Functional Effect Of A De Novo Mutation In BK Channel Associated With Epilepsy

Epilepsy is one of the most common neurological disorders,characterized by recurrent and spontaneous epileptic seizures.An epileptic seizure is a temporary disruption of brain function due to the hypersynchronous,abnormal firing of cortical neurons.Epilepsy causes profound morbidity and mortality,while the etiology and pathogenesis of epilepsy are not very clear.There are many possible causes for epilepsy,including genetic,acquired and environmental factors as well as their interactions.Epilepsy can be caused by mutations in single genes that code for ion channels,such as voltage-activated ion channels,the nicotine acetylcholine receptor and GABAA receptor.We previously reported a KCNMA1 mutation?p.D434G?that causes a syndrome of coexistent generalized epilepsy and paroxysmal dyskinesia?GEPD?.The finding established the association between KCNMA1 and epilepsy for the first time.KCNMA1encodes the BK channel,which is a large conductance calcium-and voltage-activated potassium channel.BK channel is expressed ubiquitously in many tissues and cells and involved in many vital physiological processes.The voltage sensor of the channel is located within S4 transmembrane segment,and the calcium-binding sites are located in cytosolic RCK domains.The mechanism by which p.D434G mutation causes epilepsy is that p.D434G mutation can enhance BK channel activation by increasing the calcium sensitivity of the channel.In this study,we have identified four new KCNMA1 mutations,c.2984A>G?p.N995S?,c.3476A>G?p.N1159S?,c.1967A>C?p.E656A?and c.1554G>T?p.K518N?.Interestingly,the four new KCNMA1 mutations were all identified from five independent patients who were affected with epilepsy,but not with paroxysmal dyskinesia.However,it is unknown whether the four new mutations cause disease or not,and if so,it is important to identify the underlying molecular mechanism.Intriguingly,the c.2984A>G?p.N995S?mutation occurs as a de novo mutation in two independent epileptic patients,which provides a strong genetic evidence for the association between this mutation and epilepsy.Functional studies are an important tool to determine whether a disease-associated genomic variant is a disease-causing mutation or benign polymorphism.Therefore,we characterized the four new KCNMA1 mutations by patch clamp recordings.We introduced these mutations into an expression plasmid of BK channel and expressed these mutant BK channels in HEK293 cells.We then performed patch clamping,including macroscopic current recording and single channel current recording.We found that the p.N995S mutation shifted the GV curve of the BK channel to the negative potential direction by 59 mV at 10?M Ca²⁺,whereas the other three mutations did not affect the BK channel function.In addition,the p.N995S mutation could still shift the GV curve of the BK channel to the negative potential direction by 71 mV at 10?M Ca²⁺when the?4 subuint was present.The functional data further indicated that the p.N995S mutation was a disease-causing mutation for epilepsy.On the other hand,variants c.3476A>G?p.N1159S?,c.1967A>C?p.E656A?and c.1554G>T?p.K518N?were likely to be benign polymorphisms that do not lead to epilepsy.Given that the p.N995S mutation was located within the RCK2 domain,we next studied the calcium sensitivity of the mutant channel.The data showed that the p.N995S mutation could enhance the channel activation by a similar degree at different calcium concentrations.Moreover,the p.N995S mutation could still enhance the BK channel activation even when the channel's ability to sense calcium is abolished by mutations at the calcium-binding sites?p.2D2A/p.5D5N?.These results indicated that the p.N995S mutation did not affect the calcium sensitivity of the channel.Through the single channel current recording,we found that the p.N995S mutation could increase the single channel open probability and single channel open dwell time.Our data suggest that the p.N995S mutation enhances the allosteric coupling between voltage-sensor activation and the channel's closed-open transition,and stabilizes the open-state conformation of BK channel.In addition,paxilline,one of the BK channel selective inhibitors could block currents from both WT and p.N995S mutant channels.This result indicates that the BK channel inhibitors may be used to block BK potassium current for the treatment of epilepsy caused by abnormal increased activation of BK channel.In summary,we have identified the first de novo mutation in BK channel?p.N995S?that causes epilepsy,but not paroxysmal dyskinesia.In contrast,the previous p.D434G mutation causes both epilepsy and paroxysmal dyskinesia.Although these two mutations both enhance the activation of the BK channel,their underlying molecular mechanisms are different.The p.D434G mutation enhances BK channel activation by increasing the calcium sensitivity.Whereas,the p.N995S mutation enhances the channel activation by increasing the single channel open probability and single channel open dwell time,and does not affect the calcium sensitivity.Our studies further confirm the association between BK channel and epilepsy,providing the theoretical basis for the genetic diagnosis of epilepsy.Our data highlight the significance of the functional studies in distinguishing a genomic variant of unknown significance as a disease-causing mutation in the era of precision medicine.We suggest that BK channel is a drug target for screening BK channel inhibitors for treatment of epilepsy,and p.N995S mutation can be used to study the structure and gating mechanism of BK channel.