Functional and behavioral signatures of Kv7 activator drug subtypes

Abstract

Objective: Voltage-gated potassium channels of the KCNQ (Kv7) family are targeted by a variety of activator compounds with therapeutic potential for treatment of epilepsy. Exploration of this drug class has revealed a variety of effective compounds with diverse mechanisms. In this study, we aimed to clarify functional criteria for categorization of Kv7 activator compounds, and to compare the effects of prototypical drugs in a zebrafish larvae model. Methods: In vitro electrophysiological approaches with recombinant ion channels were used to highlight functional properties important for classification of drug mechanisms. We also benchmarked the effects of representative antiepileptic Kv7 activator drugs using behavioral seizure assays of zebrafish larvae and in vivo Ca 2+ imaging with the ratiometric Ca 2+ sensor CaMPARI. Results: Drug effects on channel gating kinetics, and drug sensitivity profiles to diagnostic channel mutations, were used to highlight properties for categorization of Kv7 activator drugs into voltage sensor-targeted or pore-targeted subtypes. Quantifying seizures and ratiometric Ca 2+ imaging in freely swimming zebrafish larvae demonstrated that while all Kv7 activators tested lead to suppression of neuronal excitability, pore-targeted activators (like ML213 and retigabine) strongly suppress seizure behavior, whereas ICA-069673 triggers a seizure-like hypermotile behavior. Significance: This study suggests criteria to categorize antiepileptic Kv7 activator drugs based on their underlying mechanism. We also establish the use of in vivo CaMPARI as a tool for screening effects of anticonvulsant drugs on neuronal excitability in zebrafish. In summary, despite a shared ability to suppress neuronal excitability, our findings illustrate how mechanistic differences between Kv7 activator subtypes influence their effects on heteromeric channels and lead to vastly different in vivo outcomes.

Medium-throughput zebrafish optogenetic platform identifies deficits in subsequent neural activity following brief early exposure to cannabidiol and Δ9-tetrahydrocannabinol

“…In general, there seems to be a good correlation between neural activity measured by CaMPARI and locomotor activity 61 , 62 . However, some findings may be more sensitive towards one assay vs another, as described above.…”

“…Thus, we believe this method is a suitable technique to measure substantial changes in neural activity in the larger parts of the zebrafish CNS. For future studies in connection to epilepsy this innovation may be particularly helpful as some studies use convulstants, PTZ or 4-AP, to simulate seizures 62 , 65 . Indeed, we found that both, PTZ and 4-AP, induce CaMPARI activity.…”

“…Zebrafish expressing CaMPARI, i.e. the Tg[ elavl3 :CaMPARI (W391F + V398L)] ua3144 line were generated as described previously 61 , 62 . We re-derived CaMPARI fish in response to a federal moratorium limiting zebrafish import into Canada 73 .…”

Medium-throughput zebrafish optogenetic platform identifies deficits in subsequent neural activity following brief early exposure to cannabidiol and Δ-9-tetrahydrocannabinol

“…Effects on motor axons will be detected by our behavioural assay. In general, there seems to be a good correlation between neural activity measured by CaMPARI and locomotor activity 61,62 . However, some findings may be more sensitive towards one assay vs the other, as described above.…”

“…We believe this method is a suitable technique to measure substantial changes in neural activity in the larger parts of the zebrafish CNS. For future studies in connection to epilepsy this innovation may be particularly helpful as some studies use convulstants, PTZ or 4-AP, to simulate seizure 62,65 . We found that both, PTZ and 4-AP, induce CaMPARI activity.…”