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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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External K+ speeds the closure of Na/K pump-channels opened by palytoxin. The axon was internally dialyzed with 94 mM Na+ and held at 0 mV throughout the measurements of (A) 22Na+ efflux and (B) net membrane current. A detailed description of the experiment is given in the text. Blue lines indicate the extrapolated baselines for 22Na+ efflux and net current. The red trace represents the measured efflux data transformed by Eqs. 1 and 2 using the best-fit n' value of 0.96 and least-squares delay obtained as described in Fig. 5. After a saturating concentration of ouabain partially reverses palytoxin action, replacement of all external Na+ with K+ fully restores baseline current and 22Na+ efflux levels in ∼10 min.
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fig6: External K+ speeds the closure of Na/K pump-channels opened by palytoxin. The axon was internally dialyzed with 94 mM Na+ and held at 0 mV throughout the measurements of (A) 22Na+ efflux and (B) net membrane current. A detailed description of the experiment is given in the text. Blue lines indicate the extrapolated baselines for 22Na+ efflux and net current. The red trace represents the measured efflux data transformed by Eqs. 1 and 2 using the best-fit n' value of 0.96 and least-squares delay obtained as described in Fig. 5. After a saturating concentration of ouabain partially reverses palytoxin action, replacement of all external Na+ with K+ fully restores baseline current and 22Na+ efflux levels in ∼10 min.

Mentions: In the experiment of Fig. 5, with 400 mM Na+o and ∼100 mM Na+i and the axon held at 0 mV, an efflux/influx ratio of 0.25 (100/400) is expected if the Ussing flux ratio exponent were 1.0. An initial estimate of n' can be garnered as follows. The peak increase in inward current caused by palytoxin was 16.0 μA cm−2 (equivalent to 165.8 pmol cm−2 s−1), and the peak increase in Na+ efflux was 40.4 pmol cm−2 s−1, giving a calculated peak change in influx equivalent to 206.2 pmol cm−2 s−1. Thus the peak response gives an efflux/influx ratio of 0.20, expected (via Eq. 1) for n' ∼ 1.14. As n' must be independent of the fraction of open pump-channels, a similar calculation can be repeated for each pair of palytoxin-induced current and flux measurements, made at 1.5-min intervals, during the activation or deactivation time course. In practice, all data pairs within a given time period were simultaneously least-squares fit to yield a single estimate for n'. The period (marked by red traces, see below) was chosen to allow reliable extrapolation of baseline (e.g., straight blue lines in Fig. 5) values of current and 22Na+ efflux and to ensure that palytoxin-induced increments in current and flux were of significant magnitude. The procedure illustrated in Fig. 5 inset was used to synchronize the current and efflux records, to account for the (imprecisely known) delay between experimental chamber and the fraction collector that collected radioactive samples. The least-squares fit was repeated for successive 5-s increments of assumed delay, to find the delay time that minimized the sum of squared deviations for the estimate of n'; in the inset of Fig. 5 this occurred for a time delay of 4.70 min and gave n' = 1.16. This method of synchronization was employed for all estimations of n' reported here. The time period over which current and flux data were analyzed to calculate n' in Figs. 5–9 and Fig. S2 is indicated by the red traces, which represent the measured efflux data, transformed using Eqs. 1 and 2 and the best-fit n' to generate a “computed current,” and plotted over the measured current after synchronization by the least-squares delay. The superposition illustrates the goodness of fit.


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)

External K+ speeds the closure of Na/K pump-channels opened by palytoxin. The axon was internally dialyzed with 94 mM Na+ and held at 0 mV throughout the measurements of (A) 22Na+ efflux and (B) net membrane current. A detailed description of the experiment is given in the text. Blue lines indicate the extrapolated baselines for 22Na+ efflux and net current. The red trace represents the measured efflux data transformed by Eqs. 1 and 2 using the best-fit n' value of 0.96 and least-squares delay obtained as described in Fig. 5. After a saturating concentration of ouabain partially reverses palytoxin action, replacement of all external Na+ with K+ fully restores baseline current and 22Na+ efflux levels in ∼10 min.
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fig6: External K+ speeds the closure of Na/K pump-channels opened by palytoxin. The axon was internally dialyzed with 94 mM Na+ and held at 0 mV throughout the measurements of (A) 22Na+ efflux and (B) net membrane current. A detailed description of the experiment is given in the text. Blue lines indicate the extrapolated baselines for 22Na+ efflux and net current. The red trace represents the measured efflux data transformed by Eqs. 1 and 2 using the best-fit n' value of 0.96 and least-squares delay obtained as described in Fig. 5. After a saturating concentration of ouabain partially reverses palytoxin action, replacement of all external Na+ with K+ fully restores baseline current and 22Na+ efflux levels in ∼10 min.
Mentions: In the experiment of Fig. 5, with 400 mM Na+o and ∼100 mM Na+i and the axon held at 0 mV, an efflux/influx ratio of 0.25 (100/400) is expected if the Ussing flux ratio exponent were 1.0. An initial estimate of n' can be garnered as follows. The peak increase in inward current caused by palytoxin was 16.0 μA cm−2 (equivalent to 165.8 pmol cm−2 s−1), and the peak increase in Na+ efflux was 40.4 pmol cm−2 s−1, giving a calculated peak change in influx equivalent to 206.2 pmol cm−2 s−1. Thus the peak response gives an efflux/influx ratio of 0.20, expected (via Eq. 1) for n' ∼ 1.14. As n' must be independent of the fraction of open pump-channels, a similar calculation can be repeated for each pair of palytoxin-induced current and flux measurements, made at 1.5-min intervals, during the activation or deactivation time course. In practice, all data pairs within a given time period were simultaneously least-squares fit to yield a single estimate for n'. The period (marked by red traces, see below) was chosen to allow reliable extrapolation of baseline (e.g., straight blue lines in Fig. 5) values of current and 22Na+ efflux and to ensure that palytoxin-induced increments in current and flux were of significant magnitude. The procedure illustrated in Fig. 5 inset was used to synchronize the current and efflux records, to account for the (imprecisely known) delay between experimental chamber and the fraction collector that collected radioactive samples. The least-squares fit was repeated for successive 5-s increments of assumed delay, to find the delay time that minimized the sum of squared deviations for the estimate of n'; in the inset of Fig. 5 this occurred for a time delay of 4.70 min and gave n' = 1.16. This method of synchronization was employed for all estimations of n' reported here. The time period over which current and flux data were analyzed to calculate n' in Figs. 5–9 and Fig. S2 is indicated by the red traces, which represent the measured efflux data, transformed using Eqs. 1 and 2 and the best-fit n' to generate a “computed current,” and plotted over the measured current after synchronization by the least-squares delay. The superposition illustrates the goodness of fit.

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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Related in: MedlinePlus