High Effectiveness in Actions of Carfilzomib on Delayed-Rectifier K+ Current and on Spontaneous Action Potentials

Figures

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  Figure 1. Effect of CFZ on I K(DR) density in GH3 cells. In these whole-cell current recordings, cells were immersed in Ca2+-free Tyrode’s solution and the recording pipette was filled with K+-containing solution. The examined cells were held at −50 mV and different voltage pulses ranging from −60 to +50 mV in 10-mV increments were applied. (A) Representative traces for I K(DR) density taken from the absence (upper; control) and presence (lower; CFZ 0.3) of 0.3 μM CFZ. The upper part in (A) indicates the voltage protocol applied. (B) Averaged current density versus voltage relationships of I K(DR) measured at the beginning (filled symbols; upper [peak]) and end (open symbols; lower [steady-state]) of each depolarizing pulse. The data points in the upper (peak) and lower (steady-state) panels were obtained from the absence (square symbols) and presence (circle symbols) of 0.3 μM CFZ, respectively. Each data point represents mean ± SEM (n = 9). Summary bar graphs in (C) and (D) respectively show the whole-cell conductances of I K(DR) at the beginning (i.e., peak conductance) and end (i.e., steady-state conductance) of depolarizing pulses ranging between 0 and +50 mV (mean ± SEM; n = 8 for…

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  Figure 2. Evaluation of the kinetics of CFZ-induced block of I K(DR) elicited by membrane depolarization in GH3 cells. The I K(DR) established by depolarizing pulse from −50 to +50 mV with a duration of 1 s (indicated in the upper part of (A)) was measured as cells were exposed to different CFZ concentrations. In (A), representative I K(DR) traces was obtained in the absence (control) and presence of 0.1 μM CFZ (CFZ 0.1) or 0.3 μM CFZ (CFZ 0.3). In (B), the time courses of relative block of I K(DR) by 0.1 μM CFZ (CFZ 0.1) and 0.3 μM CFZ (CFZ 0.3) were well fitted by single exponential with a value of 181 and 91 ms (indicated by the smooth curves). The relative block (i.e., (I control− I CFZ)/I control) was evaluated by dividing the CFZ-sensitive current by the current taken from the control (i.e., in the absence of CFZ). In (C), the reciprocal of time constant (i.e., 1/τ) of relative block versus the CFZ concentration was plotted. Data points appearing in open squares were fitted by a linear regression, indicating that there is a molecularity of one. Mean ± SEM (n = 9 for each point). (D) Concentration-dependent inhibition of I K(DR) by CFZ (mean ± SEM; n = 9 for each point). Each cell was…

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  Figure 3. Effect of CFZ on the steady-state inactivation of I K(DR) in GH3 cells. (A) Representative I K(DR) traces obtained in the absence (upper) and presence (lower) of 0.3 µM CFZ. As the examined cell was held at −50 mV, the conditioning voltage pulses with a duration of 1 s to various membrane potentials ranging from −80 to +30 mV were delivered. Following each condition pulse applied, a test pulse to +50 mV with a duration of 500 ms was then applied to elicit I K(DR). The upper part indicates the voltage protocol used. (B) Steady-state inactivation curve of I K(DR) in the absence (filled squares) and presence of 0.3 µM (open circles) and 1 µM (open squares) CFZ (mean ± SEM; n = 8 for each point). The smooth curves were least-squares fitted by a Boltzmann function (detailed under Materials and Methods).

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  Figure 4. Time course of the recovery of I K(DR) in the presence of 0.3 and 1 µM CFZ. In these experiments, GH3 cells, bathed in Ca2+-free Tyrode’s solution, were depolarized from −50 to +50 mV with a duration of 300 ms and different interpulse durations were applied. (A) Representative I K(DR) traces in the absence (upper) and presence (lower) of 0.3 µM CFZ elicited by a two-pulse protocol with different interpulse durations (indicated in the upper part). Dashed line indicates the zero current level. (B) Time courses of recovery from I K(DR) inactivation caused by 0.3 µM CFZ (open squares) and 1 µM CFZ (open circles), respectively. Smooth curves in the presence of different CFZ concentrations were fitted by single exponential. Each point represents the mean ± SEM (n = 8). Asterisks indicate the peak I K(DR) elicited by different interpulse intervals.

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  Figure 5. Effect of CFZ on I K(M) in GH3 cells. To record I K(M), we bathed cells in high-K+, Ca2+-free solution and filled the recording pipette by using K+-containing solution. (A) Representative I K(M) traces in response to membrane depolarization (indicated in the upper part). CFZ 0.3:0.3 µM CFZ; CFZ 1:1 µM CFZ. Arrowhead shows the zero current level. (B) Bar graph showing the effects of CFZ and CFZ plus ML-213 on I K(M) amplitude taken from GH3 cells (mean ± SEM; n = 8–9 for each bar). Current amplitude was measured at the end of depolarizing pulse from −50 to −20 mV. *Significantly different from control (P < 0.05) and †significantly different from 1 µM CFZ alone group (P < 0.05).

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  Figure 6. Effect of CFZ on spontaneous action potentials (APs) in GH3 cells. The experiments were conducted in cells bathed in normal Tyrode’s solution containing 1.8 mM CaCl2. As whole-cell configuration was achieved, the recording model was rapidly switched to current-clamp condition for measuring any changes in membrane potential. (A) Representative potential changes obtained in the absence (control) of CFZ and the presence of 0.3 µM CFZ (CFZ 0.3) and 1 µM CFZ (CFZ 1). (B) Bar graph depicting efficacy of CFZ to elevate the firing frequency of spontaneous APs in GH3 cells (mean ± SEM; n = 9 for each bar). *Significantly different from control (P < 0.05).

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  Figure 7. Inhibitory effect of CFZ on I K(DR) in A7r5 vascular smooth muscle cells (A) and in H9c2 cardiac cells (B). In these experiments, cells were bathed in Ca2+-free Tyrode’s solution and the pipette was filled with K+-containing solution. Superimposed I K(DR) traces recorded from either an A7r5 cell (A) or an H9c2 cell (B) were respectively obtained in the absence (control) and presence of 0.1 µM CFZ (CFZ 0.1) and 0.3 µ M CFZ (CFZ 0.3). Arrowhead indicates the zero current level, and inset shown in each panel denotes the voltage protocol applied. (C) Inactivation time course of I K(DR) in the absence and presence of 0.1 or 0.3 µM CFZ measured from H9c2 cells. Smooth curves labeled in control, CFZ 0.1 or CFZ 0.3 were least-squares fitted by single exponential.