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.

Abstract

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 (λK), 39 ± 1%, was similar to λK 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.