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Figure 1. . The D447N pore mutation prevents conduction in Shaker K+ channel, while the corresponding D292N pore mutation in hSlo channel allows conduction. (A) Outward K+ current recorded with the cut open oocyte voltage clamp, from oocytes expressing Sh-IR; the current was elicited by voltage pulses as indicated for each trace, from an HP of â90 mV. (B) Current records from an oocyte expressing Sh-IR D447N channels. The fast upward and downward deflections at the beginning and end of the voltage pulses are the gating currents recorded unsubtracted during depolarizations from HP = â90 mV to the indicated potentials. (C and D) Typical current records from inside-out membrane patches from oocytes expressing hSlo and hSlo-D292N respectively, with 1.5 μM internal free Ca2+. HP was 0 mV. Current records were obtained 2 d (hSlo) and 4 d (hSlo-D292N) after oocyte injection; the amount of cRNA for hSlo-D292N was also 10 times higher than for hSlo. Note that the same voltage step elicited a larger and faster activating ionic current in wild-type hSlo than in the D292N mutant.
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Figure 2. . Shift to more depolarized potentials of the voltage activation curve by the D292N mutation. (A and B) Current traces elicited by pulses to 250 mV followed by repolarizations to the indicated potentials, for hSlo (A) and hSlo-D292N (B). (C and D) Current traces elicited by 40-ms voltage steps to â50, 0, 50, 100, 150 mV from HP-50 mV (same patches as in A and B). (E and F) Instantaneous (Î) and steady-state (â) IâV relationships for hSlo (E) and hSlo-D292N (F). The instantaneous IâV curve was measured in tail currents 40 μs after repolarization. hSlo channels have a more pronounced voltage-dependent block at positive potentials for both steady and tail currents. On the other hand, hSlo-D292N channels have a strong reduction of the instantaneous conductance at potentials more negative than +50 mV. (G) fPo vs. voltage relationship for hSlo (â) and hSlo-D292N (Î). Averaged data points (n = 3) were fitted to a double Boltzmann distribution. The parameters of the fitting are Z1 = 1.7, Z2 = 1.1, V1/21 = 61 mV, V1/22 = 111 mV, A1 = 0.65 (hSlo) and 0.30 (hSlo-D292N), A2 = 0.35 (hSlo) and 0.70 (hSlo-D292N). Inside-out membrane patches in 120 mM symmetrical K+ and 1.5 μM free [Ca2+].
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Figure 3. . The D292N mutation reduces the coupling between voltage sensor movement and pore opening. (A and B) Current recordings from inside-out patches expressing hSlo-D292N and hSlo. The currents were elicited by a voltage pulse to 50 mV, from an HP of â50 mV, and the linear component of the current was subtracted off-line (subtracting pulse was to â150 mV from â100 mV). (B) The traces of both hSlo and hSlo-D292N superimposed and normalized to the size of the gating current. The inset (C) shows the ON gating current from B on an enlarged scale. Note that, in proportion, a similar charge displacement elicits a much smaller ionic current in hSlo-D292N than in hSlo. Recordings performed in 110 mM NMG-MES, 10 mM KMES, and 1.5 μM free Ca2+.
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Figure 4. . The QâV curves in hSlo and hSlo-D292N. (A) Gating current recordings from inside-out patches expressing hSlo, in symmetrical 120 mM TEA-MES and 1.5 μM free Ca2+, elicited by voltage pulses (shown above the recordings) from â50 mV to 150 mV. The linear capacity transients were subtracted off-line by using different SHPs, as indicated for each trace. (B) Plot of the absolute value of the time integral of OFF gating currents vs. SHP for hSlo. Identical results were obtained for hSlo-D292N. Note that for SHP negative to â50 mV, the charge remains unchanged. (C and D) Gating currents recorded from inside-out patches from oocytes expressing hSlo (C) and hSlo-D292N (D), in symmetrical 120 mM TEA-MES, with 1.5 μM free Ca2+. The currents were elicited by 10-ms voltage steps to the indicated voltages. Each trace is subtracted with a subtracting pulse from a holding of â100 mV to â150 mV. (E) Averaged and normalized QâV curves estimated from the OFF gating current for hSlo (â) and hSlo-D292N (â¢) (n = 3 ± SEM). Curves were normalized to the mean charge calculated for voltage steps to 230 mV, 240 mV, and 250 mV. The two QâV curves were fitted to a single Boltzmann distribution in the form 1/(1+exp(zF(Vhalf â V)/ RT)), where F, R, and T are the usual thermodynamic parameters. Parameters fitting were for hSlo: Vhalf = 96 mV and z = 0.77, for hSlo-D292N: Vhalf = 106 mV and z = 0.74.
