Electrophysiological characterisation of KCNQ channel modulators

Potassium (K+) ion channels are ubiquitously expressed in mammalian cells, and each channel serves a precise physiological role due to its specific biophysical characteristics and expression pattern. A few K+ channels are targets for certain drugs, and in this thesis it is suggested that the KCNQ K+ channels may be targets for neuroprotective, anti-epileptic and anti-nociceptive compounds. The importance of these channels is underscored by the fact that four out of five KCNQ channel subtypes are involved in severe human diseases. However, the pharmacology of the KCNQ channels is yet poorly understood as these channels were identified only recently. Therefore, there is a need for understanding the biophysical behavior and pharmacology of these ion channels. KCNQ channels belong to the group of voltage-activated K+ channels. The subfamily consists of KCNQ1-5, which is primarily expressed in the CNS, heart, ear, spinal cord, and epithelial cells. Specific members of this subfamily regulate neuronal excitability (through the M-current), contribute to cardiac action potential repolarisation (through the IKs-current), mediate basolateral cAMP-dependent K+ conductance important for epithelial secretion, and are important for K+-recycling in the inner ear necessary for hearing. Retigabine, a compound with anti-epileptic activities activates KCNQ2/KCNQ3 channels. In this thesis, it is shown that retigabine also activates KCNQ2-5 channels stably expressed in HEK293 cells. KCNQ channels were studied in the whole-cell configuration by the patch-clamp technique. Voltage-activated KCNQ currents were enhanced by extracellular application of retigabine, and also by the novel BK channel opener Compound 1 (( )-(5-chloro-2-metoxyphenyl)-1.3-didydroxy-3-fluoro-6-(trifluoromethyl)-2H-indol-2-one) (Gribkoff et al. 2001). The effects of retigabine and Compound 1 were studied in details, and common actions of the two compounds on KCNQ4 channels can be summarised into three main observations; 1) a hyperpolarising shift in the voltage-dependence of channel activation 2) increase in the maximal whole-cell current, and 3) increase in the time of channel deactivation. For KCNQ5 channels no shift in the voltage-dependence of activation was observed. In contrast to retigabine, Compound 1 converted the voltage-activated KCNQ4 current into an instantaneous voltage-independent one. The voltage-independent current was carried by a K+ conductance, was sensitive to linopirdine and XE991, and had a nearly linear I-V relationship. Moreover, development of the voltage-independent current did not require a preceding voltage-dependent activation of the channel. This effect of Compound 1 may have profound hyperpolarising actions on cells expressing the KCNQ4 channels in vivo.