
Figure 2. Effects of external cations on the slow decay of F1485Q Na+ channels currents. Wholecell currents were evoked as described in the legend of Fig. 1. Recordings from one cell sequentially bathed in 150 mM of the indicated monovalent cations. Normalized peak currents.


Figure 3. Slow inactivation of F1485Q channels in 10 and 150 mM [Na+]o. (A) Changes in peak current in response to changes in holding potential for two F1485Qtransfected cells bathed in either 150 (closed squares) or 10 mM Na+ (open circles). The cells were held at −140 mV for at least 10 min before the holding potential was sequentially changed every 5 min to −70, −140, +40, and −140 mV. Every 15 s during the entire protocol, a 20ms recovery pulse to −140 mV was applied followed by a 20ms test pulse to +60 mV (inset). The normalized peak current at +60 mV is plotted. (B) Current levels after 5 min at either −70 or +40 mV expressed as percent of the maximal current at a −140 mV holding potential. (C) 10–90% decay times for entry into slow inactivation at −70 and +40 mV. The 10–90% time corresponds to the time it takes for the current to decay from 10 to 90% of the maximal decay measured after 5 min at either −70 or +40 mV. (D) 10–90% times for recovery at −140 mV after 5 min of inactivation at either −70 mV or +40 mV. Numbers in parentheses are number of cells.


Figure 4. Steadystate slow and fast inactivation in high and low [Na+]o. Steadystate slow inactivation (SΘ) curves for F1485Q (A) and WT hH1a (B). Transfected cells were held at holding potentials ranging from −160 to −30 mV in 10 or 20mV increments. After 2 min at each holding potential, a 20ms recovery pulse to −140 mV and a 9ms test pulse to +60 mV were given (insets). The peak current measured at +60 mV is plotted as a fraction of the maximal current. (A) F1485Q SΘ curves. The data points are fit to the Boltzmann equation (solid lines) with midpoints at −79.0 ± 1.1 and −85.9 ± 1.7 mV and slope factors of 6.9 ± 0.4 and 6.0 ± 0.6 mV for 150 and 10 mM external Na+, respectively (n = 4 cells for 150 mM Na+ and n = 5 cells for 10 mM Na+). (B) WT SΘ curves. Bestfits to the Boltzmann equation have midpoints at −82.2 ± 2.8 and −102.0 ± 0.5 mV and slope factors of 25.4 ± 2.5 and 15.3 ± 0.6 for 150 and 0 mM Na+o, respectively (n = 3 cells for each [Na+]o). (C and D) Steadystate fast inactivation (hΘ) induced by a 50ms prepulse to the indicated voltage from a holding potential of −160 mV (see insets). Test pulse, +60 mV. The peak current at +60 mV is plotted as a fraction of the maximal current. (C) F1485Q hΘ curves. Data shown are from 3 cells sequentially bathed in 150, 10, and 150 mM Na+. The bestfit Boltzmann curves have midpoints at −73.8 ± 2.4, −78.0 ± 2.6, and −75.6 ± 3.0 mV and slope factors of 3.27 ± 0.16, 3.47 ± 0.35, and 3.53 ± 0.36 mV for 150, 10, and 150 mM Na+o, respectively. (D) WT hΘ curves. Data from 3 cells transfected with WT hH1a and successively bathed in 150, 0, and 150 mM Na+. Inactivation curves are fitted to the Boltzmann equation with midpoints at −100.3 ± 0.9, −103.4 ± 0.6, and −103.5 ± 0.4 mV and slope factors of 8.0 ± 0.2, 8.8 ± 0.3, and 7.8 ± 0.2 for 150, 0, and 150 mM Na+o, respectively. In each panel, the dashed lines are the Boltzmann curves for 10 (F1485Q) or 0 mM Na+o (WT) multiplied by 0.67 (F1485Q) or 0.84 (WT), giving the expected reduction in Popen for the decrease in [Na+]o (Townsend et al., 1997).


Figure 5. Singlechannel data analysis. (A) Ensemble averages obtained from a twochannel outsideout patch successively bathed in 150, 10, and 150 mM Na+. Currents were activated by 90ms pulses to +60 mV from a holding potential of −140 mV with a frequency of 0.5 Hz (n = 200 depolarizations for each bath solution). (B) Effects of [Na+]o on open time distributions. The normalized square root of the number of events per bin (n1/2) is plotted versus the logarithm of the open duration. The lines represent fits to single exponential distributions. Mean open times were 1.12 ± 0.03, 0.63 ± 0.01, and 0.93 ± 0.03 ms for 150, 10, and 150 mM Na+, respectively. Data from the twochannel patch shown in A. (C) Normalized first latency distributions, corrected for the number of channels, obtained for the same patch. (D) Cumulative distributions of the duration of the last (truncated) closing in a 90ms depolarization for the same patch. (E and F) Cumulative distributions of the truncated closed time conditional on whether the following record contains openings (solid line) or not (dotted line) for a singlechannel patch successively bathed in 10 (E) and 150 mM Na+ (F).


Figure 6. Maximum likelihood estimates of the rate constants for the kinetic model. Data are means ± SEM from 13 patches. P values are derived by ANOVA from the natural logarithm of the rate constants.


Figure 7. Probability of a channel being in the open (A) or the slowinactivated state (B) during a depolarization. (A) Ensemble average of bursts in 10 and 150 mM external Na+. The lines represent the open probability during a burst calculated from the twochannel patch of Fig. 5. The estimated rate constants for this patch, using the model in Fig. 6, were (150 mM Na+, first exposure): k01 = 804 ± 18 s−1, k10 = 1,915 ± 85 s−1, k12 = 1,247 ± 89 s−1, k21 = 154 ± 10 s−1, k23 = 8.06 ± 1.53 s−1; (10 mM Na+): k01 = 1,357 ± 33 s−1, k10 = 1,865 ± 80 s−1, k12 = 739 ± 63 s−1, k21 = 102 ± 10 s−1, k23 = 19.2 ± 2.6 s−1; (150 mM Na+): k01 = 860 ± 15 s−1, k10 = 2,361 ± 67 s−1, k12 = 933 ± 46 s−1, k21 = 131 ± 8 s−1, k23 = 5.65 ± 2.08 s−1. The 4state model predicts three time constants for the decay of burst open probability. These time constants were determined from the estimated rate constants by spectral expansion, and for this patch were (150 mM Na+): 0.27, 2.62, and 177.0 ms; (10 mM Na+): 0.27, 2.74, and 70.1 ms; and (150 mM Na+, first exposure): 0.25, 3.23, and 272.8 ms. (B) Probability for a channel to be in the absorbing inactivated state I3 (P(I)3) during a 90ms pulse to +60 mV. P(I)3 was calculated from the above rate constants for the twochannel patch.
