Post by Sarah Hill
What's the science?
Potassium channels play an important role in regulating the activity of neurons. They maintain a neuron in an inactive resting state by allowing positively-charged potassium ions to flow out of the cell. Researchers recently identified a subtype of potassium-channels, termed TRESK channels, as a potential therapeutic target in the treatment of migraines. During pain processing, these channels are uniquely responsible for monitoring the excitability of sensory neurons. Two different mutations in the TRESK gene, the TRESK-MT frameshift mutation and the TRESK-C110R mutation, each result in a non-functioning TRESK potassium channel; however, only the TRESK-MT mutation leads to neuronal hyperexcitability and is associated with migraine pathophysiology. How two deleterious mutations, which seem to have the same functional consequence, can have different effects is not well understood. This week in Neuron, Royal and colleagues present an explanation regarding how the MT mutation, but not the C110R mutation, promotes neuron hyperexcitability and migraine induction.
How did they do it?
The authors used a combination of biochemical, optogenetic, and behavioral methods to explore the differential effects of the TRESK-MT and TRESK-C110R mutations on the induction of migraine. They carried out single-molecule pull-down (used to study the activity of a protein) of TRESK, TRESK-MT, and TRESK-C110R to isolate and identify the subtypes of potassium channels that associate with each TRESK protein in sensory neurons. After determining the structural assembly of the normal TRESK channel complex via photobleaching step analysis, they tested its functionality using optogenetic methods (allowing them to activate or inhibit the hybrid protein complex using light). To understand how the MT and C110R mutations alter the function of the hybrid channel protein complexes, they measured the potassium current resulting from co-expression of C110R. Having identified which potassium channels interact with normal the TRESK protein, they then separately overexpressed TRESK-MT mutant channels in normal mice versus those lacking the channel subtypes of interest (TREK1/TREK2 double-knockout mice), again measuring the resulting potassium current to observe any changes in neuronal excitability.
Finally, to uncover a genetic mechanism for how the TRESK-MT mutation exerts its effects on the associated potassium channel subtypes, the authors carried out a close examination of the TRESK-MT DNA sequence and verified the functional consequences through additional biochemical and transgenic analyses. The underlying genetic mechanism — in this case the production of a second truncated TRESK protein induced by the MT mutation — was then overexpressed in rats in order to measure resulting facial mechanical pain threshold as a model of migraine disorder.
What did they find?
In the first round of experiments, the authors found that normal TRESK associates specifically with the potassium channels TREK1 or TREK2 to form a heterodimer (a protein assembly made up of two different, yet similar proteins). Functional manipulation of the protein complex using optogenetics showed that the TRESK-TREK1 and TRESK-TREK2 assemblies resulted in fully functioning hybrid potassium channels. TRESK-MT, but not TRESK-C110R, was found to inhibit the function of TREK1 and TREK2 channels. While the TRESK-C110R mutant did not co-assemble with TREK1 and TREK2, resulting in no change in the potassium currents of these channels, the TRESK-MT mutant had a strong inhibitory effect on potassium currents. This inhibition of potassium currents resulted in an increase in neuronal excitation leading to a migraine phenotype (i.e. observable migraine-like symptoms) in rodents. Importantly, no change in neuronal excitation was observed when TRESK-MT was overexpressed in TREK1/TREK2 double-knockout mice, highlighting the role of the TREK1/2 subunits in this mechanism.
Unexpectedly, single-molecule pull-down of TRESK-MT did not result in co-assembly with TREK1 or TREK2, suggesting that TRESK-MT does not directly associate with the TREK subunits to exert its inhibitory effects and promote neuronal hyperexcitability. Close examination of the TRESK-MT DNA sequence revealed that instead, alternate translation initiation (i.e. an alternate site of initiating the translation of mRNA into protein) of the TRESK gene resulted in the production of a second TRESK protein, termed MT2. Further analyses confirmed that MT2 was capable of directly associating with and inhibiting TREK1/2. Therefore, MT2, but not the original MT mutation, was responsible for inhibiting TREK1/2, thereby facilitating neuronal hyperexcitability and migraine symptoms. When MT2 was virally overexpressed, the increase in neuronal excitation led to an increase in facial pain sensitization in rats, indicating that MT2 is able to produce migraine-like symptoms..
What's the impact?
This is the first study to demonstrate that the TRESK-MT mutation, but not the C110R mutation, initiates an alternative translation initiation of the TRESK coding sequence, resulting in hyperexcitability of sensory neurons. Thus hyperexcitability of sensory neurons leads to a migraine-like phenotype in rodents. This is an important finding because it provides additional support for TRESK potassium channels as a potential therapeutic target in the treatment of migraines, and highlights the need to consider alternative translation initiation as a consequence of other disease-linked frameshift mutations.
Royal et al. Migraine-associated TRESK mutations increase neuronal excitability through alternative translation initiation and inhibition of TREK. Neuron (2018). Access the original scientific publication here.