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Figure 5. . The slow component of gating currents is reduced in the hSlo-D292N channel. (A) Gating current traces for hSlo and hSlo-D292N for a pulse from â50 mV to 150 mV. (B) ON gating currents (inset of A) with Ïfast = 0.052 ms and Ïslow = 1.1 ms; the slow component amplitude was reduced from 70% in hSlo to 25% in hSlo-D292N. (C) OFF gating currents (inset of A) with superimposed fits: Ïfast = 0.022 ms, Ïmedium = 0.083 ms, Ïslow = 0.350 ms, for both clones; the relative amplitudes of the components were Afast = 51%, Amedium = 35%, and Aslow = 14% for hSlo, and Afast = 89%, Amedium = 10%, and Aslow = 1% for hslo D292N. Gating current recordings in 120 mM TEAMES and 1.5 μM free Ca2+.
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Figure 6. . Reduction of the charge movement slow component in the hSlo-D292N channel. OFF gating currents in hSlo (A) and hSlo-D292N (B) elicited by voltage pulses from HP â50 mV to 150 mV of increasing durations, ranging from 40 μs to 10.2 ms. (C and D) OFF charge from two experiments as in A and B plotted as a function of the pulse duration. Data were normalized to the charge displaced by the 5.1-ms pulse and fitted to a bi-exponential function with a single fast (40 μs) and slow (1.1 ms) time constant. The charge movement slow component was reduced from 55% in hSlo to 26% in hSlo-D292N. Gating current recordings in 120 mM TEAMES and 1.5 μM free Ca2+.
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Figure 7. . OFF gating current components with different pulse durations. (A and B) Superimposed OFF gating currents following different pulse durations (0.04 ms to 10.2 ms) in hSlo and hSlo-D292N as shown in Fig. 6 (A and B). Note the more prominent slow components in hSlo. (C and D) Selected traces from A and B with superimposed fits to the sum of three exponential functions. (D) Relative amplitudes of fast, slow, and medium components as function the pulse durations for hSlo (open symbols) and hSlo-D292N (filled symbols) with Ïfast = 0.017 ms, Ïmedium = 0.062 ms, and Ïslow = 0.278 ms for both clones. Note that the amplitude of the slow component was greatly diminished in hSlo-D292N. (E) Average values in hSlo (n = 6) and hSlo-D292N (n = 9) of the relative amplitude of fast, medium, and slow OFF gating current components at â50 mV following a 10.2-ms pulse to 150 mV. Time constant values were: hSlo, Ïfast = 0.020 ± 0.003 ms, Ïmedium = 0.062 ± 0.01 ms, and Ïslow = 0.371 ± 0.140 ms; hSlo-D292N, Ïfast = 0.019 ± 0.003 ms, Ïmedium = 0.059 ± 0.007 ms, and Ïslow = 0.333 ± 0.054 ms.
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Figure 8. . The Po at limiting [Ca2+]i at 0 mV is significantly lower in hSlo-D292N than in hSlo. (A) Single channel activity from inside out patches of oocytes expressing hSlo (left) and hSlo-D292N (right) channels. Recordings at 0 mV, in asymmetric K+ concentration ([K+]o = 5 mM, [K+]i = 120 mM) with 400 μM free [Ca2+]i. (B) Total point histograms from the experiment in A. (C) Po vs. [Ca2+]i plot at 0 mV for hSlo and hSlo-D292N. Each point is the average of three patches of hSlo channels and six membrane patches for hSlo-D292N channels. The data were fitted to the Hill equation (Po = MaxPo/(1+(K1/2/[Ca])n)), with n = 1.3, MaxPo = 0.06 D292N, and n = 1.7, MaxPo = 0.47 for WT. K1/2 = 19.5 μM for both channels.
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Figure 9. . Variance analysis shows a reduced limiting Po for the mutant hSlo-D292N channels. (A and B) Superimposed K+ current traces (100) recorded with pulses from â50 mV to 250 mV in 120 symmetrical K-MES with 1.5 μM free Ca2+ from hSlo (A) and hSlo-D292N (B). (C and D) Varianceâmean current plots in two other patches for hSlo (C) and hSlo D292N (D). Data points were fitted to Ï2(t) = I(t)i â (I(t)2/N), where Ï2 is the variance, I the mean current, i the single channel current, and N the number of channels. Values were for hSlo POmax = 0.72, i = 39 pA, and N = 419, and for hSlo-D292N POmax = 0.26, i = 19 pA, and N = 1,000.
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