Na+/K+-pump ligands modulate gating of palytoxin-induced ion channels

Abstract

The Na+/K+ pump is a ubiquitous P-type ATPase that binds three cytoplasmic Na+ ions deep within its core where they are temporarily occluded before being released to the extracellular surface. The 3Na+/2K+ -exchange transport cycle is completed when two extracellular K+ ions bind and become temporarily occluded within the protein and subsequently released to the cytoplasm. Coupling of Na+ -ion occlusion to phosphorylation of the pump by ATP and of K+ -ion occlusion to its dephosphorylation ensure the vectorial nature of net transport. The occluded-ion conformations, with binding sites inaccessible from either side, represent intermediate states in these alternating-access descriptions of transport. They afford protection against potentially catastrophic effects of inadvertently allowing simultaneous access from both membrane sides. The marine toxin, palytoxin, converts Na+/K+ pumps into nonselective cation channels, possibly by disrupting the normal strict coupling between opening of one access pathway in the Na+/K+ ATPase and closing of the other. We show here that gating of the channels in palytoxin-bound Na+/K+ pumps in excised membrane patches is modulated by the pump's physiological ligands: cytoplasmic application of ATP promotes opening of the channels, and extracellular replacement of Na+ ions by K+ ions promotes closing of the channels. This suggests that, despite the presence of bound palytoxin, certain partial reactions of the normal Na+/K+ -transport cycle persist and remain capable of effecting the conformational changes that control access to the pump's cation-binding sites. These findings affirm the alternating-access model of ion pumps and offer the possibility of examining ion occlusion/deocclusion reactions in single pump molecules.

Figure 1

Alternating-gate model of the Post-Albers (9, 10) transport cycle of the Na⁺/K⁺ pump represented in cartoon form as a channel with two gates never simultaneously open. Extra- (OUT) and intracellular (IN) surfaces of the membrane (yellow) are indicated, as are E2 states (Upper; external gate may open) and E1 states (Lower; internal gate may open). Occluded states with both gates shut (Upper Right, Lower Left) follow binding of 2 external K⁺, and of 3 internal Na⁺ and subsequent phosphorylation, respectively. ATP acts with low affinity to speed opening of the internal gate and concomitant K⁺ deocclusion, and acts with high affinity to phosphorylate the pump.

Figure 2

ATP increases P_o of PTX-induced channels in outside-out patches from myocytes, bathed by ≈150 mM Na solutions and held at −40 mV; dotted lines mark zero current (I = 0). (A Upper) With 5 mM internal ATP three channels were activated (within ≈4 s) by 25 pM PTX; the channels gated with high P_o after PTX withdrawal. (Lower) Continuation of the top record showing gating of the same channels for a longer period, and opening of more channels after reapplication of PTX. (B Upper) Without pipette ATP, 2 nM PTX elicited (within ≈60 s) opening of one channel, which showed low P_o after PTX removal (P_o = 0.19 ± 0.04, n = 4; from patches with apparently only one channel recorded for >4 min after PTX washout). (Lower) Continued gating of the same channel in absence of PTX (12 min of record omitted), and increase in the number active channels (still with low P_o) after PTX readdition.

Figure 3

ATP, AMPPNP, and ADP similarly enhance PTX-induced current in inside-out patches from HEK293 cells held at −20 mV, and bathed in ≈150 mM Na⁺ solutions, with 100 nM PTX in the pipette. (A) Increasing [MgATP] at the cytoplasmic surface progressively augmented macroscopic current; large vertical deflections reflect collection of conductance data. (B) Comparable influence of MgAMPPNP recorded from a different patch. (C) Effect of addition and removal of 1 mM MgADP. (D) Dose-response curves for current enhancement by all three nucleotides. Least squares fits (omitted for clarity) to the Hill equation yielded parameters: for ATP (black circles), K_ATP = 21 ± 3 μM, n_H = 0.8 ± 0.1 (n = 4); for AMPPNP (open triangles), K_AMPPNP = 59 ± 6 μM, n_H = 1 ± 0.1 (n = 3); for ADP (gray inverted triangles) K_ADP = 37 ± 4 μM, n_H = 1 ± 0.1 (n = 3).

Figure 4

External K⁺ closes PTX-induced channels in an outside-out HEK293 patch, held at −20 mV, in the absence of ATP. (A) After activation by 100 nM PTX, macroscopic current decline upon PTX withdrawal in external Na⁺ was slow (τ = 2600 s; blue fit line), but was accelerated upon replacing 160 mM Na⁺ with 160 mM K⁺ (double exponential fit, red line, gives A_f = A_s = 0.5, τ_f = 8 s, and τ_s = 61 s). Returning to Na⁺ after 300 s in K⁺ was without effect until PTX was reapplied. (B) Distinction between closing and PTX unbinding in the same patch as in A; brief substitution of K⁺ for Na⁺ produced rapid, partially reversible, current decay. (Inset) Rate of PTX unbinding in K⁺ estimated from the plot of residual current amplitude in Na⁺ solution against cumulative time spent in K⁺ (exponential fit, red line, yields τ = 57 s).

Figure 5

(A) The K⁺ congener Cs⁺ closes PTX-induced channels in an outside-out myocyte patch held at −40 mV, bathed by Na⁺ solutions but with no internal ATP. The few channels activated by 1 nM PTX remained active after PTX washout until replacement of 160 mM external Na⁺ by 160 mM Cs⁺ quickly reduced the P_o to ≈0. Channels slowly reopening to the initial current level after reapplication of Na⁺ indicates that PTX did not unbind; the ≈1-pA baseline shift on switching to and from Cs⁺ solution is an artifact due to the change of seal current. (B) After brief application of 200 nM PTX in 160 mM Na⁺ solution (not shown) to the same patch as in A, 160 mM Cs⁺ (thick trace) or 160 mM K⁺ (thin trace) elicited rapid decay of the 200- to 250-pA current with similar time courses. (C) Influence of pump phosphorylation on closing of PTX-induced channels by 160 mM K⁺. Normalized current traces from three outside-out patches from HEK293 cells with or without pipette nucleotide as indicated. After brief exposure to 100 nM PTX in Na⁺ (not shown), switching to K⁺ promoted channel closing that was fast without nucleotide (0 ATP trace) or with 2 mM MgAMPPNP, but very slow with 5 mM MgATP.