Palytoxin-induced Effects on Partial Reactions of the Na,K-ATPase

Abstract

The interaction of palytoxin with the Na,K-ATPase was studied by the electrochromic styryl dye RH421, which monitors the amount of ions in the membrane domain of the pump. The toxin affected the pump function in the state P-E2, independently of the type of phosphorylation (ATP or inorganic phosphate). The palytoxin-induced modification of the protein consisted of two steps: toxin binding and a subsequent conformational change into a transmembrane ion channel. At 20°C, the rate-limiting reaction had a forward rate constant of 105 M−1s−1 and a backward rate constant of about 10−3 s−1. In the palytoxin-modified state, the binding affinity for Na+ and H+ was increased and reached values between those obtained in the E1 and P-E2 conformation under physiological conditions. Even under saturating palytoxin concentrations, the ATPase activity was not completely inhibited. In the Na/K mode, ∼50% of the enzyme remained active in the average, and in the Na-only mode 25%. The experimental findings indicate that an additional exit from the inhibited state exists. An obvious reaction pathway is a slow dephosphorylation of the palytoxin-inhibited state with a time constant of ∼100 s. Analysis of the effect of blockers of the extracellular and cytoplasmic access channels, TPA+ and Br2-Titu3+, respectively, showed that both access channels are part of the ion pathway in the palytoxin-modified protein. All experiments can be explained by an extension of the Post-Albers cycle, in which three additional states were added that branch off in the P-E2 state and lead to states in which the open-channel conformation is introduced and returns into the pump cycle in the occluded E2 state. The previously suggested molecular model for the channel state of the Na,K-ATPase as a conformation in which both gates between binding sites and aqueous phases are simultaneously in their open state is supported by this study.

Maitotoxin converts the plasmalemmal Ca2+ pump into a Ca2+-permeable nonselective cation channel

“…Interestingly, the concentration required for inhibition of Ca 2ϩ -ATPase activity (micromolar) was 1,000-fold greater than the concentration needed for activation of channel activity (nanomolar). Similar observations for the effect on PTX on NKA have been explained by the different apparent affinities of the toxin for the various conformational states assumed by the pump during the normal transport cycle (3,18). In particular, elevations of extracellular K ϩ or the absence of ATP at the cytoplasmic surface greatly reduced the apparent affinity of the NKA for PTX.…”

“…Furthermore, PTX-induced single channels of 10-pS conductance were observed following reconstitution of the NKA in planar lipid bilayers following in vitro expression (19). PTX also inhibits ATPase activity associated with the NKA, although this occurs at concentrations much higher than those needed to induce channel activity (18). Together, these results provide strong evidence that the NKA is the receptor for PTX.…”

“…Depending on the ionic conditions, PTX increases channel activity with K 0.5 in the nanomolar range (3). In sharp contrast, inhibition of NKA activity by PTX generally occurs with a K 0.5 in the micromolar range (18). Furthermore, in both assays, the apparent affinity of PTX for the NKA is dramatically reduced by the presence of The explanation for this shift in PTX potency derives from the fact that it is the E 2 -P form of the NKA that exhibits the highest apparent affinity for PTX.…”

Caveolae respond to cell stretch and contribute to stretch-induced signaling

“…Control cells were not mechanically stimulated and remained in the chamber for the same period of time as the stretch group. Calculation of the lipid raft turnover rate constant k was calculated based on the loss of Alexa Fluor 555 over time according to: F5F 0 exp(-kt), where F is the fluorescence level at any time point, F 0 the fluorescence level at 0 minutes, k the turnover rate constant and t is time (Harmel and Apell, 2006).…”

Peering into an ATPase ion pump with single-channel recordings

“…position 806 in TM6) are accessible to MTSET C without palytoxin (Takeuchi et al 2008). Fourth, blockers of access channels to ion-binding sites in unmodified Na C ,K C -ATPase pumps similarly impede cation movement in palytoxin-bound pumps (Harmel & Apell 2006). Fifth, MTSET C -accessible positions in palytoxin-bound pump-channels map reasonably onto unmodified pump structures (figure 5g,h; Guennoun & Horisberger 2002;Horisberger et al 2004;Reyes & Gadsby 2006;Takeuchi et al 2008) and include sites expected to interact with transported ions (Nielsen et al 1998;Ogawa & Toyoshima 2002;Einholm et al 2005Einholm et al , 2007Morth et al 2007).…”