Limits...
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.

Show MeSH

Related in: MedlinePlus

The Na+/TMA+ biionic potential is independent of the absolute concentration of these permeant ions. (A and B) Outside-out patches from giant fiber lobe neurons held at −50 mV with pipettes containing 550 (A) or 55 (B) mM Na+. Top, horizontal bars designate bath ionic composition and application of palytoxin (10 nM). Bottom, current trace; the dotted line indicates the zero current level, and the brief vertical deflections indicate episodes in which 50-ms-long voltage pulses (−120 to +40 mV in 10-mV increments) were applied. (C) Palytoxin-induced I-V relationships with bath solutions containing 550 mM Na+ (from A; filled circles, c-b), 550 mM TMA+ (from A; open circles, d-a), 55 Na+ (from B; filled triangles, g-f) and 55 TMA+ (from B; open triangles, h-e). (D) Changes in Vrev (VrevTMA+o − VrevNa+o) at three different permeant ion concentrations. Mean values (number of experiments given in parentheses) were −58 ± 2, −57 ± 3, and −57 ± 1 mV for permeant ion concentrations of 55, 165, and 550 mM, respectively.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2085370&req=5

fig3: The Na+/TMA+ biionic potential is independent of the absolute concentration of these permeant ions. (A and B) Outside-out patches from giant fiber lobe neurons held at −50 mV with pipettes containing 550 (A) or 55 (B) mM Na+. Top, horizontal bars designate bath ionic composition and application of palytoxin (10 nM). Bottom, current trace; the dotted line indicates the zero current level, and the brief vertical deflections indicate episodes in which 50-ms-long voltage pulses (−120 to +40 mV in 10-mV increments) were applied. (C) Palytoxin-induced I-V relationships with bath solutions containing 550 mM Na+ (from A; filled circles, c-b), 550 mM TMA+ (from A; open circles, d-a), 55 Na+ (from B; filled triangles, g-f) and 55 TMA+ (from B; open triangles, h-e). (D) Changes in Vrev (VrevTMA+o − VrevNa+o) at three different permeant ion concentrations. Mean values (number of experiments given in parentheses) were −58 ± 2, −57 ± 3, and −57 ± 1 mV for permeant ion concentrations of 55, 165, and 550 mM, respectively.

Mentions: Because TMA+ is somewhat more permeant than NMG+ in palytoxin-bound pump-channels (Artigas and Gadsby, 2004), we paired internal Na+ with external TMA+ (instead of NMG+) to more accurately measure reversal potentials. The reversal potential in nominally symmetrical Na+ solutions ([Na+] = 55, 165, or 550 mM) averaged 0.1 ± 0.7 mV (n = 11). We determined biionic potentials in each patch as the reversal potential shift (ΔVrev) on quickly switching from the Na+-containing external solution to one containing the same concentration of TMA+. Fig. 3 (A and B) shows examples of palytoxin-induced currents activated in outside-out patches initially exposed to symmetrical solutions containing 550 mM Na+ (A) or 55 mM Na+ (B). The corresponding, roughly linear, palytoxin-induced current–voltage curves, with reversal potentials close to 0 mV (Fig. 3 C, filled symbols), were obtained by subtraction of currents recorded during voltage-step episodes marked b and c, and f and g, respectively. Equivalent palytoxin-induced current–voltage curves at the same internal Na+ concentrations, but after switching to external solutions containing 550 mM (Fig. 3 A) or 55 mM (Fig. 3 B) TMA+, were obtained by subtracting currents from episodes a and d, and e and h, respectively, which yielded reversal potentials negative to −50 mV. On average, ΔVrev upon replacing external Na+ with TMA+ (Fig. 3 D) was the same at ion concentrations of 55 mM (−58 ± 2 mV), 165 mM (−57 ± 3 mV), and 550 mM (−57 ± 1 mV), indicating that PTMA/PNa was concentration independent over this range, with a mean value of 0.102 ± 0.004 (n = 11).


