Charge Translocation by the Na +/K +-ATPase Investigated on Solid Supported Membranes: Rapid Solution Exchange with a New Technique

Abstract

Adsorption of Na +/K +-ATPase containing membrane fragments from pig kidney to lipid membranes allows the detection of electrogenic events during the Na +/K +-ATPase reaction cycle with high sensitivity and time resolution. High stability preparations can be obtained using solid supported membranes (SSM) as carrier electrodes for the membrane fragments. The SSMs are prepared using an alkanethiol monolayer covalently linked to a gold surface on a glass substrate. The hydrophobic surface is covered with a lipid monolayer (SAM, self-assembled monolayer) to obtain a double layer system having electrical properties similar to those of unsupported bilayer membranes (BLM). As we have previously shown (Seifert et al., 1993, Biophys. J. 64:384–391), the Na +/K +-ATPase on a SSM can be activated by photolytic release of ATP from caged ATP. In this publication we show the first results of a new technique which allows rapid solution exchange at the membrane surface making use of the high mechanical stability of SSM preparations. Especially for substrates, which are not available as a caged substance—such as Na + and K +—this technique is shown to be capable of yielding new results. The Na +/K +-ATPase was activated by rapid concentration jumps of ATP and Na + (in the presence of ATP). A time resolution of up to 10 ms was obtained in these experiments. The aim of this paper is to present the new technique together with the first results obtained from the investigation of the Na +/K +-ATPase. A comparison with data taken from the literature shows considerable agreement with our experiments.