figures. (D) Duration of the inter-pulse interval did not affect the extent of inhibition. Currents were elicited as in the panel C. After development of inhibition at 0.2 Hz in the presence of 10 µM celecoxib, the frequency of stimulation was first switched to 0.1 Hz for 10 pulses, and then to 1 Hz for 60 pulses; average of 3 cells.
Figure 3.
Modification of hERG gating by celecoxib.
(A) Expanded first 200 ms from Fig. 2A shows a transient increase in hERG amplitude in the presence of celecoxib at 15 ms (vertical line) after the onset of pulse; gray traces indicate the range of SE. (B) Examples of hERG in control elicited by 4 s pulses to −60 mV after 4 s pre-pulses to voltages between −60 and +40 mV in 10 mV increments. (C) Normalized voltage-current relations for the sustained (end of a 4 s pre-pulse) and transient (maximal amplitude during the first 20 ms of pulse) components of hERG in control and at 10 µM celecoxib; currency signs mark statistical significance for circles, asterisks – for triangles. (D) Voltage-dependence of peak tail currents plotted against the pre-pulse voltage in control and at 10 µM celecoxib.
Figure 4.
Celecoxib accelerated kinetics of activation, inactivation and re-activation of the hERG channels.
(A) Examples of currents recorded during envelope-of-tails protocol to measure rates of activation at +20 mV in control and at 10 µM celecoxib. The protocol consisted of a variable duration (between 10 and 3,000 ms) pre-pulse to +20 mV followed by a test pulse to −60 mV. (B) Voltage dependence of the τact. (C) Examples of inactivating currents in control and at 10 µM celecoxib. The protocol consisted of a 2 s first pre-pulse to +60 mV, a 10 ms second pre-pulse to −100 mV, and a test pulse between −20 and +60 mV in 10 mV increments. (D) Voltage dependence of the τinact. (E) An example of re-activating current in control evoked by a 2 s pre-pulse to +40 mV followed by a 50 ms test pulse between −130 and +10 mV in 10 mV increments. (F) Voltage dependence of the τreact.
Figure 5.
Effects of celecoxib on the KCNQ1/MinK and KCNQ1 channels.
KCNQ1/MinK (A, B) and KCNQ1 (C, D) currents were elicited by voltage pulses between −80 mV and +40 mV in 10 mV increments (HP = −80 mV). (E) Dose-response relations for the KCNQ1/MinK current at the end of 2 s pulse and for the peak KCNQ1 current (at +40 mV); number of experiments varied between 3 and 18. (F) Celecoxib modulated closed channels; currents were elicited by 0.1 Hz train consisting of 1 s pulses to +40 mV. After 10 pulses in control, the stimulation was paused and 3 µM celecoxib rapidly washed in (arrow). Stimulation was resumed after 5 min exposure to celecoxib.
Figure 6.
Effects of celecoxib on SCN5A and KCND3.
(A–B) SCN5A currents were evoked by pulses to −20 mV from a HP of −70 mV (A) or −100 mV (B) in control and in the presence of different concentrations of celecoxib. (C) Inhibition of KCND3 channels. The currents were evoked by pulses to +20 mV from a HP of −80 mV. (D) Dose-response relations for inhibition of peak SCN5A and KCND3 currents; letters in the legend refer to the corresponding panels. A–C, legends are coded as in the panel A.