Sodium Flux Ratio in Na/K Pump-Channels Opened by Palytoxin

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 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 K 0.5 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.

The ion pathway through the opened Na+,K+-ATPase pump

“…That ∼6-Å wide × ∼12-Å long MTSET + , MTSES - , and MTSACE pass through palytoxin-bound Na + ,K + pump-channels corroborates the findings that these channels conduct N-methyl-D-glucamine ions (diameter ≥7 Å) only ∼50 fold more slowly than Na + ions22, and that their measured27 Na + flux ratio exponent28 is ∼1.0, implying little interaction between Na + ions in a queue along the principal pathway that passes through site II. Occupancy by a second Na + ion of site I, off the main pathway but linked to it via a connection narrow enough to preclude reactivity with MTSET + , could account for suggested average pump-channel occupancy by two Na + ions23.…”

CryoEM structure of the human SLC4A4 sodium-coupled acid-base transporter NBCe1

“…Specifically, a double mutation of residues flanking the central Cl − in the E. coli ClC 2Cl − /H + exchanger converts the transporter into a channel likely by stabilizing conformations where inner and outer gates are open 34 . The Na + -K + -ATPase (the ATP-dependent Na + /K + -exchanger) is converted into a cation channel when bound to palytoxin likely by uncoupling the inner and outer gates 35 . Finally, naturally occurring mutations in AE1 block its anion exchange activity and uncover a monovalent cation conductance 30 , 36 .…”

On the effect of hyperaldosteronism-inducing mutations in Na/K pumps

“…These slow turnover values contrast with the much faster transport rates in ion channels (10 7 –10 8 s −1 ). Thus, none of these Na/K pump α1 mutants can be considered bona fide channels, like those induced by binding of the Na/K pump–specific marine toxin palytoxin, which opens a pump channel that transports millions of cations per second ( Artigas and Gadsby, 2004 ; Rakowski et al, 2007 ). Rather, these mutations appear to destabilize ion-occlusion reactions, an effect resembling the alterations produced by deletion or mutation of the α-subunit C-terminal end, which also induce leaky pumps under near-physiological Na + o ( Yaragatupalli et al, 2009 ; Meier et al, 2010 ; Vedovato and Gadsby, 2010 ).…”