XE991 and Linopirdine Are State-Dependent Inhibitors for Kv7/KCNQ Channels that Favor Activated Single Subunits

Abstract

M-channel inhibitors, especially XE991, are being used increasingly in animal experiments; however, insufficient characterization of XE991 at times confounds the interpretation of results when using this compound. Here, we demonstrate that XE991 and linopirdine are state-dependent inhibitors that favor the activated-subunit of neuronal Kv7/KCNQ channels. We performed patch-clamp experiments on homomeric Kv7.2 or heteromeric Kv7.2/3 channels expressed in Chinese hamster ovary cells to characterize XE991 and linopirdine. Neither inhibitor was efficacious around the resting membrane potential of cells in physiologic conditions. Inhibition of Kv7.2 and Kv7.2/3 channels by XE991 was closely related with channel activation. When the voltage dependence of activation was left-shifted by retigabine or right-shifted by the mutation, Kv7.2(R214D), the shift in half-activation voltage proportionally coincided with the shift in the half-effective potential for XE991 inhibition. Inhibition kinetics during XE991 wash-in was facilitated at depolarized potentials. Ten-minute washout of XE991 resulted in ∼30% current recovery, most of which was attributed to surface transport of Kv7.2 channels. Linopirdine also exhibited similar inhibition characteristics, with the exception of near- complete current recovery after washout at depolarized potentials. Inhibition kinetics of both XE991 and linopirdine was not as sensitive to changes in voltage as would be predicted by open- channel inhibition. Instead, they were well explained by binding to a single activated subunit. The characteristics of XE991 and linopirdine should be taken into account when these M-channel inhibitors are used in experiments.

Attenuating M‐current suppression in vivo by a mutant Kcnq2 gene knock‐in reduces seizure burden and prevents status epilepticus–induced neuronal death and epileptogenesis

“…This duration of XE991 effect is consistent with published pharmacokinetic studies . In addition, our recent study shows that XE991 is an irreversible inhibitor for Kv7 channels, suggesting that recovery is due to removal of XE991 from the system rather than washout of XE991 from Kv7 channels . Our FJC staining results suggest that recovery of M‐current after XE991 inhibition, which occurs after status epilepticus, is still sufficient to prevent neuronal degeneration.…”

“…31,32 In addition, our recent study shows that XE991 is an irreversible inhibitor for Kv7 channels, suggesting that recovery is due to removal of XE991 from the system rather than washout of XE991 from Kv7 channels. 33 Our FJC staining results suggest that recovery of M-current after XE991 inhibition, which occurs after status epilepticus, is still sufficient to prevent neuronal degeneration. Nonetheless, the neuroprotective effects in Kv7.2(S559A) mice were unexpected because we initially assumed that postseizure neuronal degeneration was due to excitotoxicity as a result of status epilepticus.…”

Differential contributions of Ca2+‐activated K+ channels and Na+/K+‐ATPases to the generation of the slow afterhyperpolarization in CA1 pyramidal cells

“…One hypothetical possibility is that their voltage of activation is shifted negatively due to increases in cellular PIP 2 associated with repetitive firing (Kim, Duignan, Hawryluk, Soh, & Tzingounis, ). Here we show that blocking K V 7 channels with XE991, whose voltage‐dependent efficacy in blocking these channels dynamically matches their voltage range of activation (Greene, Kang, & Hoshi, ), does not affect the sAHP in CA1 pyramidal cells. Therefore we conclude that K V 7 channels do not contribute to the sAHP in these neurons.…”

KCNQ/M-channels regulate mouse vagal bronchopulmonary C-fiber excitability and cough sensitivity

“…Upon washout of XE991, the inhibition of currents observed at voltages ≥-5 mV was largely relieved. However, the inhibitory effects of XE991 on currents recorded at voltages negative to -15 mV (including the currents reactivated by steps from -60 mV to V h ) and the corresponding I tail could not be reversed by washout of the drug, suggesting that these currents are mediated by M-channels, which have been showed to be irreversibly blocked by XE991 (21,22). These effects are well illustrated with total XE991-sensitive currents, obtained by digital subtraction of currents recorded in the presence of XE991 from those recorded under control conditions; reversible XE991-sensitive currents, obtained by digital subtraction of currents recorded in the presence of XE991 from those recorded after washout of the drug; and irreversible XE991-sensitive currents, derived by digital subtraction of currents recorded after washout of XE991 from those under the control condition (Figure 2A, lower panels).…”