Characterization of a novel high-potency positive modulator of K(v)7 channels.

K(v)7 channel activators decrease neuronal excitability and might potentially treat neuronal hyperexcitability disorders like epilepsy and mania. Here we introduce NS15370 ((2-(3,5-difluorophenyl)-N-[6-[(4-fluorophenyl)methylamino]-2-morpholino-3-pyridyl]acetamide)hydrochloride, an in vitro high-potency chemical analogue of retigabine, without effects on GABA(A) receptors. NS15370 activates recombinant homo- and heteromeric K(v)7.2-K(v)7.5 channels in HEK293 cells at sub-micromolar concentrations (EC50~100 nM, as quantified by a fluorescence based Tl⁺-influx assay). In voltage clamp experiments NS15370 exhibits a complex, concentration-dependent mode-of-action: At low concentrations it accelerates voltage-dependent activation rates, slows deactivations, and increases steady-state current amplitudes. Quantified by the peak-tail current method, the V½ value of the steady-state activation curve is shifted towards hyperpolarized potentials at concentrations ~100 times lower than retigabine. However, in contrast to retigabine, NS15370 also introduces a distinct time-dependent current decrease, which eventually, at higher concentrations, causes suppression of the current at depolarized potentials, and an apparent "cross-over" of the voltage-activation curve. In brain slices, NS15370 hyperpolarizes and increases spike frequency adaptation of hippocampal CA1 neurons and the compound reduces the autonomous firing of dopaminergic neurons in the substantia-nigra pars compacta. NS15370 is effective in rodent models of hyperexcitability: (i) it yields full protection against mouse 6 Hz seizures and rat amygdala kindling discharges, two models of partial epilepsia; (ii) it reduces (+)-MK-801 hydrogen maleate (MK-801)-induced hyperactivity as well as chlordiazepoxide (CDP)+d-amphetamine (AMP)-induced hyperactivity, models sensitive to classic anti-psychotic and anti-manic treatments, respectively. Our findings with NS15370 consolidate neuronal K(v)7 channels as targets for anti-epileptic and psychiatric drug development.

Inhibitory gating modulation of small conductance Ca2+-activated K+ channels by the synthetic compound (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphtylamine (NS8593) reduces afterhyperpolarizing current in hippocampal CA1 neurons.

SK channels are small conductance Ca(2+)-activated K(+) channels important for the control of neuronal excitability, the fine tuning of firing patterns, and the regulation of synaptic mechanisms. The classic SK channel pharmacology has largely focused on the peptide apamin, which acts extracellularly by a pore-blocking mechanism. 1-Ethyl-2-benzimidazolinone (1-EBIO) and 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309) have been identified as positive gating modulators that increase the apparent Ca(2+) sensitivity of SK channels. In the present study, we describe inhibitory gating modulation as a novel principle for selective inhibition of SK channels. In whole-cell patch-clamp experiments, the compound (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphtylamine (NS8593) reversibly inhibited recombinant SK3-mediated currents (human SK3 and rat SK3) with potencies around 100 nM. However, in contrast to known pore blockers, NS8593 did not inhibit (125)I-apamin binding. Using excised patches, it was demonstrated that NS8593 decreased the Ca(2+) sensitivity by shifting the activation curve for Ca(2+) to the right, only slightly affecting the maximal Ca(2+)-activated SK current. NS8593 inhibited all the SK1-3 subtypes Ca(2+)-dependently (K(d) = 0.42, 0.60, and 0.73 microM, respectively, at 0.5 microM Ca(2+)), whereas the compound did not affect the Ca(2+)-activated K(+) channels of intermediate and large conductance (hIK and hBK channels, respectively). The site of action was accessible from both sides of the membrane, and the NS8593-mediated inhibition was prevented in the presence of a high concentration of the positive modulator NS309. NS8593 was further tested on mouse CA1 neurons in hippocampal slices and shown to inhibit the apaminand tubocurarine-sensitive SK-mediated afterhyperpolarizing current, at a concentration of 3 microM.