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-Q(0)) by BTEA, 4.0 +/- 0.3 mM, was much lower than the K-Q(0) 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 (lambda(k)), 39 +/- 1%, was similar to lambda(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 verb, 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.