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

Townsend C, Hartmann HA, Horn R - J. Gen. Physiol. (1997)

Bottom Line: 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).There is little effect of permeant ion concentration on activation kinetics at depolarized voltages.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA.

ABSTRACT
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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Effect of external [Na+] on F1485Q tail currents. (A)  Families of currents elicited by 9-ms depolarizations to voltages  ranging from −80 to +70 mV. Holding potential, −140 mV. Currents shown were recorded from two cells bathed in either 150  mM Na+ (left) or 150 mM Cs+ at 8.0 and 7.1°C, respectively. (B)  Tail currents were recorded from −80 to +70 mV after a brief depolarization (1.5 ms) to 0 mV from a holding potential of −140  mV. Currents recorded from the same two cells shown in A at the  same temperatures.
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Figure 10: Effect of external [Na+] on F1485Q tail currents. (A) Families of currents elicited by 9-ms depolarizations to voltages ranging from −80 to +70 mV. Holding potential, −140 mV. Currents shown were recorded from two cells bathed in either 150 mM Na+ (left) or 150 mM Cs+ at 8.0 and 7.1°C, respectively. (B) Tail currents were recorded from −80 to +70 mV after a brief depolarization (1.5 ms) to 0 mV from a holding potential of −140 mV. Currents recorded from the same two cells shown in A at the same temperatures.

Mentions: Whole-cell voltage clamp recordings were carried out as previously reported (O'Leary and Horn, 1994). Sylgard-coated (Dow-Corning Corp., Midland, MI), fire-polished pipettes of Corning 8161 glass were used. Currents were filtered at 5 kHz with an Axopatch 200A patch clamp amplifier (Axon Instruments Inc., Burlingame, CA). Data were acquired with pCLAMP6 and the Digidata 1200 interface (Axon Instruments). Cells were dialyzed at least 10 min before recording data. Series resistance was <2 MΩ after 80% compensation. The pipette solution consisted of (in mM) 140 NaF, 10 NaCl, 5 EGTA, and 10 Cs-HEPES (pH 7.4). The 10 mM Na+ bath solution contained (in mM) 10 NaCl, 140 NMG-OH (N-methyl-d-glucamine hydroxide), 2 KCl, 1.5 CaCl2, 1 MgCl2, 10 HEPES (titrated to pH 7.4 with methanesulfonic acid). The 150 mM Na+ bath solution contained (in mM) 150 NaCl, 2 Kcl, 1.5 CaCl2, 1 MgCl2, and 10 HEPES (titrated to pH 7.4 with NaOH). In other solutions the NaCl was replaced by NMG-methanesulfonate, or by the chloride salts of Li+, K+, Rb+, Cs+, NMG, Tris(hydroxymethyl)-aminomethane, tetramethylammonium, choline, or hydrazinium. We found no difference between NMG -methanesulfonate and NMG-Cl in our experiments. The hydrazinium chloride solution was made by substituting 150 mM hydrazinium monochloride for 150 mM NaCl in the bath solution, and then titrating the solution to pH 7.4 with measured amounts of liquid hydrazine (Aldrich Chemical Co., Milwaukee, WI). Assuming a pK of 7.97 for the hydrazinium ion, we calculate the total concentration of cationic hydrazinium in our solution to be 138 mM. All solution components were purchased either from Aldrich or Sigma Chemical Co. (St. Louis, MO). Corrections were made for liquid junction potentials. After seal formation, cells were gently lifted off the bottom of the dish to allow better access to the entire cell surface. Bath solutions were exchanged by application of a desired solution to single cells with a Sigmacoted (Sigma) Corning Pyrex macropipette (Yang et al., 1996). Most experiments were performed at room temperature (19–21°C). The tail current experiments of Fig. 10 were done at a reduced temperature effected by Peltier devices regulated by a TC-10 Temperature Controller (Dagan Corp., Minneapolis, MN).


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

Townsend C, Hartmann HA, Horn R - J. Gen. Physiol. (1997)

Effect of external [Na+] on F1485Q tail currents. (A)  Families of currents elicited by 9-ms depolarizations to voltages  ranging from −80 to +70 mV. Holding potential, −140 mV. Currents shown were recorded from two cells bathed in either 150  mM Na+ (left) or 150 mM Cs+ at 8.0 and 7.1°C, respectively. (B)  Tail currents were recorded from −80 to +70 mV after a brief depolarization (1.5 ms) to 0 mV from a holding potential of −140  mV. Currents recorded from the same two cells shown in A at the  same temperatures.
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Figure 10: Effect of external [Na+] on F1485Q tail currents. (A) Families of currents elicited by 9-ms depolarizations to voltages ranging from −80 to +70 mV. Holding potential, −140 mV. Currents shown were recorded from two cells bathed in either 150 mM Na+ (left) or 150 mM Cs+ at 8.0 and 7.1°C, respectively. (B) Tail currents were recorded from −80 to +70 mV after a brief depolarization (1.5 ms) to 0 mV from a holding potential of −140 mV. Currents recorded from the same two cells shown in A at the same temperatures.
Mentions: Whole-cell voltage clamp recordings were carried out as previously reported (O'Leary and Horn, 1994). Sylgard-coated (Dow-Corning Corp., Midland, MI), fire-polished pipettes of Corning 8161 glass were used. Currents were filtered at 5 kHz with an Axopatch 200A patch clamp amplifier (Axon Instruments Inc., Burlingame, CA). Data were acquired with pCLAMP6 and the Digidata 1200 interface (Axon Instruments). Cells were dialyzed at least 10 min before recording data. Series resistance was <2 MΩ after 80% compensation. The pipette solution consisted of (in mM) 140 NaF, 10 NaCl, 5 EGTA, and 10 Cs-HEPES (pH 7.4). The 10 mM Na+ bath solution contained (in mM) 10 NaCl, 140 NMG-OH (N-methyl-d-glucamine hydroxide), 2 KCl, 1.5 CaCl2, 1 MgCl2, 10 HEPES (titrated to pH 7.4 with methanesulfonic acid). The 150 mM Na+ bath solution contained (in mM) 150 NaCl, 2 Kcl, 1.5 CaCl2, 1 MgCl2, and 10 HEPES (titrated to pH 7.4 with NaOH). In other solutions the NaCl was replaced by NMG-methanesulfonate, or by the chloride salts of Li+, K+, Rb+, Cs+, NMG, Tris(hydroxymethyl)-aminomethane, tetramethylammonium, choline, or hydrazinium. We found no difference between NMG -methanesulfonate and NMG-Cl in our experiments. The hydrazinium chloride solution was made by substituting 150 mM hydrazinium monochloride for 150 mM NaCl in the bath solution, and then titrating the solution to pH 7.4 with measured amounts of liquid hydrazine (Aldrich Chemical Co., Milwaukee, WI). Assuming a pK of 7.97 for the hydrazinium ion, we calculate the total concentration of cationic hydrazinium in our solution to be 138 mM. All solution components were purchased either from Aldrich or Sigma Chemical Co. (St. Louis, MO). Corrections were made for liquid junction potentials. After seal formation, cells were gently lifted off the bottom of the dish to allow better access to the entire cell surface. Bath solutions were exchanged by application of a desired solution to single cells with a Sigmacoted (Sigma) Corning Pyrex macropipette (Yang et al., 1996). Most experiments were performed at room temperature (19–21°C). The tail current experiments of Fig. 10 were done at a reduced temperature effected by Peltier devices regulated by a TC-10 Temperature Controller (Dagan Corp., Minneapolis, MN).

Bottom Line: 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).There is little effect of permeant ion concentration on activation kinetics at depolarized voltages.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus