Pore- and voltage sensor-targeted KCNQ openers have distinct state-dependent actions.

Ion channels encoded by KCNQ2-5 generate a prominent K+ conductance in the central nervous system, referred to as the M current, which is controlled by membrane voltage and PIP2. The KCNQ2-5 voltage-gated potassium channels are targeted by a variety of activating compounds that cause negative shifts in the voltage dependence of activation. The underlying pharmacology of these effects is of growing interest because of possible clinical applications. Recent studies have revealed multiple binding sites and mechanisms of action of KCNQ activators. For example, retigabine targets the pore domain, but several compounds have been shown to influence the voltage-sensing domain. An important unexplored feature of these compounds is the influence of channel gating on drug binding or effects. In the present study, we compare the state-dependent actions of retigabine and ICA-069673 (ICA73, a voltage sensor-targeted activator). We assess drug binding to preopen states by applying drugs to homomeric KCNQ2 channels at different holding voltages, demonstrating little or no association of ICA73 with resting states. Using rapid solution switching, we also demonstrate that the rate of onset of ICA73 correlates with the voltage dependence of channel activation. Retigabine actions differ significantly, with prominent drug effects seen at very negative holding voltages and distinct voltage dependences of drug binding versus channel activation. Using similar approaches, we investigate the mechanistic basis for attenuation of ICA73 actions by the voltage-sensing domain mutation KCNQ2[A181P]. Our findings demonstrate different state-dependent actions of pore- versus voltage sensor-targeted KCNQ channel activators, which highlight that subtypes of this drug class operate with distinct mechanisms.

Four drug-sensitive subunits are required for maximal effect of a voltage sensor-targeted KCNQ opener.

KCNQ2-5 (Kv7.2-Kv7.5) channels are strongly influenced by an emerging class of small-molecule channel activators. Retigabine is the prototypical KCNQ activator that is thought to bind within the pore. It requires the presence of a Trp side chain that is conserved among retigabine-sensitive channels but absent in the retigabine-insensitive KCNQ1 subtype. Recent work has demonstrated that certain KCNQ openers are insensitive to mutations of this conserved Trp, and that their effects are instead abolished or attenuated by mutations in the voltage-sensing domain (VSD). In this study, we investigate the stoichiometry of a VSD-targeted KCNQ2 channel activator, ICA-069673, by forming concatenated channel constructs with varying numbers of drug-insensitive subunits. In homomeric WT KCNQ2 channels, ICA-069673 strongly stabilizes an activated channel conformation, which is reflected in the pronounced deceleration of deactivation and leftward shift of the conductance-voltage relationship. A full complement of four drug-sensitive subunits is required for maximal sensitivity to ICA-069673-even a single drug-insensitive subunit leads to significantly weakened effects. In a companion article (see Yau et al. in this issue), we demonstrate very different stoichiometry for the action of retigabine on KCNQ3, for which a single retigabine-sensitive subunit enables near-maximal effect. Together, these studies highlight fundamental differences in the site and mechanism of activation between retigabine and voltage sensor-targeted KCNQ openers.

Sequence determinants of subtype-specific actions of KCNQ channel openers.

KEY POINTS: Retigabine is a KCNQ voltage-gated potassium channel opener that was recently approved as an add-on therapeutic for patients with drug-resistant epilepsy. Retigabine exhibits very little specificity between most KCNQ channel subtypes, and there is interest in generating more potent and specific KCNQ channel openers. The present study describes the marked specificity of ICA069673 for KCNQ2 vs. KCNQ3, and exploits this property to investigate determinants of KCNQ subtype specificity. ICA069673 acts on a binding site in the voltage-sensing domain that is distinct from the putative retigabine site in the channel pore. ICA069673 has two separable effects on KCNQ channel activity. We identify two channel residues required for subtype specificity of KCNQ channel openers and show that these are sufficient to generate ICA069673 sensitivity in KCNQ3. ABSTRACT: Retigabine (RTG) is the first approved anti-epileptic drug that acts via activation of voltage-gated potassium channels, targeting KCNQ channels that underlie the neuronal M-current. RTG exhibits little specificity between KCNQ2-5 as a result of conservation of a Trp residue in the pore domain that binds to the drug. The RTG analogue ICA-069673 ('ICA73') exhibits much stronger effects on KCNQ2 channels, including a large hyperpolarizing shift of the voltage-dependence of activation, an ∼2-fold enhancement of peak current and pronounced subtype specificity for KCNQ2 over KCNQ3. Based on ICA73 sensitivity of chimeric constructs of the transmembrane segments of KCNQ2 and KCNQ3, this drug appears to interact with the KCNQ2 voltage sensor (S1-S4) rather than the pore region targeted by RTG. KCNQ2 point mutants in the voltage sensor were generated based on KCNQ2/KCNQ3 sequence differences, and screened for ICA73 sensitivity. These experiments reveal that KCNQ2 residues F168 and A181 in the S3 segment are essential determinants of ICA73 subtype specificity. Mutations at either position in KCNQ2 abolish the ICA73-mediated gating shift, but preserve RTG sensitivity. Interestingly, A181P mutant channels show little ICA73-mediated gating shift but retain current potentiation by the drug. Mutations (L198F and P211A), which introduce these critical KCNQ2 residues at corresponding positions in KCNQ3, transplant partial ICA73 sensitivity. These findings demonstrate that RTG and ICA73 act via distinct mechanisms, and also reveal specific residues that underlie subtype specificity of KCNQ channel openers.

An automated LC method for the small-scale purification of organic molecules derived from combinatorial libraries.

This paper presented the development of an automated HPLC small-scale purification method for single bead compounds derived from combinatorial libraries. The method was found to produce higher and more consistent recoveries of purified compounds as compared to conventional manual HPLC purification. Using the manual method, the average percentage recovery of one synthetic compound was determined to be 24% and the coefficients of variation (C.V.%) of recovery were found to be greater than 38%. Using the automated system, the average percentages recovery of a standard compound at 600 and 1000 micromol l(-1) were determined to be 72.63+/-10.17% and 81.34+/-4.39%, respectively. This represented an approximate 3-folds increase in percentage recovery compared to that of the manual small-scale purification process. It was also found that the C.V.% of recovery were less than 15% at both concentration levels. The development of this automated method was found to be straightforward. The importance and implications of this study were discussed.