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Position-dependent attenuation by Kv1.6 of N-type inactivation of Kv1.4-containing channels.
Macdara Bodeker, Ahmed Al-Sabi, Marie Le Berre, J Oliver Dolly
Assembly of distinct α subunits of voltage-gated K+ channels (Kv1) into tetramers underlies the diversity of their outward currents in neurons. Kv1.4-containing channels normally exhibit N-type rapid inactivation, mediated through an N-terminal inactivation ball (NIB); this can be over-ridden if associated with a Kv1.6 α subunit, via its N-type inactivation prevention (NIP) domain. Herein, NIP function was shown to require positioning of Kv1.6 adjacent to the Kv1.4 subunit. Using a recently-devised gene concatenation, hetero-tetrameric Kv1 channels were expressed as single-chain proteins on the plasmalemma of HEK-293 cells, so their constituents could be arranged in different positions. Placing Kv1.4 and 1.6 genes together, followed by two copies of Kv1.2 yielded a K+ current devoid of fast inactivation. Mutation of critical glutamates within the NIP endowed rapid inactivation. Moreover, separating Kv1.4 and 1.6 with a copy of Kv1.2 gave a fast-inactivating K+ current with steady-state inactivation shifted to more negative potentials and exhibiting slower recovery. Alternatively, separating Kv1.4 and 1.6 with two copies of Kv1.2 yielded slowly-inactivating currents because in this concatamer Kv1.4 and 1.6 are together. These findings, also, confirm that the gene concatenation can generate K+ channels with α subunits in pre-determined positions.