Pre-steady-state charge translocation in NaK-ATPase from eel electric organ.

Abstract

Time-resolved measurements of charge translocation and phosphorylation kinetics during the pre-steady state of the NaK-ATPase reaction cycle are presented. NaK-ATPase-containing microsomes prepared from the electric organ of Electrophorus electricus were adsorbed to planar lipid bilayers for investigation of charge translocation, while rapid acid quenching was used to study the concomitant enzymatic partial reactions involved in phosphoenzyme formation. To facilitate comparison of these data, conditions were standardized with respect to pH (6.2), ionic composition, and temperature (24°C). The different phases of the current generated by the enzyme are analyzed under various conditions and compared with the kinetics of phosphoenzyme formation. The slowest time constant (,~l = 8 s -1) is related to the influence of the capacitive coupling of the adsorbed membrane fragments on the electrical signal. The relaxation time associated with the decaying phase of the electrical signal (,~l = 10-70 s -l) depends on ATP and caged ATP concentration. It is assigned to the ATP and caged ATP binding and exchange reaction. A kinetic model is proposed that explains the behavior of the relaxation time at different ATP and caged ATP concentrations. Control measurements with the rapid mixing technique confirm this assignment. The rising phase of the electrical signal was analyzed with a kinetic model based on a condensed Albers-Post cycle. Together with kinetic information obtained from rapid mixing studies, the analysis suggests that electroneutral ATP release, ATP and caged ATP binding, and exchange and phospholylation are followed by a fast electrogenic EIP --~ E2P transition. At 24°C and pH 6.2, the rate constant for the EIP --* E2P transition in NaK-ATPase from eel electric organ is > 1,000 s -1.

Effect of chaotropic anions on the sodium transport by the Na,K-ATPase

“…The activation energy of s 1 , E a , which represents the rising phase of the current transient, was 98 kJ/mol in the standard solution, whereas an E a of 65 kJ/mol was obtained for s 2 characterizing the falling phase. These values are in good agreement with previously reported results of measurements performed in the same model system under similar conditions (Fendler et al 1993), where the activation energies were reported to be 101 kJ/mol and 68 kJ/mol, respectively. The amplitude of short circuit current decreased and its kinetics was slowed down in the presence of choline iodide (Fig.…”

“…The conformation transition P-E 1 /P-E 2 is supposed to be the slowest step of the ion transfer (Sokolov et al 1998b). Although this interpretation is most widely adopted (Holmgren et al 2000;Sokolov et al 1998a;Wuddel and Apell 1995), there is an alternative point of view that the conformation transition controls the rising phase of the current signal (Fendler et al 1993).…”

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

“…The asymmetric location of the binding site of the third sodium ion means that the cytoplasmic access channel for this ion should be rather shallow with a dielectric coefficient of 0.25 [24]. Therefore, electric signals associated with the transport in the cytoplasmic access channel evaded detection for a long time.…”

“…Electrogenic ion transport in the Na + ,K + -ATPase can be studied in a system consisting of a lipid bilayer (BLM) and small membrane fragments that contain ion-pumps and adsorb to the bilayer surface in an oriented manner [15,[24][25][26][27][28]. The BLM serves as capacitive electrode transmitting the electrical signals that arise due to electrogenic transport in the membrane fragments to an external detection circuit.…”

“…These considerations allow determining the voltage drop inside the protein, ϕ c and ϕ e , with respect to the voltage U applied between both aqueous solutions: (24) where (25) The same coefficients define the relation of the ion movements in the access channels to the current measured in the external circuit, I. The charge movements inside the protein correspond to recharging of one of both internal capacitors, C c and C e .…”