Influence of sodium concentration on changes of membrane capacitance associated with the electrogenic ion transport by the Na,K-ATPase

Abstract

Electrogenic ion transport by the Na,K-ATPase was investigated in a model system of protein-containing membrane fragments adsorbed to a lipid bilayer. Transient Na + currents were induced by photorelease of ATP from inactive caged ATP. This process was accompanied by a capacitance change of the membrane system. Two methods were applied to measure capacitances in the frequency range 1 to 6000 Hz. The frequency dependent capacitance increment, ∆C, was of sigmoidal shape and decreased at high frequencies. The midpoint frequency, f 0 , depended on the ionic strength of the buffer. At 150 mM NaCl f 0 was about 200 Hz and decreased to 12 Hz at high ionic strength (1 M). At low frequencies (f K f 0 ) the capacitance increment became frequency independent. It was, however, dependent on Na + concentration and on the membrane potential which was generated by the charge transferred. A simple model is presented to analyze the experimental data quantitatively as a function of two parameters, the capacitance of the adsorbed membrane fragments, C P , and the potential of maximum capacitance increment, ψ 0 . Below 5 mM Na + a negative capacitance change was detected which may be assigned to electrogenic Na + binding to cytoplasmic sites. It could be shown that the results obtained by experiments with the presented alternating current method contain the information which is determined by current-relaxation experiments with cell membranes.

Assignment of charge movements to electrogenic reaction steps of Na,K-ATPase by analysis of salt effects on the kinetics of charge movements

“…According to The lines through the data points are ®ts with an exponential function, corresponding to a regression line when plotted on a linear concentration axis these considerations, in the earlier studies the frequency dependence of the capacitance increment was ®tted by a Lorentzian function to determine the characteristic parameter x 0 . However, in the present work we measured the capacitance and conductance increments in the lower frequency range in more detail than previously (Sokolov et al 1998b). These measurements indicated, however, that the capacitance increment cannot be described satisfactorily by the simple Lorentzian of Eq.…”

“…As demonstrated recently (Sokolov et al 1998b), the frequency dependencies of the capacitance and conductance increments may be described by a Lorentzian function in the case when the alternating current aects the charge translocation of a single step of the transport process:…”

“…The reaction Na 3 E 1 ÀP P À E 2 Na 3 is the rate-limiting step in the observed partial reactions After the indicated period of time, the membrane was exposed to UV light¯ashes to produce ATP-concentration jumps by a fast release of ATP from caged ATP, which caused transient currents associated with the transfer of positive charge by the Na,K-ATPase from the bulk phase towards the BLM. A modi®cation of the detection electronics allowed the determination of minor variations of the capacitance and conductance of the compound membrane system (Sokolov et al 1992(Sokolov et al , 1998b. Electrical signals were recorded after the UV¯ash.…”

“…The in¯u-ence of dierent anions on the conformational stability of Na,K-ATPase was clearly demonstrated, and the effectiveness of the anions was in agreement with the Hofmeister series (Post and Suzuki 1991). Recently we demonstrated that in a concentrated NaCl solution the kinetics of the electrogenic transport was notably slowed and the eect was comparable when Na + ions were replaced by choline + (Sokolov et al 1998b). The salt eect on the investigated electrogenic transport correlates with the eect on the¯uorescence changes of the dye RH421, which can be used to observe the action of Na,K-ATPase (Ganea et al 1999).…”

“…Lipid bilayer membranes (BLMs) with absorbed Na,K-ATPase-containing membrane fragments proved to be a convenient experimental system Fendler et al 1985). In such a set-up the ion pumps are capacitively coupled to the BLM, and it is possible to detect simultaneously transient currents generated by a fast release of ATP from caged ATP and by an externally applied alternating voltage (Sokolov et al 1992(Sokolov et al , 1994(Sokolov et al , 1998a(Sokolov et al , 1998b. The interpretation of transient currents from ATP-jump experiments was based on a mathematical model of the Albers-Post cycle Heyse et al 1994;Sokolov et al 1998a;Wuddel and Apell 1995), whereas the interpretation of the voltage-jump experiments was based on a phenomenological``access channel '' model (Rakowski 1993).…”