Abstracts

Rationale: Although depression is a very common comorbidity in epilepsy, the basis for this is not known. Ankyrin-G (AnkG) is a molecular scaffold for two classes of ion channels that are both mutated in epilepsy and the targets of approved anti-epileptic drugs (AEDs). Voltage-gated sodium (NaV) and KCNQ2/3 channels are densely clustered and colocalized via binding to AnkG at AISs and nodes of Ranvier. These channels are crucial for action potential (AP) generation, propagation, and modulation. Genetic variation in ANK3, encoding Ankyin-G, is associated with bipolar disease, and NaV blocking AEDs are very commonly used as mood stabilizers. As an effort to understand the mechanisms linking epilepsy and mood disorder, we analyzed the molecular basis for AnkG binding of KCNQ and NaV channels. Methods: AnkG contains variably spiced N-terminal (NT) region of 10-39 amino acids followed by an ~800 residue membrane binding (MB) domain containing 24 concatenated ankyrin repeats. We generated antibodies against AnkG NT regions useful for immunostaining and western blotting. We constructed families of AnkG MB domain fragments and expressed these in mammalian cells. We constructed fusion proteins linking glutathione-S-transferase (GST) and regions of KCNQ2, KCNQ3 and NaV1.2 previously shown to contain AnkG binding motifs. We performed in solution pull-down and cell-based membrane surface redistribution assays to detect interaction between the channels and various regions of AnkG. We refined the results by studying the effect of specific mutations introduced into minimal binding regions. Results: An AnkG fragment, consisting of the NT region and the first 7 ankyrin repeats, was sufficient to bind both NaV and KCNQ channels. R7 was required for binding of KCNQ3, but not for binding NaV1.2. The presence of the 39 residue NT region of Ankyrin G significantly inhibited the binding of the KCNQ2/3 channel to AnkG, but not that of the NaV channel.Conclusions: The most N-terminal ~200 amino acids of AnkG, a giant, variably spliced protein of up to 4400 amino acids, contains the principal binding sites for KCNQ2/3 and NaV channels. The sites appear to be overlapping but non-identical. Biophysical and electrophysiological data indicate that the balance of these two channel types strongly influences action potential generation at the axon initial segment. Control of this ratio may be a useful therapeutic mechanism for epilepsy and associated mood disturbance. Identification of the binding regions makes possible screening for agents capable of modulating binding and thus neuronal excitability.