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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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Apparent affinity for palytoxin. (A) Outside-out patch from a giant fiber lobe neuron held at −50 mV. Top, horizontal bars designate bath ionic composition and the concentration of palytoxin used. Bottom, current trace. The dotted line indicates the zero current level. Palytoxin concentrations were increased when approximately steady current levels were reached at each dose. The brief vertical deflections in the current record indicate I-V measurements. (B) Membrane current traces in response to 50 ms voltage steps from −100 to 100 mV (displayed here in 40-mV increments) in external solutions containing NMG+ or Na+ before (a and b) and after complete treatment with palytoxin (c and d). (C) Average palytoxin-induced current (I after palytoxin minus I before palytoxin) during the last 5 ms of every pulse under each ionic condition plotted against the applied voltage. The relative permeability of NMG+ (PNMG/PNa) was 0.024 ± 0.002 as calculated from the large shift in reversal potential (ΔVrev = −94 ± 2 mV). (D) Normalized and averaged dose response curve for palytoxin obtained from six patches. The solid line represents a Michaelis-Menten equation fit with a K0.5 = 74 ± 21 pM.
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fig2: Apparent affinity for palytoxin. (A) Outside-out patch from a giant fiber lobe neuron held at −50 mV. Top, horizontal bars designate bath ionic composition and the concentration of palytoxin used. Bottom, current trace. The dotted line indicates the zero current level. Palytoxin concentrations were increased when approximately steady current levels were reached at each dose. The brief vertical deflections in the current record indicate I-V measurements. (B) Membrane current traces in response to 50 ms voltage steps from −100 to 100 mV (displayed here in 40-mV increments) in external solutions containing NMG+ or Na+ before (a and b) and after complete treatment with palytoxin (c and d). (C) Average palytoxin-induced current (I after palytoxin minus I before palytoxin) during the last 5 ms of every pulse under each ionic condition plotted against the applied voltage. The relative permeability of NMG+ (PNMG/PNa) was 0.024 ± 0.002 as calculated from the large shift in reversal potential (ΔVrev = −94 ± 2 mV). (D) Normalized and averaged dose response curve for palytoxin obtained from six patches. The solid line represents a Michaelis-Menten equation fit with a K0.5 = 74 ± 21 pM.

Mentions: With symmetrical [Na+], and ATP in the pipette, exposure of outside-out patches to stepwise increasing concentrations of palytoxin caused a progressive inward current increase at −50 mV, reflecting eventual transformation of every Na+/K+ pump in the patch into a cation channel (Fig. 2 A). Complete replacement of external Na+ with NMG+ after that transformation caused a large outward current shift (Fig. 2, A and B), due to the slower permeation of the larger NMG+ cation through palytoxin-bound pump-channels. Current flowing through the palytoxin-bound pump-channels was estimated by appropriate subtraction of currents averaged over the final 5 ms of 50-ms steps to voltages between −100 and +100 mV (in 20-mV increments), in external Na+ and in external NMG+, before (Fig. 2 B, a and b) and after (Fig. 2 B, c and d) exposure to palytoxin. The resulting palytoxin-induced current is shown plotted against voltage in Fig. 2 C. From the large negative shift of reversal potential (ΔVrev = −94 ± 2 mV, n = 3) upon replacing external Na+ with NMG+, the relative permeability PNMG/PNa was calculated to be 0.024 ± 0.002. The smaller outward current at large positive potentials suggests that extracellular NMG+ impaired the outward flow of Na+ ions through palytoxin-bound pump-channels.


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)

Apparent affinity for palytoxin. (A) Outside-out patch from a giant fiber lobe neuron held at −50 mV. Top, horizontal bars designate bath ionic composition and the concentration of palytoxin used. Bottom, current trace. The dotted line indicates the zero current level. Palytoxin concentrations were increased when approximately steady current levels were reached at each dose. The brief vertical deflections in the current record indicate I-V measurements. (B) Membrane current traces in response to 50 ms voltage steps from −100 to 100 mV (displayed here in 40-mV increments) in external solutions containing NMG+ or Na+ before (a and b) and after complete treatment with palytoxin (c and d). (C) Average palytoxin-induced current (I after palytoxin minus I before palytoxin) during the last 5 ms of every pulse under each ionic condition plotted against the applied voltage. The relative permeability of NMG+ (PNMG/PNa) was 0.024 ± 0.002 as calculated from the large shift in reversal potential (ΔVrev = −94 ± 2 mV). (D) Normalized and averaged dose response curve for palytoxin obtained from six patches. The solid line represents a Michaelis-Menten equation fit with a K0.5 = 74 ± 21 pM.
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fig2: Apparent affinity for palytoxin. (A) Outside-out patch from a giant fiber lobe neuron held at −50 mV. Top, horizontal bars designate bath ionic composition and the concentration of palytoxin used. Bottom, current trace. The dotted line indicates the zero current level. Palytoxin concentrations were increased when approximately steady current levels were reached at each dose. The brief vertical deflections in the current record indicate I-V measurements. (B) Membrane current traces in response to 50 ms voltage steps from −100 to 100 mV (displayed here in 40-mV increments) in external solutions containing NMG+ or Na+ before (a and b) and after complete treatment with palytoxin (c and d). (C) Average palytoxin-induced current (I after palytoxin minus I before palytoxin) during the last 5 ms of every pulse under each ionic condition plotted against the applied voltage. The relative permeability of NMG+ (PNMG/PNa) was 0.024 ± 0.002 as calculated from the large shift in reversal potential (ΔVrev = −94 ± 2 mV). (D) Normalized and averaged dose response curve for palytoxin obtained from six patches. The solid line represents a Michaelis-Menten equation fit with a K0.5 = 74 ± 21 pM.
Mentions: With symmetrical [Na+], and ATP in the pipette, exposure of outside-out patches to stepwise increasing concentrations of palytoxin caused a progressive inward current increase at −50 mV, reflecting eventual transformation of every Na+/K+ pump in the patch into a cation channel (Fig. 2 A). Complete replacement of external Na+ with NMG+ after that transformation caused a large outward current shift (Fig. 2, A and B), due to the slower permeation of the larger NMG+ cation through palytoxin-bound pump-channels. Current flowing through the palytoxin-bound pump-channels was estimated by appropriate subtraction of currents averaged over the final 5 ms of 50-ms steps to voltages between −100 and +100 mV (in 20-mV increments), in external Na+ and in external NMG+, before (Fig. 2 B, a and b) and after (Fig. 2 B, c and d) exposure to palytoxin. The resulting palytoxin-induced current is shown plotted against voltage in Fig. 2 C. From the large negative shift of reversal potential (ΔVrev = −94 ± 2 mV, n = 3) upon replacing external Na+ with NMG+, the relative permeability PNMG/PNa was calculated to be 0.024 ± 0.002. The smaller outward current at large positive potentials suggests that extracellular NMG+ impaired the outward flow of Na+ ions through palytoxin-bound pump-channels.

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