Summary: A new study sheds light on the biological mechanism behind some genetic forms of epilepsy.
Source: Linkoping University
Sometimes, even the alteration of a single nucleotide in a gene can cause serious disease. In a young boy with epilepsy, this kind of mutation has not just affected the functioning of the protein in question – it could also curb the functioning of several closely related proteins.
This has been shown in a study, published in the journal PNAS, by researchers in Sweden and the USA. The study sheds light on the molecular biology behind some forms of genetic epilepsy.
In this study, the researchers found a previously unknown mutation in a child with epilepsy. He has a very subtle change – a single nucleotide that is different – in the gene KCNA2, which produces an ion channel protein. Ion channels are proteins that form pores in the cell surface membrane.
When the channels open, they allow the flow of specific electrically charged ions into or out of the cell. This triggers or terminates electrical impulses in cells, such as nerve or muscle cells. Thus, ion channels are important for, among other things, brain function.
Ordinarily, there is a balance in the brain between signals that increase cell activity and ones which suppress it. In epilepsy, the balance is disturbed, and the nerve cells send signals in an uncontrolled manner. The ion channel which has mutated in the patient normally has a dampening effect on nerve signaling.
“What is interesting with this ion channel is that both mutations that increase function and those that decrease function have been connected to epilepsy. It seems that you just need the right amount of activity, otherwise the risk to develop seizures increases”, says Antonios Pantazis, associate professor at the Department of Biomedical and Clinical Sciences, and the Wallenberg Center for Molecular Medicine at Linköping University.
Using various experimental methods, the research team found that cells could produce the mutant channel proteins, but they could not transport them to their surface membrane. As mutant channels stayed trapped inside the cell, there was no measurable ion channel activity.
There are two copies of most of our genes. The patient had one mutated copy and one normal one, so one would expect that he had 50% of functional ion channels. But when the scientists reproduced this condition in the lab, it turned out that the channel activity was as low as 20 percent, compared to normal. The mutation apparently decreased the function of the normal proteins, too: a “dominant negative” mutation.
To understand how this happened, we need to consider that this ion channel comprised four interconnected proteins. The researchers showed that proteins from the mutant gene can connect with proteins made by the normal copy, and also keep them trapped inside the cell.
And here comes the crux of the story: there are, in fact, several ion-channel genes related to KCNA2. Proteins made from these different genes often mix together to form ion channels. When the scientists mixed proteins from mutant KCNA2 with those of related gene KCNA4they found that KCNA4 proteins were also prevented from transporting to the cell surface.
Pantazis explains that when different channel proteins combine with each other to create mixed ion channels, this contributes to the great diversity and complexity of nerve cell signaling. This diversity is important for the processes that we associate with the functioning of the brain, such as thoughts, consciousness and the ability to imagine.
Yet this ability of channel proteins to combine also confers a disadvantage, as the brain becomes vulnerable to single, dominant-negative mutations, which can disrupt the function of several ion channels, and cause neurological disease, as in this story.
“Studying the effects of mutations on ion channels can give us vital clues about the mechanisms of disease and potential therapeutic strategies”, says Michelle Nilsson, a PhD student in Pantazis’ research group, and principal author on the study.
“Since these mutations can have unexpected effects on ion-channel function, studying them can also lead to new discoveries about how our bodies work at the molecular level,” Antonios Pantazis says.
Funding: The study has been funded by the Knut and Alice Wallenberg Foundation through the Wallenberg Center for Molecular Medicine (WCMM) at Linköping University, and the Swedish Research Council.
About this genetics and epilepsy research news
Abstract: Karin Soderlund Leifler
Source: Linkoping University
Contact: Karin Söderlund Leifler – Linköping University
Picture: The image is in the public domain
OriginalResearch: Open access.
“An epilepsy-associated K1.2 charge-transfer-center mutation impairs K1.2 and K1.4 trafficking” by Michelle Nilsson et al. PNAS
An epilepsy-associated K1.2 charge-transfer-center mutation odds K1.2 and K1.4 trafficking
We report on a heterozygous KCNA2 variant in a child with epilepsy. KCNA2 K-encodesV1.2 subunits, which form homotetrameric potassium channels and participate in heterotetrameric channel complexes with other KV1-family subunits, regulating neuronal excitability.
The mutation causes substitution F233S at the KV1.2 charge transfer center of the voltage-sensing domain. Immunocytochemical trafficking assays showed that KV1.2(F233S) subunits are trafficking deficient and reduce the surface expression of wild-type KV1.2 and KV1.4: a dominant-negative phenotype extending beyond KCNA2likely profoundly disturbing electrical signaling.
Yet some KV1.2(F233S) trafficking was rescued by wild-type KV1.2 and KV1.4 subunits, likely in permissible heterotetrameric stoichiometries: electrophysiological studies utilizing applied transcriptomics and concatemer constructs support that up to one or two KV1.2(F233S) subunits can participate in trafficking-capable heterotetramers with wild-type KV1.2K goldV1.4, respectively, and that both early and late events along the biosynthesis and secretion pathway odd trafficking.
These studies suggested that F233S causes a depolarizing shift of ∼48 mV on KV1.2 voltage dependence. Optical tracking of the KV1.2(F233S) voltage-sensing domain (rescued by wild-type KV1.2K goldV1.4) revealed that it operates with modestly perturbed voltage dependence and retains pore coupling, evidenced by off-charge immobilization.
The equivalent mutation in the Shaker K+ channel (F290S) was reported to modestly affect trafficking and strongly affect function: an ∼80-mV depolarizing shift, disrupted voltage sensor activation and pore coupling.
Our work exposes the multigenic, molecular etiology of a variant associated with epilepsy and reveals that charge-transfer-center disruption has different effects in KV1.2 and Shaker, the archetypes for potassium channel structure and function.