Abnormal or synchronous neuronal activity in the brain leads to epileptic seizures that, when repeated or prolonged, can cause neuronal damage resulting in delayed psychomotor development, intellectual disability and other neurological disorders. In neurons, action potentials are terminated by the inactivation of Na+ channels and by the repolarizing outward currents triggered by activation of K+ channels. One of the major potassium current in neurons is the muscarine-regulated M-current, a non-inactivating slow current that is activated at subthreshold voltages. The M-current, which is generated from the heteromerization of KCNQ2 and KCNQ3 channels (IKM), activates in the time frame of action potential initiation, providing a crucial role in controlling neuronal excitability. The slow kinetics of activation and deactivation of the IKM (KCNQ2/KCNQ3) channel regulates the membrane potential and impedes repetitive neuronal firing. A growing number of inherited mutations have been found in the IKM channel that cause a wide spectrum of early-onset epileptic disorders ranging from benign familial neonatal seizures to severe epileptic encephalopathies. I will determine the molecular mechanisms by which a set of epileptic-inducing mutations in KCNQ2 and KCNQ3 cause malfunction of the IKM channel. I will use a fluorescence assay, voltage clamp fluorometry (VCF), to simultaneously measure voltage sensor movement and gate opening during IKM channel activation in these mutations. Knowing the mechanisms that lead to defective channel function is essential to study how to modulate and ultimately restore function of these mutated channel. Furthermore, the IKM channel is an attractive pharmacological target to treat hyperexcitability-related diseases, such as epilepsy, because increasing the M-current stabilizes the resting and subthreshold membrane potential, thereby reducing membrane excitability. Lipophilic compounds, such as polyunsaturated fatty acids (PUFAs), have been shown to modulate neuronal function. In particular, PUFAs have been shown to improve the outcomes of epilepsy, therefore constituting very promising anti-epileptic agents. However, the molecular mechanism of action of PUFAs is unknown. For example, it is unknown whether PUFAs affect the voltage sensor movement, gate movement, or both. This is important because knowing the channel region where PUFAs act will allow designing PUFAs derivatives to more specifically tackle the IKM channel. Based on the molecular mechanism for each epileptic-inducing IKM channel mutation, I will assess, using VCF, which type of PUFAs variants would be most suitable to restore physiological channel activity in order to develop an antiepileptic drug. I will also use induced pluripotent stem cell (iPSC)-derived neurons, a simple but powerful model, to test the efficacy of PUFAs on the derived neurons expressing mutated KCNQ2 and KCNQ3 to test both the mechanistic implications of the proposed work and the therapeutic potential of the PUFAs. The anticipated results of these experiments will provide the basis to mechanistically understand how different mutations cause IKM channel defects, and should show proof-of-concept that PUFAs can act as antiepileptic drugs. This would be a milestone toward mutation-specific treatments of epilepsy and other neurological disorders caused by mutations in the IKM channel.
A growing number of inherited mutations have been found in the IKM channels that cause epilepsy in patients. I have recently identified that PUFAs act on IKM channels and have antiepileptic effect when applied to heterologous expressed channels. I will here test more variants of these compounds on heterologously expressed channels and on induced pluripotent stem cells (iPSC)-derived neurons expressing wt IKM an mutated channels in order to develop an antiepileptic drug that will lead to better treatments of epileptic encephalopathy and other neurological disorders.
|Lassuthova, Petra; Rebelo, Adriana P; Ravenscroft, Gianina et al. (2018) Mutations in ATP1A1 Cause Dominant Charcot-Marie-Tooth Type 2. Am J Hum Genet 102:505-514|
|Barro-Soria, Rene; Ramentol, Rosamary; Liin, Sara I et al. (2017) KCNE1 and KCNE3 modulate KCNQ1 channels by affecting different gating transitions. Proc Natl Acad Sci U S A 114:E7367-E7376|
|Barro-Soria, Rene; Liin, Sara I; Larsson, H Peter (2017) Using fluorescence to understand ? subunit-NaV channel interactions. J Gen Physiol :|