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)

The Na+/TMA+ biionic potential is independent of the absolute concentration of these permeant ions. (A and B) Outside-out patches from giant fiber lobe neurons held at −50 mV with pipettes containing 550 (A) or 55 (B) mM Na+. Top, horizontal bars designate bath ionic composition and application of palytoxin (10 nM). Bottom, current trace; the dotted line indicates the zero current level, and the brief vertical deflections indicate episodes in which 50-ms-long voltage pulses (−120 to +40 mV in 10-mV increments) were applied. (C) Palytoxin-induced I-V relationships with bath solutions containing 550 mM Na+ (from A; filled circles, c-b), 550 mM TMA+ (from A; open circles, d-a), 55 Na+ (from B; filled triangles, g-f) and 55 TMA+ (from B; open triangles, h-e). (D) Changes in Vrev (VrevTMA+o − VrevNa+o) at three different permeant ion concentrations. Mean values (number of experiments given in parentheses) were −58 ± 2, −57 ± 3, and −57 ± 1 mV for permeant ion concentrations of 55, 165, and 550 mM, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2085370&req=5

fig3: The Na+/TMA+ biionic potential is independent of the absolute concentration of these permeant ions. (A and B) Outside-out patches from giant fiber lobe neurons held at −50 mV with pipettes containing 550 (A) or 55 (B) mM Na+. Top, horizontal bars designate bath ionic composition and application of palytoxin (10 nM). Bottom, current trace; the dotted line indicates the zero current level, and the brief vertical deflections indicate episodes in which 50-ms-long voltage pulses (−120 to +40 mV in 10-mV increments) were applied. (C) Palytoxin-induced I-V relationships with bath solutions containing 550 mM Na+ (from A; filled circles, c-b), 550 mM TMA+ (from A; open circles, d-a), 55 Na+ (from B; filled triangles, g-f) and 55 TMA+ (from B; open triangles, h-e). (D) Changes in Vrev (VrevTMA+o − VrevNa+o) at three different permeant ion concentrations. Mean values (number of experiments given in parentheses) were −58 ± 2, −57 ± 3, and −57 ± 1 mV for permeant ion concentrations of 55, 165, and 550 mM, respectively.
Mentions: Because TMA+ is somewhat more permeant than NMG+ in palytoxin-bound pump-channels (Artigas and Gadsby, 2004), we paired internal Na+ with external TMA+ (instead of NMG+) to more accurately measure reversal potentials. The reversal potential in nominally symmetrical Na+ solutions ([Na+] = 55, 165, or 550 mM) averaged 0.1 ± 0.7 mV (n = 11). We determined biionic potentials in each patch as the reversal potential shift (ΔVrev) on quickly switching from the Na+-containing external solution to one containing the same concentration of TMA+. Fig. 3 (A and B) shows examples of palytoxin-induced currents activated in outside-out patches initially exposed to symmetrical solutions containing 550 mM Na+ (A) or 55 mM Na+ (B). The corresponding, roughly linear, palytoxin-induced current–voltage curves, with reversal potentials close to 0 mV (Fig. 3 C, filled symbols), were obtained by subtraction of currents recorded during voltage-step episodes marked b and c, and f and g, respectively. Equivalent palytoxin-induced current–voltage curves at the same internal Na+ concentrations, but after switching to external solutions containing 550 mM (Fig. 3 A) or 55 mM (Fig. 3 B) TMA+, were obtained by subtracting currents from episodes a and d, and e and h, respectively, which yielded reversal potentials negative to −50 mV. On average, ΔVrev upon replacing external Na+ with TMA+ (Fig. 3 D) was the same at ion concentrations of 55 mM (−58 ± 2 mV), 165 mM (−57 ± 3 mV), and 550 mM (−57 ± 1 mV), indicating that PTMA/PNa was concentration independent over this range, with a mean value of 0.102 ± 0.004 (n = 11).

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