XB-ART-16304J Gen Physiol July 1, 1997; 110 (1): 23-33.
Effect of alkali metal cations on slow inactivation of cardiac Na+ channels.
Human heart Na+ channels were expressed transiently in both mammalian cells and Xenopus oocytes, and Na+ currents measured using 150 mM intracellular Na+. The kinetics of decaying outward Na+ current in response to 1-s depolarizations in the F1485Q mutant depends on the predominant cation in the extracellular solution, suggesting an effect on slow inactivation. The decay rate is lower for the alkali metal cations Li+, Na+, K+, Rb+, and Cs+ than for the organic cations Tris, tetramethylammonium, N-methylglucamine, and choline. In whole cell recordings, raising [Na+]zero from 10 to 150 mM increases the rate of recovery from slow inactivation at -140 mV, decreases the rate of slow inactivation at relatively depolarized voltages, and shifts steady-state slow inactivation in a depolarized direction. Single channel recordings of F1485Q show a decrease in the number of blank (i.e., null) records when [Na+]0 is increased. Significant clustering of blank records when depolarizing at a frequency of 0.5 Hz suggests that periods of inactivity represent the sojourn of a channel in a slow-inactivated state. Examination of the single channel kinetics at +60 mV during 90-ms depolarizations shows that neither open time, closed time, nor first latency is significantly affected by [Na+]0. However raising [Na+]0 decreases the duration of the last closed interval terminated by the end of the depolarization, leading to an increased number of openings at the depolarized voltage. Analysis of single channel data indicates that at a depolarized voltage a single rate constant for entry into a slow-inactivated state is reduced in high [Na+]0, suggesting that the binding of an alkali metal cation, perhaps in the ion-conducting pore, inhibits the closing of the slow inactivation gate.
PubMed ID: 9234168
PMC ID: PMC2229358
Article link: J Gen Physiol
Genes referenced: tbx2
Article Images: [+] show captions
|Figure 2. Effects of external cations on the slow decay of F1485Q Na+ channels currents. Whole-cell 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 F1485Q-transfected 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 20-ms recovery pulse to −140 mV was applied followed by a 20-ms 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. Steady-state slow and fast inactivation in high and low [Na+]o. Steady-state 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 20-mV increments. After 2 min at each holding potential, a 20-ms recovery pulse to −140 mV and a 9-ms 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. Best-fits 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) Steady-state fast inactivation (hΘ) induced by a 50-ms 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 best-fit 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. Single-channel data analysis. (A) Ensemble averages obtained from a two-channel outside-out patch successively bathed in 150, 10, and 150 mM Na+. Currents were activated by 90-ms 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 two-channel 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 90-ms 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 single-channel 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 slow-inactivated 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 two-channel 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 4-state 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 90-ms pulse to +60 mV. P(I)3 was calculated from the above rate constants for the two-channel patch.|