Fig 1.
Nifedipine reversibly inhibited macroscopic Kv2.1 currents.
Whole-cell Kv2.1 currents were elicited during 500 ms voltage steps to potentials ranging from -80 to +60 mV in the absence of nifedipine. (B) Exposure to 50 μM nifedipine significantly reduced the Kv2.1 currents. (C) After washout, Kv2.1 currents were partial and significant recovery. (D) The time course of inhibitory actions of nifedipine (n = 6). (E) Current-voltage (I-V) curves of the absence, presence and washout of 50 μM nifedipine (n = 11). (F) Statistical analysis of nifedipine effects at +60 mV (n = 11). *P <0.05 compared with control group and ** P <0.05 compared with the group of nifedipine treatment.
Fig 2.
Concentration–response curves for the effects of nifedipine on wild type and Y380R mutant of Kv2.1 channels.
The protocol of stimulation is identical to that in Fig 1. Whole-cell currents at +60 mV were plotted against the concentration of nifedipine. Solid lines were fit to the data of wild type Kv2.1 currents using a Hill equation with IC50 value of 37.5 μM and h value of 0.9 (n = 8). For Y380R-Kv2.1 currents, the value of IC50 is 15.2 μM and value of h is 1.1 (n = 7).
Fig 3.
Effects of nifedipine on the activation of Kv2.1 currents.
(A) The voltage dependence of activation time constant was altered by the different concentrations of nifedipine (n = 9). (B) Activation curves of Kv2.1 currents during each test pulse in the absence and presence of different levels of nifedipine were normalized to maximal currents (I/Imax) and plotted against conditioning prepulse potential. Data are fitted with a Boltzmann equation (n = 7).
Fig 4.
The blockage of Kv2.1 currents by nifedipine in a voltage-dependent manner.
(A) The representative current traces were exhibited in the absence and presence of 10, 50 and 100 μM nifedipine. Currents were generated by a 15 s voltage pulse to +60 mV from the holding potential of -80 mV. (B) Different levels of nifedipine inhibited Kv2.1 currents in a voltage-dependent way (n = 8). (C) The voltage-dependent accelerations of inactivation time constants (τinact) were induced by different levels of nifedipine. τinact was determined by fitting the current elicited by protocol described above using single exponential function (n = 9).
Fig 5.
Effects of Nifedipine on the Kv2.1 current deactivation.
(A) The representative tail currents of Kv2.1 deactivation were shown for control, 10 μM and 50 μM nifedipine. The tail currents were generated by 500 ms voltage steps to potentials from -110 mV to -40 mV following a conditioning prepulse to +40 mV. (B) The voltage dependence of deactivation time constants of Kv2.1 before and after application of 10 and 50 μM nifedipine. The deactivation time constant was acquired by fitting tail currents using a mono-exponential decay function (n = 9).
Fig 6.
Nifedipine induced a large left shift of the inactivation curve of Kv2.1 currents.
(A) The schematic illustrated the three-pulse protocol employed to elicit the currents for building the inactivation curve (top panel). Representative Kv2.1 currents generated by three-pulse protocol in the absence (middle panel) and presence (bottom panel) of nifedipine were shown. (B) The inactivation curves of Kv2.1 currents in the absence and presence of 50 μM nifedipine were constructed by plotting the ration of currents (I3/I1) against the P2 voltage. The steady-state inactivation data were well described by a single Boltzmann with V1/2 of—11.4 mV and k of 8.5 mV in control, as well as V1/2 of—38.5 mV and k of 10.1 mV in the presence of 50 μM nifedipine (n = 8).
Fig 7.
Nifedipine block was unaffected by intracellular K+ concentration.
(A) Exposure to 50 μM nifedipine significantly accelerated the inactivation time course of Kv2.1 currents in the presence of 140 and 70 mM pipette K+. However, there was not significant difference for inactivation time course between 140 and 70 mM pipette K+ (n = 9). (B) The percentage block of Kv2.1 currents by 50 μM nifedipine in the presence of 70 and 140 mM intracellular K+ (n = 9).
Fig 8.
Nifedipine shorten the recovery of Kv2.1 currents.
(A) Typical recovery waveforms of Kv2.1 currents were recorded in the absence (upper panel) and presence of 50 μM nifedipine (bottom panel). Currents were elicited by a 100 ms test potential to +40 mV following a 3 s conditioning prepulse to +40 mV with varying recovery intervals from 100 ms to 15 s. The experimental protocol is illustrated between the records. (B) Normalized recovery currents were plotted for control and the presence of 50 μM nifedipine. Data were fitted by a mono-exponential function to obtain the recovery time constant (n = 7).