Electrogenic transport of sodium ions in cytoplasmic and extracellular ion access channels of Na+,K+-ATPase probed by admittance measurement technique

Abstract

-Electrogenic movements of sodium ions in cytoplasmic and extracellular access channel of the Na + ,K + -ATPase have been studied by the admittance measurement technique which allows the detection of small changes of the membrane capacitance and conductance induced by phosphorylation of the ion pump. The measurements were carried out on a model system consisting of a bilayer lipid membrane, to which membrane fragments with ion pumps were adsorbed that contain the ion pumps in high density. Small changes of the membrane capacitance and conductance were induced by a fast release of ATP from caged ATP. The effect was measured at various frequencies and in solutions with different Na + concentrations. The experimentally observed frequency dependences were explained using a theoretical model assuming that Na + movement through the cytoplasmic access channel occurs in one step and through the extracellular access channel, in two steps. The phosphorylation of the protein by ATP leads to a block of the cytoplasmic access channel and an opening the extracellular access channel. The disappearance of electrogenic Na + movements on the cytoplasmic side produces a negative change of capacitance and conductance, while the emergence of extracellular Na + movements generates a positive change. Fitting the experimental dependences of capacitance and conductance by theoretical curves allowed the determination equilibrium and kinetic parameters of sodium transport in the access channels.

Electrogenic binding of ions at the cytoplasmic side of the Na+,K+-ATPase

“…This model is a generalization of the model of electrogenic transport of ions in the cytoplasmic access channel of the Na + ,K + ATPase developed earlier [13]. The model is based on the assumption that the sites of the Na + ,K + ATPase accessible from cytoplasmic side can bind not only sodium ions but also protons.…”

“…The quantitative description of this process can be considerably simplified when the fre quency of alternating voltage is low enough to assume that the ion distribution between binding sites and solution is at or close to equilibrium. As was supposed in [19], the sites are charged negatively, so that the net charge Q of the sites at equilibrium is defined by the amount of free sites which are occupied neither by ] are the sodium and proton concentra tions in the solution, K Na and K H are the sodium and proton dissociation constants, respectively, α is the fraction of the applied ac voltage, ϕ, that affects the distribution of ions in the channel (it is proportional to so called "dielectric coefficient", which is determined by the relative length of the channel [13]); β = e/kT, e is the elementary charge, k is the Boltzmann's con stant, and T, absolute temperature.…”

“…It is affected by the voltage applied to the membrane, and the ion movement from the aqueous phase to the binding site creates an elec tric current [12][13][14]. Using fluorescent dyes it has been demonstrated that the Na + ,K + ATPase can bind not only sodium and potassium ions from the cyto plasmic side but also protons, and there is a competi [7,15,16].…”

Mechanistic principles of ion transport in the Na,K-ATPase

“…In the E 2 conformation the reverse exchange is performed with the extracellular medium. In the "occluded" states, (Na 3 )E 1 -P, E 2 (K) 2 , and ATP · E 2 (K) 2 [32].…”

“…A recent study demonstrated that the access channels are impenetrable on the side where the gate is closed, i.e. the external access in E 1 and the cytoplasmic access in P-E 2 [32]. .…”