Article Title: Inactivation gating of Kv7.1 channels does not involve concerted cooperative subunit interactions
Figure Lengend Snippet: Characterization of the WT and L273F mutant Kv7.1 concatenated tetrameric channels. A, Top panel, scheme of the Kv7.1 concatenated tetrameric channel construct (Con), where subunits D1, D2, D3 and D4 are connected by flexible linkers. Each linker (8 glycines) harbors a unique restriction site. B, conductance-voltage relations of WT monomeric and WT concatenated tetrameric Kv7.1 constructs in the absence or presence of KCNE1. Data were fitted with a single Boltzmann function. C, Western blot showing lysates from HEK293 transfected with empty vector (Mock), KCNQ1 monomeric and concatenated tetrameric constructs. D, location of residue L273 in the S5 α helical segment of the Kv7.1 pore domain as mapped in the recent cryo-EM structure of the frog Kv7.1 (Sun, 2017). E, representative current traces of L273F Kv7.1 monomeric and concatenated tetrameric mutant constructs. CHO cells were held at −90 mV and the membrane voltage was stepped for 3 s from −60 mV to +60 mV in 10 mV increments and then repolarized for 1.5 s to −60 mV. F, conductance-voltage relations of the WT Kv7.1 concatemer, L273F Kv7.1 monomer and L273F Kv7.1 concatemer. Data were fitted with a single Boltzmann function.
Article Snippet: Equal amounts of lysate proteins were resolved by 8% SDS-PAGE and blots were reacted using rabbit anti-Kv7.1 antibodies (Alomone Labs).
Techniques: Mutagenesis, Construct, Western Blot, Transfection, Plasmid Preparation, Cryo-EM Sample Prep