Interaction of Riluzole with the Closed Inactivated State of Kv4.3 Channels

Abstract

The effect of riluzole on Kv4.3 was examined using the wholecell patch-clamp technique. Riluzole inhibited the peak amplitude of Kv4.3 in a reversible, concentration-dependent manner with an IC 50 of 115.6 M. Under control conditions, a good fit for the inactivation of Kv4.3 currents to a double exponential function, with the time constants of the fast component ( f ) and the slow component ( s ), was obtained. f was not altered by riluzole at concentrations up to 100 M, but s became slower with increasing riluzole concentration, resulting in the crossover of the currents. The inhibition increased steeply with increasing channel activation at more positive potentials. In the full activation voltage range positive to ϩ30 mV, however, no voltagedependent inhibition was found. Riluzole shifted the voltage dependence of the steady-state inactivation of Kv4.3 in the hyperpolarizing direction in a concentration-dependent manner. However, the slope factor was not affected by riluzole. The K i for riluzole for interacting with the inactivated state of Kv4.3 was estimated from the concentration-dependent shift in the steady-state inactivation curve and was determined to be 1.2 M. Under control conditions, closed state inactivation was fitted to a single exponential function. Riluzole caused a substantial acceleration in the closed state inactivation. In the presence of riluzole, the recovery from inactivation was slower than under control conditions. Riluzole induced a significant use-dependent inhibition of Kv4.3. These results suggest that riluzole inhibits Kv4.3 by binding to the closed inactivated state of the channels and that the unbinding of riluzole occurs from the closed state during depolarization.

Rosiglitazone inhibits Kv4.3 potassium channels by open‐channel block and acceleration of closed‐state inactivation

“…Figure 1A shows the superimposed Kv4.3 channel currents that resulted from a 500 ms depolarizing pulse to +40 mV under control conditions and in the presence of rosiglitazone. Under control conditions, the Kv4.3 currents were activated to maximum and then were rapidly inactivated as reported previously (Ohya et al, 1997;Ahn et al, 2006;Kim et al, 2007). Rosiglitazone not only reduced the peak amplitude of the Kv4.3 currents but also altered the time course of the current decay.…”

“…The cells were transfected with cDNA encoding Kv4.3 channels using the Lipofectamine reagent (Invitrogen, Grand Island, NY, USA), as described previously (Ahn et al, 2006). The stable cell line expressing K v4.3 channels was maintained in a humidified atmosphere at 37°C with 5% CO2-enriched air.…”

Inhibition of Kv4.3 by genistein via a tyrosine phosphorylation-independent mechanism

“…Figure 1A shows the superimposed Kv4.3 currents expressed in CHO cells under control conditions and in the presence of various concentrations of genistein. Under control conditions, Kv4.3 currents were activated to a peak and then were rapidly inactivated during a 500-ms pulse of ϩ40 mV at 10-s intervals, as described previously (2,22,30). In the presence of genistein, inhibition of Kv4.3 currents was substantiated by a concentration-dependent reduction in peak current amplitude.…”

“…The Kv4.3 cDNA was stably transfected into CHO cells (American Type Culture Collection, Manassas, VA) using the lipofectamine reagent (Invitrogen, Grand Island, NY), as described previously (2,30). CHO cells were cultured in Iscove's modified Dulbecco's medium (Invitrogen), supplemented with 10% fetal bovine serum, 2 mM glutamine, 0.1 mM hypoxanthine, and 0.01 mM thymidine, under a 95% humidified air-5% CO 2 environment at 37°C.…”

Open Channel Block of A-Type, Kv4.3, and Delayed Rectifier K+ Channels, Kv1.3 and Kv3.1, by Sibutramine

“…The K v 4.3, K v 1.3, and K v 3.1 cDNA were stably transfected into CHO cells (American Type Culture Collection, Manassas, VA) using the Lipofectamine reagent (Invitrogen, Grand Island, NY) as described previously (Hahn et al, 1996;Choi et al, 1999;Ahn et al, 2006). CHO cells were maintained in Iscove's modified Dulbecco's medium (Invitrogen) supplemented with 10% fetal bovine serum, 0.1 mM hypoxanthine, and 0.01 mM thymidine in a humidified 5% CO 2 incubator at 37°C.…”