Quaternary Organic Amines Inhibit Na,K Pump Current in a Voltage-dependent Manner: Direct Evidence of an Extracellular Access Channel in the Na,K-ATPase

The effects of organic quaternary amines, tetraethylammonium (TEA) chloride and benzyltriethylammonium (BTEA) chloride, on Na,K pump current were examined in rat cardiac myocytes superfused in extracellular Na -free solutions and whole-cell voltage-clamped with patch electrodes containing a high Na -salt solution. Extracellular application of these quaternary amines competitively inhibited extracellular K (K o ) activation of Na,K pump current; however, the concentration for half maximal inhibition of Na,K pump current at 0 mV ( K 0 Q ) by BTEA, 4.0 0.3 mM, was much lower than the K 0 Q for TEA, 26.6 0.7 mM. Even so, the fraction of the membrane electric field dissipated during K o activation of Na,K pump current ( K ), 39 1%, was similar to K determined in the presence of TEA (37 2%) and BTEA (35 2%), an indication that the membrane potential ( V M ) dependence for K o activation of the Na,K pump current was unaffected by TEA and BTEA. TEA was found to inhibit the Na,K pump current in a V M -independent manner, i.e., inhibition of current dissipated 4 2% of the membrane electric field. In contrast, BTEA dissipated 40 5% of the membrane electric field during inhibition of Na,K pump current. Thus, BTEA inhibition of the Na,K-ATPase is V M -dependent. The competitive nature of inhibition as well as the similar fractions of the membrane electric field dissipated during K o -dependent activation and BTEA-dependent inhibition of Na,K pump current suggest that BTEA inhibits the Na,K-ATPase at or very near the enzyme’s K o binding site(s) located in the membrane electric field. Given previous findings that organic quaternary amines are not occluded by the Na,K-ATPase, these data clearly demonstrate that an ion channel–like structure provides access to K o binding sites in the enzyme. key words: Na,K-ATPase • K binding • tetraethylammonium chloride • benzyltriethylammonium chloride I N T R O D U C T I O N The stoichiometry of ion transport by the Na,K-ATPase under physiologic conditions, 3 Na extruded from the cell in exchange for the uptake of 2 K per ATP hydrolyzed (Glynn, 1985; Rakowski et al., 1989), results in one positive charge being moved across the cell membrane per enzyme transport cycle (De Weer et al., 1988; Glynn and Karlish, 1990; Läuger, 1991; Vasilets and Schwarz, 1993; Rakowski et al., 1997). This movement of positive charge can be measured as a membrane depolarization upon inhibition of the Na,K-ATPase with cardiac glycosides or as cardiac glycoside–inhibitable, extracellular K (K o )-dependent steady-state outward current (Na,K pump current) under voltage-clamp conditions where other ionic membrane currents are blocked or inactivated (Cooke et al., 1974; Isenberg and Trautwein, 1974; Eisner and Lederer, 1980; De Weer et al., 1984; Gadsby et al., 1985; Ishizuka et al., 1996; Berlin and Peluffo, 1997). The net movement of positive charge during the enzyme transport cycle also requires that the rate of at least one reaction step is dependent on the membrane potential (Läuger, 1991). The finding that Na,K pump current is membrane potential ( V M ) dependent in extracellular Na (Na o ) and K o -containing salt solutions confirmed this theoretical requirement and demonstrated that under some conditions the V M -dependent reaction(s) can become rate limiting for steady-state ion transport (Hansen et al., 1981; Läuger, 1991). In fact, Na,K pump current has a bell-shaped dependence on V M in the presence of nonsaturating concentrations of Na o and K o (Lafaire and Schwarz, 1986; Bielen et al., 1991; Rakowski et al., 1991; Sagar and Rakowski, 1994; Berlin and Peluffo, 1997). Such a biphasic relationship necessitates the presence of at least two V M -dependent reaction steps in the transport cycle (Läuger, 1991). One V M -dependent reaction of the Na,K-ATPase was identified with Na -dependent transport steps by demAddress correspondence to Joshua R. Berlin, Department of Pharmacology and Physiology, UMDNJ-New Jersey Medical School, 185 S. Orange Avenue. P.O. Box 1709, Newark, NJ 07101-1709. Fax: (973) 972-7950; email: [email protected] Abbreviations used in this paper: BTEA, benzyltriethylammonium ion; NMG, N-methyl-D-glucamine; TMA, tetramethylammonium ion; TPA, tetrapropylammonium ion. on A ril 5, 2017 D ow nladed fom Published February 23, 2004