|
Figure 1. . Summary of S4 accessibility determinations in Shaker and KvAP channels. O and I denote accessibility to outside and inside solutions; x, no effect; Ø and I, not accessible to outside and inside, respectively. (A) Accessibility of substituted histidine residues to hydrogen ions in Shaker (Starace and Bezanilla, 2001). (B) Accessibility of Shaker substituted cysteine residues to MTSET; data summarized from Table I of Bezanilla (2000). (C) Sensitivity of biotinylated KvAP cysteine mutants to avidin, from Jiang et al. (2003).
|
|
Figure 2. . Calibration of the decay of MTSET activity. Recordings of F373C currents were made from four successive inside-out patches obtained from the same oocyte. (A) Ionic current amplitude during pulses to +80 mV as a function of time of exposure to 1.2 mM MTSET solution. In the four experiments, the MTSET solution was aged 12, 21, 62, and 93 min. Single-exponential fits (solid curves) yielded decay rates of 3.41, 2.00, 0.46, and 0.46 s−1, respectively. (B). The decay rates are plotted as a function of the age of the MTSET. The time constant from this fit was 26 min. This time constant was used in Eq. 1 to correct the loss of MTSET activity due to hydrolysis.
|
|
Figure 3. . MTSET effects on ionic currents of Shaker WTT and F370C mutant channels. (A) Activation curves of WTT and F370C channels, before and after 10.8 mM·s exposure to MTSET. Plotted is the normalized tail current at −100 mV following depolarizations to the indicated voltages. Boltzmann fits yielded V1/2 = −51 ± 1 mV and z = 3.8 ± 0.4 for WTT, and V1/2 = −54 ± 1 mV and z = 6.4 ± 0.8 for F370C. After MTSET exposure, the WTT currents were essentially unchanged with V1/2 = −55 ± 0.8 mV and z = 3.6 ± 0.4. Tail currents in F370C channels were reduced from ∼1 nA to a residual <5 pA for all tested depolarizations. (B) F370C currents in an inside-out patch exposed to 0.5 mM MTSET (1 min old). Depolarizations were given to +80 mV from a −80 mV holding potential every 830 ms; currents from sweeps 1, 4, 7, and 19 are shown. (C) The elicited current is plotted as a function of cumulative MTSET exposure. There is no MTSET effect on WTT Shaker channels (circles). (D) Scaled activation time course of the F370C current sweeps shown in B. The rising phase slows down as the MTSET modification progresses. (E) The corresponding scaled tail currents. The deactivation time courses of the three sweeps are indistinguishable. In all experiments, the computed MTSET exposure was corrected for hydrolysis as described in materials and methods.
|
|
Figure 4. . Description of the MTSET modification effects at +80 mV. (A) Five representative F370C channel current sweeps recorded from the same inside-out patch as in Fig. 3 B. Fits to the activation time courses (smooth curves) were computed as sums of two exponential components. (B) The two exponential components u(t) and m(t), which are interpreted to be the normalized channel open probabilities of unmodified channels and one-subunit modified channels. (C) The amplitudes I0 and I1 of the two components, obtained from fits to sweeps at various times during the MTSET modification. The decays of the first component amplitude (circles) and the second component amplitude (squares) were fitted simultaneously to Eqs. 2, 4, and 5, yielding the subunit modification rate k = 0.165 ± 0.007 mM−1·s−1 and the amplitudes A = 366 ± 9 pA and B = 136 ± 0.4 pA. The total current amplitude (triangles) can alternatively be fitted by a single exponential with a decay rate of 0.46 ± 0.02 mM−1·s−1 (dashed curve).
|
|
Figure 5. . Two components of activation in partially modified F370C channels. Before modification, a Boltzmann fit yields V1/2 = −63.8 ± 0.8 mV and z = 4.27 ± 0.5. After partial MTSET modification (7.7 mM·s), there is an additional, right-shifted component of activation, with z = 0.8 ± 0.2 and V1/2 estimated to be ∼+100 mV.
|
|
Figure 6. . F370C channel gating currents and the effect of MTSET. (A) The voltage dependence of charge movement in W434F and F370C/W434F channels is very similar. Boltzmann fits of integrated on currents yielded V1/2 = −55 mV and z = 3.4, and V1/2 = −53 mV and z = 4.2, respectively. The on currents were recorded from depolarizations of 10 ms duration. (B) Four gating current sweeps recorded from an inside-out patch with 0.2 mM, 15-min-old MTSET perfused. The sweep number and exposure duration are given for each trace. Upward off transients result from errors in the P/4 subtraction, and the unstable baseline due to increased leakage current is visible in the bottom traces. (C) Charges were integrated from on gating currents and are plotted against the MTSET exposure. (D) Expanded on current transients from the sweeps in B. The smallest trace (73 s of exposure, or 7.5 mM·s) contains only 20% of the initial charge movement. There is a trend toward faster decays with MTSET exposure, as can be seen in the plot of decay time constants in the inset. In all traces, leakage currents were subtracted using the P/4 protocol with a leak holding potential of −120 mV.
|
|
Figure 7. . Decay of ionic and gating currents with MTSET modification. (A) Time course of decay of F370C ionic currents under exposure to MTSET. Data are shown for four patches using +40 mV test pulses, along with a fifth patch (open squares, same data as in Fig. 4) using test pulses to +80 mV. The average decay rate was 0.63 ± 0.18 mM−1·s−1, while the rate estimated from the most stable recording (filled circles) yielded a rate of 0.81 ± 0.02 mM−1·s−1. (B) Decay of on gating charge under exposure to MTSET. Data are shown from four patches using test pulses to +40 mV. The average decay rate was 0.17 ± 0.1 mM−1·s−1 while the most stable recording (filled circles) had a decay rate of 0.21 mM–1·s–1.
|
