FIGURE 1.
Single-channel recordings of the in vitro transcription and translation-purified Kv7.3 PM reconstituted in droplet lipid bilayers. A, Western blotting of Kv7.3 PM proteoliposomes purified by nickel affinity. Lane 1, Mr standards. Lane 3, flow-through (FT). Lane 5, 10 mm imidazole wash (W). Lane 7, 300 mm imidazole eluate (E) of the purified Kv7.3 PM used for reconstitution. The purified protein migrates as a monomer with an apparent Mr of ∼15. B–E, representative current versus voltage (IV) relationship of the Kv7.3 PM (left panels) at V = 150 mV (B), V = 100 mV (C), V = 50 mV (D), and V = 30 mV (E) recorded in 0.5 m KCl. Bursting activity is readily discerned in the center panels, in which the currents are displayed at a faster time resolution. The corresponding normalized all-point current histograms (right panels) for the entire analyzed record are fitted to the Gaussian curve. c and o denote the closed and open states of the channel, respectively. Kv7.3 PM exhibits a single-channel slope conductance of 27 ± 3 pS.