Inactivation gating of Kv7.1 channels does not involve concerted cooperative subunit interactions

Figures

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  Figure 1.. 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.

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  Figure 2.. Expression of concatenated Kv7.1 subunits bearing an increasing number of L273F mutations. A, representative current traces of the following constructs: ConD2L273F (ConLFLL), ConD2,4L273F (ConLFLF), ConD1,2L273F (ConFFLL), ConD,2,3,4L273F (ConLFFF) and ConD1,2,3,4L273F (ConFFFF). CHO cells were held at −90 mV and the membrane voltage was stepped for 2 s from −70 mV to +50 mV in 10 mV increments and then repolarized for 1 s to −60 mV. B, Fractional inactivation F of the different concatemer constructs was measured at +50 mV (n = 7–10). C, Time constants of inactivation relaxation measured at +50 mV and determined by one exponential fit of the inactivation traces (n = 8–14). D, conductance-voltage relations of the different concatemer constructs. Data were fitted with a single Boltzmann function (n = 7–10).

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  Figure 3.. Steady-state inactivation of the different concatemer constructs. A, representative steady-state inactivation current traces of the different concatemer constructs, where the membrane potential was stepped to varying pre-pulse voltages from −70 mV to +50 mV in increments of 10 mV and to a subsequent test pulse of +50 mV. B, The different peak currents measured at the test pulse at +50 mV were divided by the largest peak current (at +50 mV) obtained from the −70 mV prepulse. These ratios of steady-state inactivation were plotted against the prepulse voltage (n = 7–10). C, The normalized ratios of steady-state inactivation were plotted against the prepulse voltage Data were fitted with a single Boltzmann function.

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  Figure 4.. Recovery from inactivation of the different concatemer constructs. A, representative current traces of the recovery from inactivation of the different concatemer constructs. The recovery time from inactivation was determined from a −90 mV holding potential, by stepping the membrane voltage for 1.5 s to +50 mV to open the various Kv7.1 concatemer channels (first pulse), then repolarizing to −90 mV for various times to allow recovery from inactivation and stepping back to +50 mV for 0.5 s to reopen the channels (second pulse). B, The fractional recovery was calculated by the peak current of the second pulse divided by the peak of the first pulse. These ratios were plotted against the recovery times (n = 4). C, time constants of the recovery time from inactivation were calculated from an exponential fit (one way ANOVA, n = 4; *P < 0.01, **P < 0.001, ***P < 0.0001).