Probing the Extracellular Access Channel of the Na,K-ATPase
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Abstract
When the Na,K-ATPase pumps at each
turnover two K<sup>+</sup> ions
into the cytoplasm, this translocation consists of several reaction
steps. First, the ions diffuse consecutively from the extracellular
phase through an access pathway to the binding sites where they are
coordinated. In the next step, the enzyme is dephosphorylated and
the ions are occluded inside the membrane domain. The subsequent transition
to the E<sub>1</sub> conformation produces a deocclusion of the binding
sites to the cytoplasmic side of the membrane and allows in the last
steps ion dissociation and diffusion to the aqueous phase. The interaction
and competition of K<sup>+</sup> with various quaternary organic ammonium
ions have been used to gain insight into the molecular mechanism of
the ion binding process from the extracellular side in the P-E<sub>2</sub> conformation of the enzyme. Using the electrochromic styryl
dye RH421, evidence has been obtained that the access pathway consists
of a wide and water-filled funnel-like part that is accessible also
for bulky cations such as the benzyltriethylammonium ion, and a narrow
part that permits passage only of small cations such as K<sup>+</sup> and NH<sub>4</sub><sup>+</sup> in a distinct electrogenic way. Benzyltriethylammonium
ions inhibit K<sup>+</sup> binding in a competitive manner that can
be explained by a stopper-like function at the interface between the
wide and narrow parts of the access pathway. In contrast to other
quaternary organic ammonium ions, benzyltriethylammonium ions show
a specific binding to the ion pump in a position inside the access
pathway where it blocks effectively the access to the binding sites