Sodium flux ratio in Na/K pump-channels opened by palytoxin
Figure S1: Example of a control experiment to compare the time course and amplitude of membrane current and Na+ efflux, under zero-trans conditions, during an ∼40-min-long washout (starting at the solution change artifact, a), and reapplication (at b), of 200 nM TTX. (193.5Kb)
Rakowski, Robert F.
De Weer, Paul
Gadsby, David C.
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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 22Na+ 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 ~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 K0.5 for palytoxin action was ~70 pM. The pump-channels were ~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.
© 2007 Rakowski et al. This article is distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported License. The definitive version was published in Journal of General Physiology 130 (2007): 41-54, doi:10.1085/jgp.200709770.
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