Abstracts

Rationale: KCNQ potassium channels composed of KCNQ2 and KCNQ3 subunits give rise to the M-current, a slowly activating and non-inactivating voltage-dependent potassium current that limits repetitive firing of action potentials. KCNQ channels are enriched at the surface of axons and axonal initial segments, the sites for action potential generation and modulation. Their enrichment at the axonal surface is impaired by mutations in KCNQ2 carboxy-terminal tail that cause benign familial neonatal convulsion (BFNC) and myokymia, suggesting that their correct surface distribution and density at the axon is crucial for their control of neuronal excitability. However, the underlying molecular mechanisms remain elusive. Methods: To test the hypothesis that preferential targeting of KCNQ channels to the axonal surface is regulated by calmodulin (CaM), surface immunostaining was performed in rat dissociated hippocampal cultured neurons transfected with KCNQ3 containing an extracellular HA tag (HA-KCNQ3) and KCNQ2, or trafficking reporter protein CD4 (cluster of differentiation 4) fused to the carboxy(C)-terminal tail of KCNQ2 (CD4-KCNQ2). The KCNQ2 interaction with CaM was blocked by introducing BFNC mutations or mutations in the CaM binding IQ motif in KCNQ2, whereas CaM activity is enhanced or inhibited by overexpression of wild type or dominant negative calcium-binding incompetent CaM (CaM1234).Results: Here we demonstrate that the KCNQ2 carboxy-terminal tail is sufficient to target CD4 proteins to the axonal surface whereas such targeting is reduced by the overexpression of CaM1234. Furthermore, inhibition of CaM binding to KCNQ2 C-terminal tail abolishes axonal surface expression of CD4 fusion proteins by retaining them in the endoplasmic reticulum. Importantly, inhibition of CaM binding also disrupts the enrichment of heteromeric KCNQ2/KCNQ3 channels at the axonal surface by blocking their trafficking from the endoplasmic reticulum to the axon. Conclusions: These findings collectively reveal CaM as a critical player that modulates trafficking and enrichment of KCNQ channels to the neuronal axon. Some of the mutations we have tested are associated with BFNC, making a strong case for impaired CaM binding and subsequent loss or reduction of axonal KCNQ channels to be one etiology of neuronal hyperexcitability. Furthermore, de novo mutations in CaM-binding domains of KCNQ2 are recently reported to be associated with severe symptomatic neonatal epileptic encephalopathy including drug-resistant Ohtahara syndrome. Presently, whole-cell patch clamp recording of action potential firing is being conducted in neurons expressing wild type or mutant KCNQ2 to better understand the effect of these CaM-binding deficient mutations on neuronal excitability. Hence, this study will foster future studies to investigate how CaM regulates the cellular machineries that control ER retention and/or export of KCNQ channels and explore therapeutic means to combat epilepsy by modulating these fundamental cellular pathways to enhance axonal surface expression of KCNQ channels.