Abstracts

Rationale: KCNQ2 and KCNQ3 are voltage-gated potassium channel subunits underlying the M-current (IM) that regulates neuronal excitability. Mutations in KCNQ2 cause a broad spectrum of neonatal-onset epilepsy, from mild and transient (benign familial neonatal seizures, BFNS) to quite severe (KCNQ epileptic encephalopathy, Q2EE). Changes in the distribution of KCNQ2/3 channels throughout development may play a role in KCNQ genotype-phenotype relationships. Localization and concentration of KCNQ2/3 channels at the axon initial segment (AIS) tightly regulates normal firing patterns. However, determining the level of expression of KCNQ2 across developmental stages is challenging due to the overall low abundance of the protein and the very different biochemical characteristics of subcellular locations it occupies during its biosynthesis and trafficking. Methods: We performed KCNQ2 immunofluorescence labeling in parallel using no fixation, low fixation, and standard aldehyde fixation in mouse brain sections obtain across development from newborn to adult. We used surface biotinylation and immunoblotting assays to compare the efficiency of membrane trafficking of wild-type and mutant KCNQ2, expressed as a homotetramer and as a heterotetramer with KCNQ3. We used western blots of neuronal homogenates to measure total KCNQ2 protein in vivo. Results: In mouse sections, KCNQ2 and KCNQ3 are detectable at AISs of pyramidal neurons (PyNs) in neocortex and the hippocampal CA3 region at P7, and appear to increase in concentration thereafter. Western blots of forebrain homogenates of mice show that full length KCNQ2 polypeptide is visible at P4, and increases in abundance over development. KCNQ3, however, shows relatively high protein intensities at P4 until adulthood. In neocortical pyramidal neurons, careful quantification shows that KCNQ2/3 is expressed in an increasing gradient within the distal two-thirds of the AIS, and is most concentrated at the distal tip, the location where action potentials initiate. Additionally, immunofluorescence microscopy performed tissue sections from transgenic mice overexpressing the dominant negative mutant G279S revealed an aberrant labeling pattern: KCNQ2 was completely absent at the AIS and was retained at intracellular puncta in the soma and dendrites. KCNQ3 was partially redistributed to these puncta.Conclusions: KCNQ2 and KCNQ3 proteins were detected at AISs in rodent at time points roughly equivalent to human birth and labeling intensity increased with age. Some mutations may act by preventing surface trafficking and AIS concentration. Since such effects that may not be easily revealed through heterologous expression, further development of in vivo models is warranted.