Biophysical characterization of KV3.1 potassium channel activating compounds.

The effect of two positive modulators, RE1 and EX15, on the voltage-gated K(+) channel Kv3.1 was investigated using the whole-cell patch-clamp technique on HEK293 cells expressing Kv3.1a. RE1 and EX15 increased the Kv3.1 currents in a concentration-dependent manner with an EC50 value of 4.5 and 1.3µM, respectively. However, high compound concentrations caused an inhibition of the Kv3.1 current. The compound-induced activation of Kv3.1 channels showed a profound hyperpolarized shift in activation kinetics. 30µM RE1 shifted V1/2 from 5.63±0.31mV to -9.71±1.00mV and 10µM EX15 induced a shift from 10.77±0.32mV to -15.11±1.57mV. The activation time constant (Tauact) was reduced for both RE1 and EX15, with RE1 being the fastest activator. The deactivation time constant (Taudeact) was also markedly reduced for both RE1 and EX15, with EX15 inducing the most prominent effect. Furthermore, subjected to depolarizing pulses at 30Hz, both compounds were showing a use-dependent effect resulting in a reduction of the compound-mediated effect. However, during these conditions, RE1- and EX15-modified current amplitudes still exceeded the control condition amplitudes by up to 200%. In summary, the present study introduces the first detailed biophysical characterization of two new Kv3.1 channel modifying compounds with different modulating properties.

Underlying mechanism of regulatory actions of diclofenac, a nonsteroidal anti-inflammatory agent, on neuronal potassium channels and firing: an experimental and theoretical study.

Diclofenac (DIC), a nonsteroidal anti-inflammatory drug, is known to exert anti-nociceptive and anti-convulsant actions; however, its effects on ion currents, in neurons remain debatable. We aimed to investigate (1) potential effects of diclofenac on membrane potential and potassium currents in differentiated NSC-34 neuronal cells and dorsal root ganglion (DRG) neurons with whole-cell patch-clamp technology, and (2) firing of action potentials (APs), using a simulation model from hippocampal CA1 pyramidal neurons based on diclofenac's effects on potassium currents. In the NSC-34 cells, diclofenac exerted an inhibitory effect on delayed-rectifier K⁺ current (I(KDR)) with an IC50 value of 73 µM. Diclofenac not merely inhibited the I(KDR) amplitude in response to membrane depolarization, but also accelerated the process of current inactivation. The inhibition by diclofenac of IK(DR) was not reversed by subsequent application of either naloxone. Importantly, diclofenac (300 µM) increased the amplitude of M-type K⁺ current (I)(KM)), while flupirtine (10 µM) or meclofenamic acid (10 µM) enhanced it effectively. Consistently, diclofenac (100 µM) increased the amplitude of I(KM) and diminished the I(KDR) amplitude, with a shortening of inactivation time constant in DRG neurons. Furthermore, by using the simulation modeling, we demonstrated the potential electrophysiological mechanisms underlying changes in AP firing caused by diclofenac. During the exposure to diclofenac, the actions on both I(KM) and I(KDR) could be potential mechanism through which it influences the excitability of fast-spiking neurons. Caution needs to be made in attributing the effects of diclofenac primarily to those produced by the activation of I(KM).