title: Position-dependent attenuation by Kv1.6 of N-type inactivation of Kv1.4-containing channels.
abstract: Assembly of distinct α subunits of Kv1 (voltage-gated K(+) channels) into tetramers underlies the diversity of their outward currents in neurons. Kv1.4-containing channels normally exhibit N-type rapid inactivation, mediated through an NIB (N-terminal inactivation ball); this can be over-ridden if associated with a Kv1.6 α subunit, via its NIP (N-type inactivation prevention) domain. Herein, NIP function was shown to require positioning of Kv1.6 adjacent to the Kv1.4 subunit. Using a recently devised gene concatenation, heterotetrameric Kv1 channels were expressed as single-chain proteins on the plasmalemma of HEK (human embryonic kidney)-293 cells, so their constituents could be arranged in different positions. Placing the 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, correlating with similar inactivation kinetics seen for Kv1.4-(1.2)(3). Alternatively, separating Kv1.4 and 1.6 with two copies of Kv1.2 yielded slow-inactivating currents, because in this concatamer Kv1.4 and 1.6 should be together. These findings also confirm that the gene concatenation can generate K(+) channels with α subunits in pre-determined positions.
authors.affiliation: International Centre for Neurotherapeutics, Dublin City University, Glasnevin, Ireland.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Al-Sabi
authors.forename: Seshu
authors.initials: S
authors.lastname: Kaza
authors.forename: Marie
authors.initials: M
authors.lastname: Le Berre
authors.forename: Liam
authors.initials: L
authors.lastname: O'Hara
authors.forename: MacDara
authors.initials: M
authors.lastname: Bodeker
authors.forename: Jiafu
authors.initials: J
authors.lastname: Wang
authors.forename: J Oliver
authors.initials: JO
authors.lastname: Dolly
mesh_terms: D000818:Animals; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D051665:Kv1.4 Potassium Channel / Q000378:metabolism*; D051667:Kv1.6 Potassium Channel / Q000737:chemistry / Q000378:metabolism*; D016296:Mutagenesis / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism; D051381:Rats
chemical_list: C064390:Kcna6 protein, rat; D051665:Kv1.4 Potassium Channel; D051667:Kv1.6 Potassium Channel; D021122:Protein Subunits; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins