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Sodium flux ratio in Na/K pump-channels opened by palytoxin.

Rakowski RF, Artigas P, Palma F, Holmgren M, De Weer P, Gadsby DC - J. Gen. Physiol. (2007)

Bottom Line: The pump-channels were approximately 40-50 times less permeable to N-methyl-d-glucamine (NMG(+)) than to Na(+).In giant axons, the Ussing flux ratio exponent (n') for Na(+) movements through palytoxin-bound pump-channels, over a 100-400 mM range of external [Na(+)] and 0 to -40 mV range of membrane potentials, averaged 1.05 +/- 0.02 (n = 28).These findings are consistent with occupancy of palytoxin-bound Na(+)/K(+) pump-channels either by a single Na(+) ion or by two Na(+) ions as might be anticipated from other work; idiosyncratic constraints are needed if the two Na(+) ions occupy a single-file pore, but not if they occupy side-by-side binding sites, as observed in related structures, and if only one of the sites is readily accessible from both sides of the membrane.

View Article: PubMed Central - PubMed

Affiliation: Marine Biological Laboratory, Woods Hole, MA 02543, USA. rakowski@ohio.edu

ABSTRACT
Palytoxin binds to Na(+)/K(+) pumps in the plasma membrane of animal cells and opens an electrodiffusive cation pathway through the pumps. We investigated properties of the palytoxin-opened channels by recording macroscopic and microscopic currents in cell bodies of neurons from the giant fiber lobe, and by simultaneously measuring net current and (22)Na(+) efflux in voltage-clamped, internally dialyzed giant axons of the squid Loligo pealei. The conductance of single palytoxin-bound "pump-channels" in outside-out patches was approximately 7 pS in symmetrical 500 mM [Na(+)], comparable to findings in other cells. In these high-[Na(+)], K(+)-free solutions, with 5 mM cytoplasmic [ATP], the K(0.5) for palytoxin action was approximately 70 pM. The pump-channels were approximately 40-50 times less permeable to N-methyl-d-glucamine (NMG(+)) than to Na(+). The reversal potential of palytoxin-elicited current under biionic conditions, with the same concentration of a different permeant cation on each side of the membrane, was independent of the concentration of those ions over the range 55-550 mM. In giant axons, the Ussing flux ratio exponent (n') for Na(+) movements through palytoxin-bound pump-channels, over a 100-400 mM range of external [Na(+)] and 0 to -40 mV range of membrane potentials, averaged 1.05 +/- 0.02 (n = 28). These findings are consistent with occupancy of palytoxin-bound Na(+)/K(+) pump-channels either by a single Na(+) ion or by two Na(+) ions as might be anticipated from other work; idiosyncratic constraints are needed if the two Na(+) ions occupy a single-file pore, but not if they occupy side-by-side binding sites, as observed in related structures, and if only one of the sites is readily accessible from both sides of the membrane.

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Determination of the flux ratio exponent n' for Na/K pump-channels opened by palytoxin. The axon was internally dialyzed with 98 mM Na+ and superfused with 400 mM Na+, and held at 0 mV. Simultaneous measurements of unidirectional 22Na+ efflux (top trace) and net current (bottom trace) were used to estimate the flux ratio exponent n' for Na/K pump-channels opened by 20 nM palytoxin. The inset figure illustrates the procedure to account for the delay between current measurements and the fraction collector that collected radioactive samples. Least-squares fits of Eq. 1 to the current and flux data were repeated for successive 5-s increments of delay, to find the delay time that minimized the sum of squared deviations for the estimate of n'. In this experiment, a time delay of 4.70 min resulted in a minimum in the sum of squares and gave an estimate for n' of 1.16. Baseline values (blue lines) for 22Na+ efflux and net current were used to correct measured values in the determinations of n'. The red trace represents the measured efflux data transformed by Eqs. 1 and 2 using the best-fit n' and delay value obtained from the fits. In the presence of external Na+, saturating concentrations of ouabain fully reverse palytoxin-induced 22Na+ efflux in ∼100 min. Vertical current displacements are in response to voltage staircases, with 0.5 mV steps, from −5 to 5 mV applied to monitor membrane conductance during the decline in current and flux.
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fig5: Determination of the flux ratio exponent n' for Na/K pump-channels opened by palytoxin. The axon was internally dialyzed with 98 mM Na+ and superfused with 400 mM Na+, and held at 0 mV. Simultaneous measurements of unidirectional 22Na+ efflux (top trace) and net current (bottom trace) were used to estimate the flux ratio exponent n' for Na/K pump-channels opened by 20 nM palytoxin. The inset figure illustrates the procedure to account for the delay between current measurements and the fraction collector that collected radioactive samples. Least-squares fits of Eq. 1 to the current and flux data were repeated for successive 5-s increments of delay, to find the delay time that minimized the sum of squared deviations for the estimate of n'. In this experiment, a time delay of 4.70 min resulted in a minimum in the sum of squares and gave an estimate for n' of 1.16. Baseline values (blue lines) for 22Na+ efflux and net current were used to correct measured values in the determinations of n'. The red trace represents the measured efflux data transformed by Eqs. 1 and 2 using the best-fit n' and delay value obtained from the fits. In the presence of external Na+, saturating concentrations of ouabain fully reverse palytoxin-induced 22Na+ efflux in ∼100 min. Vertical current displacements are in response to voltage staircases, with 0.5 mV steps, from −5 to 5 mV applied to monitor membrane conductance during the decline in current and flux.

Mentions: At high (400 mM) external Na+, responses to 20 nM palytoxin applied to an axon, held at 0 mV, and with ∼100 mM internal Na+, appeared larger (Fig. 5) than expected from the results in Na+-free NMG+ external solution (e.g., Fig. 4). In the axon of Fig. 5, before its accelerating action was precipitously interrupted by addition of 100 μM ouabain, palytoxin activated >35 (compared with ∼15) pmol cm−2 s−1 of 22Na efflux within 10 min, and a net current (≥15 μA cm−2) that was larger than predicted, assuming independence. The smaller response in Fig. 4 likely reflects partial block of palytoxin-bound pump-channels by external NMG+, as observed in outside-out patches (Fig. 2 C). A further contrast with Fig. 4 is the order-of-magnitude slower reversal of palytoxin action upon its withdrawal in the presence of saturating concentrations of ouabain (1 mM was no more effective than 100 μM; Fig. 5). The palytoxin pump complex is evidently stabilized at high external [Na+] (compare Artigas and Gadsby, 2004).


Sodium flux ratio in Na/K pump-channels opened by palytoxin.

Rakowski RF, Artigas P, Palma F, Holmgren M, De Weer P, Gadsby DC - J. Gen. Physiol. (2007)

Determination of the flux ratio exponent n' for Na/K pump-channels opened by palytoxin. The axon was internally dialyzed with 98 mM Na+ and superfused with 400 mM Na+, and held at 0 mV. Simultaneous measurements of unidirectional 22Na+ efflux (top trace) and net current (bottom trace) were used to estimate the flux ratio exponent n' for Na/K pump-channels opened by 20 nM palytoxin. The inset figure illustrates the procedure to account for the delay between current measurements and the fraction collector that collected radioactive samples. Least-squares fits of Eq. 1 to the current and flux data were repeated for successive 5-s increments of delay, to find the delay time that minimized the sum of squared deviations for the estimate of n'. In this experiment, a time delay of 4.70 min resulted in a minimum in the sum of squares and gave an estimate for n' of 1.16. Baseline values (blue lines) for 22Na+ efflux and net current were used to correct measured values in the determinations of n'. The red trace represents the measured efflux data transformed by Eqs. 1 and 2 using the best-fit n' and delay value obtained from the fits. In the presence of external Na+, saturating concentrations of ouabain fully reverse palytoxin-induced 22Na+ efflux in ∼100 min. Vertical current displacements are in response to voltage staircases, with 0.5 mV steps, from −5 to 5 mV applied to monitor membrane conductance during the decline in current and flux.
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Related In: Results  -  Collection

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fig5: Determination of the flux ratio exponent n' for Na/K pump-channels opened by palytoxin. The axon was internally dialyzed with 98 mM Na+ and superfused with 400 mM Na+, and held at 0 mV. Simultaneous measurements of unidirectional 22Na+ efflux (top trace) and net current (bottom trace) were used to estimate the flux ratio exponent n' for Na/K pump-channels opened by 20 nM palytoxin. The inset figure illustrates the procedure to account for the delay between current measurements and the fraction collector that collected radioactive samples. Least-squares fits of Eq. 1 to the current and flux data were repeated for successive 5-s increments of delay, to find the delay time that minimized the sum of squared deviations for the estimate of n'. In this experiment, a time delay of 4.70 min resulted in a minimum in the sum of squares and gave an estimate for n' of 1.16. Baseline values (blue lines) for 22Na+ efflux and net current were used to correct measured values in the determinations of n'. The red trace represents the measured efflux data transformed by Eqs. 1 and 2 using the best-fit n' and delay value obtained from the fits. In the presence of external Na+, saturating concentrations of ouabain fully reverse palytoxin-induced 22Na+ efflux in ∼100 min. Vertical current displacements are in response to voltage staircases, with 0.5 mV steps, from −5 to 5 mV applied to monitor membrane conductance during the decline in current and flux.
Mentions: At high (400 mM) external Na+, responses to 20 nM palytoxin applied to an axon, held at 0 mV, and with ∼100 mM internal Na+, appeared larger (Fig. 5) than expected from the results in Na+-free NMG+ external solution (e.g., Fig. 4). In the axon of Fig. 5, before its accelerating action was precipitously interrupted by addition of 100 μM ouabain, palytoxin activated >35 (compared with ∼15) pmol cm−2 s−1 of 22Na efflux within 10 min, and a net current (≥15 μA cm−2) that was larger than predicted, assuming independence. The smaller response in Fig. 4 likely reflects partial block of palytoxin-bound pump-channels by external NMG+, as observed in outside-out patches (Fig. 2 C). A further contrast with Fig. 4 is the order-of-magnitude slower reversal of palytoxin action upon its withdrawal in the presence of saturating concentrations of ouabain (1 mM was no more effective than 100 μM; Fig. 5). The palytoxin pump complex is evidently stabilized at high external [Na+] (compare Artigas and Gadsby, 2004).

Bottom Line: The pump-channels were approximately 40-50 times less permeable to N-methyl-d-glucamine (NMG(+)) than to Na(+).In giant axons, the Ussing flux ratio exponent (n') for Na(+) movements through palytoxin-bound pump-channels, over a 100-400 mM range of external [Na(+)] and 0 to -40 mV range of membrane potentials, averaged 1.05 +/- 0.02 (n = 28).These findings are consistent with occupancy of palytoxin-bound Na(+)/K(+) pump-channels either by a single Na(+) ion or by two Na(+) ions as might be anticipated from other work; idiosyncratic constraints are needed if the two Na(+) ions occupy a single-file pore, but not if they occupy side-by-side binding sites, as observed in related structures, and if only one of the sites is readily accessible from both sides of the membrane.

View Article: PubMed Central - PubMed

Affiliation: Marine Biological Laboratory, Woods Hole, MA 02543, USA. rakowski@ohio.edu

ABSTRACT
Palytoxin binds to Na(+)/K(+) pumps in the plasma membrane of animal cells and opens an electrodiffusive cation pathway through the pumps. We investigated properties of the palytoxin-opened channels by recording macroscopic and microscopic currents in cell bodies of neurons from the giant fiber lobe, and by simultaneously measuring net current and (22)Na(+) efflux in voltage-clamped, internally dialyzed giant axons of the squid Loligo pealei. The conductance of single palytoxin-bound "pump-channels" in outside-out patches was approximately 7 pS in symmetrical 500 mM [Na(+)], comparable to findings in other cells. In these high-[Na(+)], K(+)-free solutions, with 5 mM cytoplasmic [ATP], the K(0.5) for palytoxin action was approximately 70 pM. The pump-channels were approximately 40-50 times less permeable to N-methyl-d-glucamine (NMG(+)) than to Na(+). The reversal potential of palytoxin-elicited current under biionic conditions, with the same concentration of a different permeant cation on each side of the membrane, was independent of the concentration of those ions over the range 55-550 mM. In giant axons, the Ussing flux ratio exponent (n') for Na(+) movements through palytoxin-bound pump-channels, over a 100-400 mM range of external [Na(+)] and 0 to -40 mV range of membrane potentials, averaged 1.05 +/- 0.02 (n = 28). These findings are consistent with occupancy of palytoxin-bound Na(+)/K(+) pump-channels either by a single Na(+) ion or by two Na(+) ions as might be anticipated from other work; idiosyncratic constraints are needed if the two Na(+) ions occupy a single-file pore, but not if they occupy side-by-side binding sites, as observed in related structures, and if only one of the sites is readily accessible from both sides of the membrane.

Show MeSH
Related in: MedlinePlus