Gagnon DG , Bezanilla F .

GM30376 NIGMS NIH HHS , R01 GM030376 NIGMS NIH HHS , R37 GM030376 NIGMS NIH HHS

	Figure 1. The homotetrameric Shaker quadruple mut R362Q/R365Q/R368N/R371Q expresses poorly at the plasma membrane in Xenopus oocytes. (A) Ionic current (top) and confocal image (bottom) of a typical oocyte injected with VENUS-Shaker zH4 IR (monomers). (B) Ionic current (top) and confocal image (bottom) of a typical oocyte injected with the VENUS-Shaker quadruple mut R362Q/R365Q/R368N/R371Q (monomers). Cut-open voltage clamp measurements. The internal and external solutions contained 120 and 12 mM K+, respectively.
	Figure 2. Ionic currents and G-V curve of Shaker tetramers wt (4wt), with two (2wt/2mut) or three subunits (1wt/3mut) with neutralized S4 gating charges. (A; Top) Scaled ionic currents from oocytes expressing Shaker zH4 IR (black line) or our tetramer 4wt (red line) for selected membrane potentials, as indicated. The maximal current recorded at +80 mV was around 5 µA for each oocyte. (Bottom) Averaged normalized conductance (G)-membrane potential (V) relation taken from the tail currents, Shaker zH4 IR (n = 4; filled black squares) and tetramer 4wt (n = 4; filled red circles). Holding membrane potential was −90 mV, and each depolarization was preceded by a 20-ms prepulse to −120 mV. The internal and external solutions contained 120 and 12 mM K+, respectively. (B) Ionic currents from oocytes expressing 4wt, 2wt/2mut, or 1wt/3mut heterotetramers. The voltage pulse protocol is shown on top. The HP was −90 mV. Pulse from −140 to +40 mV in 10-mV increments was applied, and the tail currents were measured at −50 mV for 4wt and at −80 mV for 2wt/2mut and 1wt/3mut. (C) Normalized G-V curve for 4wt (filled squares), 2wt/2mut (filled circles), and 1wt/3mut (filled triangles). The lines represent the global fit of a simple Boltzmann distribution for 1wt/3mut and Boltzmann distribution taken to the “nth” power for 2wt/2mut and 4wt, all sharing the parameters V1/2 and z. The HP was −90 mV for 4wt and −120 mV for 2wt/2mut and 1wt/3mut, and depolarization from −70 to +40 mV was applied for 4wt and from −120 to +40 mV for 2wt/2mut and 1wt/3mut. The conductance was measured using the tail currents. The internal and external solutions both contained 120 mM K+. Cut-open voltage clamp measurements.
	Figure 3. Slow inactivation occurs with the same time constant but with shifted voltage dependence in the heterotetramers compared with the 4wt tetramer. (A) Time course of slow inactivation during a 100-s pulse. Current traces, at the indicated voltages, from representative oocytes expressing 4wt (left), 2wt/2mut (middle), and 1wt/3mut (right). The weighted time constants represent an average from four to five experiments. The internal and external solutions contained 120 and 12 mM K+, respectively. (B) Voltage dependence of slow inactivation. The normalized peak tail current at +40 mV is plotted as a function of the HP and fitted to a Boltzmann distribution: Gm = Gmin + Gmax/(1 + exp[−z(V1/2 −Vm)F/RT]). 4wt data are shown as filled squares, 2wt/2mut data are shown as filled circles, and 1wt/3mut data are shown as filled triangles. Cut-open voltage clamp measurements. The internal and external solutions both contained 120 mM K+.
	Figure 4. Time constant of activation and deactivation. (A) Deactivation time constants of heterotetramers compared with the 4wt tetramer. The currents were fitted with two exponentials. The weighted time constant is shown, and the inset represents the contribution of the fast time constant. 4wt data are shown as filled squares (n = 5), 2wt/2mut data are shown as filled circles (n = 8), and 1wt/3mut data are shown as filled triangles (n = 4). (B) Activation time constants of the heterotetramers compared with 4wt. The currents could be fitted with a single exponential for Vm lower than 0 mV but required two exponentials in the positive region. The weighted time constant is shown, and the inset represents the contribution of the fast component in the positive region. 4wt data are shown as filled squares (n = 3), 2wt/2mut data are shown as filled circles (n = 4), and 1wt/3mut data are shown as filled triangles (n = 5). The HP was −90 mV for 4wt and −120 mV for 2wt/2mut and 1wt/3mut. Note the log scale vertically. The applied voltage routine is shown in the top panel. Points are connected with lines for clarity. Cut-open voltage clamp measurements. For activation time constants, the experiments were performed at room temperature and in symmetrical 120 mM K+ to have larger currents in the negative region of voltage. Deactivation time constants were measured at 6–8°C; the internal and external solutions contained 120 and 50 mM K+, respectively.
	Figure 5. The Cole-Moore shift is reduced in the heterotetramers compared with 4wt. (A) The family of K+ current traces from representative oocytes expressing 4wt (top), 2wt/2mut (middle), and 1wt/3mut (bottom). The pulse protocol is represented in the top panel. The currents are the ones measured a +40 mV in each case, preceded by various prepulses in 20-mV increments. For 4wt, the 20-ms prepulse ranges from −160 to −40 mV, and for 2wt/2mut, it ranges from −180 to −40 mV (20-ms duration). For 4wt and 2wt/2mut, HP was −90 mV, and subtracting HP was −120 mV. For 1wt/3mut, HP was either −120 or −100 mV, and subtracting HP was −160 mV. In that case, a 200-ms prepulse (from −200 to −70 mV) was applied. The red line represents the fit to the data for the first 10 ms of the lower trace with Eq. 1. (B) The averaged exponent n of the fit of the data indicated by the red line in part A to Eq. 1 for the heterotetramers compared with 4wt. The number of oocytes is shown in parentheses. The dotted lines are located at an abscissa value of 4wt (7.5 ± 1.5); half of this value and a quarter of this value. Cut-open voltage clamp measurements. Experiments were performed at 6–8°C. The internal and external solutions contained 120 and 12 mM K+, respectively. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
	Figure 6. Variance–mean plot for the heterotetramers compared with the 4wt. (A) Fluctuation analysis from an ensemble of current traces (n = 300 pulses) for a 20-ms depolarization to +70 mV, followed by repolarization to −120 mV in a cell-attached patch of an oocyte expressing 4wt. The data were fitted with Eq. 2, and the single-channel current i and the number of channels N in the patches were estimated. The corresponding conductance was estimated to be of 13 and 27 pS at +70 and −120 mV, respectively. The number of channels in the patch was 213. Eq. 3 allows for the calculation of the Pomax (maximal open probability), which was calculated to be 0.73 (n = 8). (B) Fluctuation analysis from a cell-attached patch of an oocyte expressing 2wt/2mut. 450 current traces of 20-ms depolarizations to +70 mV and repolarizations to −120 mV were used for noise analysis. The number of channels in the patch was estimated at 374, the single-channel conductance to 11.7 and 25 pS at +70 and −120 mV, respectively, and the Pomax to 0.67 (n = 7). (C) Fluctuation analysis from an oocyte expressing 1wt/3mut in a cell-attached configuration. 600 current traces of a 20-ms depolarization to +70 mV and repolarization to −180 mV were averaged and analyzed. The single-channel conductance was estimated at 10.8 and 22 pS at +70 and −180 mV, respectively. The number of channels in the patch could not be estimated accurately because of the open probability of 0.47 (n = 3). The black lines are the data, and the red lines are the fitted curve. Patch clamp experiments. The external and internal solutions both contained 120 mM K+.
	Figure 7. Single-channel activity of 1wt/3mut reveals subconductance levels in a range of voltage at which the wt Shaker subunit is closed. (A) Single-channel recordings measured in an oocyte expressing 4wt from an HP of −40 to +70 mV. (B) All-points histograms from the selected traces shown in A at +70 mV (left) and −40 mV (right). (C) Single-channel current amplitude versus voltage relationship of single channels and macro patches of 4wt (n = 6–15; n = 7 of single-channel recordings). (D) Single-channel recordings taken from an oocyte expressing 1wt/3mut at +70 and −70 mV. (E) All-points histograms from selected traces as shown in D at +70 mV (middle) and −70 mV (right). (F) Single-channel current amplitude versus voltage relationship of single channels and macro patches of 1wt/3mut (n = 6–9; n = 6 from single-channel recordings). (G) Ensemble average of the single-channel recordings from the patch shown in D for depolarizations from an HP of −70 to +70 mV (black line) and from an HP −100 to −70 mV (red line). (H) Single-channel recordings taken from another patch of an oocyte expressing 1wt/3mut from an HP of −40 to −70 mV. (I) All-points histogram of the recording shown in H. The dotted lines indicate the level of current in the closed or open channel. Patch clamp experiments. The external and the internal solutions both contained 120 mM K+.
	Figure 8. The double mutation T449V-I470C in one wt subunit impairs slow inactivation in 1wt/3mut, but not in 4wt. (A) Time course of slow inactivation. Representative current traces from oocytes expressing Shaker IR TVIC injected as monomers (left), wtTVIC/3wt (middle), and wtTVIC/3mut (right) from an HP of −90 mV to voltages from −20 to +60 mV in 20-mV intervals for 100 s. Cut-open voltage clamp measurements. The internal and external solutions contained 120 and 12 mM K+, respectively. (B) Ratio of the remaining current after a 100-s depolarizing pulse over the peak current (I100s/Ipeak) for Shaker TVIC injected as monomers (open black squares), 4wt (filled black squares), wtTVIC/3wt (filled red squares), 1wt/3mut (filled black circles), and wtTVIC/3mut (filled red circles).
	Figure 9. Predictions of the model for ionic currents in symmetrical 120 mM K+ at room temperature (19–20°C) for 4wt, 2wt/2mut, and 1wt/3mut compared with experimental data. Experimental data are shown on the left, and the right panel illustrates the predictions of the kinetic model illustrated in Scheme 1. The artifacts of pulse subtraction and compensation measured at the beginning of the pulses (first ms) are not shown. The HP was −90 mV, and steps were elicited to the annotated Vm. The rate constants and equivalent valence in electronic charge units are shown in Table I.

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