Townsend C et al. (1997), Anomalous effect of permeant ion concentration ...

XB-ART-16305

J Gen Physiol 1997 Jul 01;1101:11-21. doi: 10.1085/jgp.110.1.11.

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Anomalous effect of permeant ion concentration on peak open probability of cardiac Na+ channels.

Townsend C , Hartmann HA , Horn R .

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Human heart Na+ channels were expressed transiently in both mammalian cells and Xenopus oocytes, and Na+ currents measured using 150 mM intracellular Na+. Decreasing extracellular permeant ion concentration decreases outward Na+ current at positive voltages while increasing the driving force for the current. This anomalous effect of permeant ion concentration, especially obvious in a mutant (F1485Q) in which fast inactivation is partially abolished, is due to an alteration of open probability. The effect is only observed when a highly permeant cation (Na+, Li+, or hydrazinium) is substituted for a relatively impermeant cation (K+, Rb+, Cs+, N-methylglucamine, Tris, choline, or tetramethylammonium). With high concentrations of extracellular permeant cations, the peak open probability of Na+ channels increases with depolarization and then saturates at positive voltages. By contrast, with low concentrations of permeant ions, the open probability reaches a maximum at approximately 0 mV and then decreases with further depolarization. There is little effect of permeant ion concentration on activation kinetics at depolarized voltages. Furthermore, the lowered open probability caused by a brief depolarization to +60 mV recovers within 5 ms upon repolarization to -140 mV, indicative of a gating process with rapid kinetics. Tail currents at reduced temperatures reveal the rapid onset of this gating process during a large depolarization. A large depolarization may drive a permeant cation out of a site within the extracellular mouth of the pore, reducing the efficiency with which the channel opens.

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	Figure 5. Effect of extracellular Na+, Cs+, and Li+ on Popen of F1485Q Na+ channels. Popen (Po) was calculated from whole-cell peak currents (Fig. 2) and single-channel i-V relations (Figs. 3 and 4) as described in methods. NMG replaced Na+ in the 10 mM Na+ bath solution. Data are means Â± SEM normalized to the maximum Po obtained in 150 mM Na+o.
	Figure 2. Effects of external cations on peak Na+ current-voltage relations. Currents were activated as described in the legend of Fig. 1. Normalized peak currents for WT- (A, n = 5) and F1485Q- (B, n = 3) transfected cells sequentially bathed in 10, 150, and 10 mM Na+. (C, D, and E) Current-voltage relations for F1485Q-transfected cells successively bathed in (in mM): 10 Na+ (140 Cs+), 150 Na+, and 10 Na+ (140 Cs+) (C, n = 3); 150 NMG, 150 Cs+, and 150 NMG (D, n = 5); 150 NMG, 150 Li+, and 150 NMG (E, n = 4); or 150+ Na, 150 Li+, and 150 Na+ (F, n = 3). Intracellular [Na+] was 150 mM in all cases.
	Figure 3. Effects of [Na+]o on single-F1485Q channel currents. Selected single-channel current recordings from outside-out patches bathed in 10 (A) and 150 mM Na+ (B). Internal [Na+] was 150 mM. Currents were activated by 90-ms depolarizations (arrow) to voltages ranging from +20 to +80 mV (as indicated to the left of the traces) from a holding potential of â140 mV. The dotted lines represent the closed level. (C) Single-channel current-voltage relations in 10 (n = 2) and 150 mM (n = 4) Na+o. Data points for 150 mM Na+ were fit by linear regression, yielding an estimate of the GHK permeability (solid line). The dotted line represents single-channel currents for 10 mM Na+o as predicted by the GHK current equation.
	Figure 4. Effects of external cations on single-channel outward currents. Currents were evoked as described in the legend of Fig. 3. (A) Selected single-channel recordings obtained in the presence of 150 mM NMG, 150 mM Cs+, and 150 mM Li+ in the bath solution (V = +60 mV). (B) Current-voltage relations for 150 mM NMGo (n = 2), 150 mM Cs+o (n = 3), and 150 mM Li+o (n = 3). Data points were fit to straight lines with slopes of 27, 31, and 35 pS for NMG, Cs+, and Li+, respectively.
	Figure 1. WT and F1485Q hH1a Na+ channel currents in 10 and 150 mM [Na+]o. Currents were elicited by 9-ms depolarizations to voltages ranging from â80 to +70 mV in 10-mV increments from a holding potential of â140 mV and at a frequency of 0.5 Hz. Each panel shows families of Na+ currents obtained for one cell transfected with either WT (A) or F1485Q (B) Na+ channels and successively bathed in 10, 150, and 10 mM external Na+. Intracellular [Na+] was 150 mM.
	Figure 6. Effects of impermeant cations on normalized P-V relationships. Peak Popen (Po) was determined from whole-cell and single-channel current-voltage relations as described in methods. Data from F1485Q-transfected cells bathed in 150 mM of the indicated cations. In each panel, the dotted line corresponds to the P-V curve obtained with 150 mM Na+o. Data are means Â± SEM with maximums normalized to unity.
	Figure 7. Effect of hydrazinium on the I-V relationship and channel open probability. Currents were evoked as described in the legend of Fig. 1 except that 5-mV increments were used between each pulse. (A) I-V relationships obtained from F1485Q-transfected cells were successively bathed in Na+, NMG, Na+, hydrazinium, and Na+ (150 mM except [hydrazinium]o â¼138 mM, see methods). Data are means Â± SEM from 3 cells. (B) Peak Popen (Po) versus voltage relations for cells bathed in hydrazinium (138 mM, n = 3, see methods). The dotted line corresponds to the P-V curve obtained with 150 mM Na+o. Data are means Â± SEM with maximums normalized to unity.
	Figure 8. Lack of effect of [Na+]o on activation kinetics. (A) Scaled Na+ currents from a cell bathed sequentially in Na+, Cs+, and Na+ (150 mM). V = +60 mV, â140 mV holding potential. (B) Time to peak versus voltage for cells successively bathed in Na+, Cs+, and Na+ (150 mM). Currents were evoked as described in the legend of Fig. 1 except that 5-mV increments were used. Data are means Â± SEM from three cells.
	Figure 9. Rapid kinetics of recovery after alteration of Popen. Recovery at â140 mV after a 0.8-ms prepulse to +60 (A) or +20 mV (B, see insets). 16 0.8-ms recovery test pulses to +20 mV were given 0.1â18.1 ms after the prepulse (superimposed traces). Data are from one cell successively bathed in 150 mM Na+ and Cs+.

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Armstrong, Charge movement associated with the opening and closing of the activation gates of the Na channels. 1974, Pubmed