Quaternary Benzyltriethylammonium Ion Binding to the Na,K- ATPase: a Tool to Investigate Extracellular K + Binding Reactions †

Ion binding followed rapidly by occlusion is a mechanism utilized by the Na,K-ATPase and many P-type ATPases to minimize futile ATP hydrolysis cycles during transport. Enzyme conformations associated with ion occlusion are relatively stable from a thermodynamic standpoint as ion occluded states of the enzyme can be maintained for extended periods under the appropriate conditions (1–3). Ion bound but not occluded states of the enzyme, on the other hand, appear to be unstable and have been more difficult to study (4). Nonetheless, Forbush (3) showed that release of the K+ congener, Rb+, from the ion-occluded enzyme occurred by a sequential, ordered reaction, in agreement with data from Glynn et al. (2). Likewise, the kinetics of extracellular Na+ binding and occlusion reactions also suggested that binding and release of this ion occurs as an ordered series of diffusion-limited binding reactions separated by protein conformational rearrangements (5). Even so, to gain a greater understanding of how the Na,K-ATPase functions in its capacity as an ion transporter, it would be advantageous to study ion binding reactions in more detail and separate from the associated conformational changes that lead to ion occlusion. One obvious reason to separate ion binding from subsequent reactions would be to answer unambiguously the question of how the membrane electric field affects ion transport. Ion binding kinetics are influenced by electric field strength (5). The problem with previous studies is that ion binding cannot be separated from subsequent enzyme conformational changes. Secondly, the ability to study ion binding independently of occlusion should open the door to more detailed studies of the factors affecting ion binding reactions. Forbush (6) previously suggested that ion binding reactions of the Na,K-ATPase could be studied with an enzyme inhibitor that interacted with ion binding sites without becoming occluded. His work suggested that organic quaternary ammonium ions could be such inhibitors; however, this conclusion was based on assumptions about how quaternary amines inhibit the Na,K-ATPase. Should these assumptions be confirmed experimentally, these compounds might prove to be valuable tools to further study ion binding by the Na,K-ATPase. Organic quaternary amines competitively inhibit extracellular K+ and Rb+ activation of ion transport by the Na,K-ATPase (7–10). Tetraethylammonium ion (TEA) has been shown to inhibit ion transport by the Na,K-ATPase in this fashion (7,9,11). Measurements of Na,K-pump current in the presence of TEA have also shown that this quaternary amine inhibits the Na,K-ATPase in a membrane potential (VM)-independent manner (9,11), i.e. irrespective of VM, inhibition decreases the apparent affinity for extracellular K+ (K+o) activation of Na,K-pump current by a similar degree (see eq. 2 in Peluffo et al. (9)). The VM independence of Na,K-pump current block by TEA leads one to the conclusion that, although TEA competes with K+o, the amine does not inhibit the enzyme at K+o binding sites because K+o dissipates 30–40 % of the membrane electric field as it binds and activates Na,K-ATPase turnover (9,12–14). Even though the relationship between electrical and physical distances in the enzyme is not known, it seems reasonable to suppose that TEA, which has the same charge and is approximately the same size of a K+ ion with a single hydration shell, would dissipate a similar fraction of the membrane electric field if it inhibited the enzyme at the same site where K+ binding occurs. Thus, like cardiac glycosides (15), TEA inhibition of the Na,K-ATPase is competitive with K+ but might reflect interaction of the blocker with a site or enzyme conformation other than that for K+ binding, as noted by Kropp and Sachs (8) for another quaternary amine, tetrapropylammonium ion. For this reason, TEA inhibition of Na,K-pump current, while interesting, would only provide limited information about ion binding and transport by the Na,K-ATPase. The benzylic analogue of TEA, benzyltriethylammonium ion (BTEA), has also been shown to inhibit Na,K-pump current in a manner that is competitive with K+o activation (9) and slow release of 86Rb+ occluded by the Na,K-ATPase (6). However, BTEA inhibits Na,K-pump current in a VM-dependent manner. Specifically, BTEA appears to produce greater current block at more negative VM. Furthermore, the fraction of the membrane electric field dissipated during BTEA inhibition is similar to the fraction of the electric field dissipated during K+o activation of ion transport (9). In other words, the binding sites for BTEA and K+ in the Na,K-ATPase appear to have similar electrical properties. Given the competitive nature of the BTEA and K+ interaction, a reasonable conclusion is that BTEA inhibits the Na,K-ATPase at or near a K+ binding site responsible for stimulating enzyme turnover (9). If this conclusion is correct, BTEA or similar compounds might become valuable probes to understand K+ binding reactions by the Na,K-ATPase and possibly as leading compounds to develop novel inhibitors of this enzyme. Should the Na,K-ATPase be unable to occlude BTEA or similar compounds, they would be the first probes to allow investigation of VM-dependent ion binding, distinct from subsequent occlusion and transport reaction steps by the Na,K-ATPase. In that case, one prediction is that a compound undergoing VM-dependent ion binding should produce movement of charge in the membrane electric field. These charge movements should only be seen under pre-steady state conditions, analogous to electroneutral K+-K+ exchange conditions in which Tl+o-dependent transient charge movements have been observed previously (12). Nonetheless, a critical distinction should exist between charge movements produced by BTEA-like compounds and those produced by Tl+ and other transported ions, such as K+ and Na+. Movement of Tl+ in the electric field is thought to be diffusion limited, so that the rate of transient charge movements reflects kinetics of slow enzyme conformational changes subsequent to ion binding (5, 12, 16,17). By comparison, if not occluded, quaternary amines would yield measurable charge movements whose rates would reflect kinetics of binding that are not diffusion-limited. The experiments in this report were therefore undertaken to test these predictions and derive information regarding ion binding reactions from such charge movements. The first high resolution structure for the Na,K-ATPase has been published (18). Nonetheless, our understanding of the conformational changes that accompany ion transport by P-type ATPases relies largely on structures for SERCA (19–21). To see if quaternary amines inform us about extracellular ion binding reactions by the Na,K-ATPase, amine binding to Na,K-ATPase was simulated with homology models, based on SERCA (22). The results of these simulations suggest why some quaternary amines inhibit the Na,K-ATPase in a VM-dependent manner. Portions of this work have appeared previously in abstract form (23–26).