Regulation of Kv1.3 Channels by a Matrix Metalloproteinase

Abstract

Matrix metalloproteinase 23 (MMP23) contains a functional K+ channel-blocking toxin domain (TxD) with structural similarity to the sea anemone toxins BgK and ShK (J. Biol. Chem. 2010.285:9124-9136). MMP23 co-localizes with and traps TxD-sensitive Kv1.3 channels intracellularly without affecting TxD-resistant Kv1.2 channels (J. Biol. Chem. 2010.285:9124-9136). Here we use C-terminal deletion analysis to define the segments of MMP23 required for regulation of human Kv1.3. Confocal microscopy showed that eGFP-Kv1.3 co-localized with dsRed-tagged MMP23 and with three deletion constructs of MMP23 (lack the IgCAM domain, IgCAM + TxD, IgCAM + TxD + Catalytic Domain), but not with dsRed (vector control), in COS-7 cells. Patch-clamp experiments revealed suppression of Kv1.3 currents by full-length dsRed-MMP23 and by the deletion construct lacking all three external domains (IgCAM+TxD+Catalytic domain), but not by dsRed. These results indicate that the N-terminal segment of MMP23 (stretching from the N-terminus, through the single transmembrane segment, and ending at the external furin-cleavage site) is sufficient for both current suppression and co-localization with Kv1.3 channels. Western blot analysis demonstrated cleavage of external loops of Kv1.3 by full-length MMP23. Thus, MMP23's N-terminal segment associates with and retains Kv1.3 intracellularly, while the TxD binds to the channel pore and positions the catalytic domain to cleave external loops of Kv1.3.