Intellectual merit. One of the major challenges in the broad field of bioenergetics is to understand how nutrients such as sugars and amino acids cross biological membranes and accumulate inside of cells. It is now understood that most living cells function like batteries. Thus, chemical reactions like respiration or ATP hydrolysis are coupled to the pumping of hydrogen ions or sodium out of cells. In this manner, cells or intracellular organelles like mitochondria create an electrochemical gradient of either hydrogen ions or sodium in which the inside of the cell is electrically negative and low in hydrogen ions or sodium relative to the outside. Given this scenario, there is then a driving force on hydrogen ions or sodium to diffuse back into the cell down their electrochemical gradients. Transport proteins embedded in the membrane like the one described in this proposal utilize the free energy released by this energetically downhill movement to drive translocation and accumulation of a specific nutrient in this case, the sugar lactose and it is known that transport of each lactose molecule across the membrane is accompanied by one hydrogen ion. It is the aim of this research to understand the molecular mechanism of this basic and universal biological process by studying the electrical properties of the lactose permease (LacY), a model for the members of the Major Facilitator Superfamily, a huge group of related membrane transport proteins. The project includes the application of an important new technique to observe the electrical properties of this transporter using newly developed solid-supported membrane electrodes.

Broader impacts. Studies on active transport and bioenergetics with LacY as the paradigm have revolutionized the field of membrane transport. From (i) the initial discovery that membrane vesicles are useful as a model system to study transport to (ii) the development of probes for quantifying electrical potentials and hydrogen ion gradients in microscopic systems to (iii) site-directed and Cys-scanning mutagenesis to (iv) purifying LacY to homogeneity in a functional state to (v) obtaining X-ray crystal structures to (vi) engineering the symporter for all manner of biochemical and spectroscopic studies, this laboratory has pioneered developments in the field for over 45 years. Most of the breakthroughs in the history of the research have been widely selected for inclusion in various textbooks, reference books and teaching materials in many languages for both undergraduate and graduate teaching worldwide. The PI will continue to speak to high school and university audiences in order to convey scientific knowledge to young people and stimulate their interest in basic science. Invited lectures in symposia, at universities and multi-disciplinary conferences nationally and internationally will serve as multiple channels to convey this novel information to society in a timely manner.

Project Report

The aim of this study is to develop an atomic-level understanding of the mechanism of lactose/H+ symport by the lactose permease of Escherichia coli (LacY), a paradigm for the Major Facilitator Superfamily (MFS), the largest family of membrane transport proteins. Members of the MFS are found in the membranes of all living cells. In addition to the insight gained regarding the mechanism of symport, the innovative methods developed by the PI to study the problem have been applied directly to many other membrane proteins in addition to other members of the Major Facilitator Superfamily. However, despite an increasing number of X-ray structures of MFS members, including seven of LacY from the PI’s laboratory, as well as the demonstration that lactose/H+ symport is driven chemiosmotically by an H+ electrochemical gradient, the mechanism by which this process operates is not yet fully understood. Among the many techniques for functional characterization of transport proteins, electrophysiology is an extremely sensitive and highly time-resolved method allowing direct measurement of charge movement. Sugar transport by LacY is coupled stoichiometrically with transport of an H+ (galactoside/H+ symport), and sugar binding requires protonation of LacY. Although the transport mechanism is electrogenic, this phenomenon cannot be studied by classical electrophysiology because LacY does not target to the plasma membrane of frog oocytes or other eukaryotic cells. Now for the first time, LacY has been studied electrophysiologically by using the novel solid-supported membrane (SSM) electrode approach. This is first SSM instrument to be installed in a laboratory in the United States. We purchased an updated version of the SSM instrument from Scientific Devices (Germany) which was designed according to Professor Klaus Fendler’s specifications. Professor Fendler visited our laboratory at UCLA and helped with installation of the instrument and the required software. Successful tests confirmed that the newly installed SSM instrument is functional. SSM technology demonstrates directly that purified, reconstituted LacY catalyses lactose-coupled H+ transport. Strikingly, the SSM also demonstrates that sugar binding itself causes a conformational change in LacY that results from charge rearrangement in the protein. As shown previously, the currents generated by lactose/H+ symport in the absence of the H+ electrochemical gradient are due specifically to the activity of LacY. Furthermore, from an analysis of the currents observed under various conditions in addition to biochemical data, it is clear that the predominant electrogenic event in ‘downhill’ sugar/H+ symport is deprotonation on the inside of the proteoliposomes. LacY mutants E325A and R302A are severely defective with respect to lactose/H+ symport, but bind galactosides well and catalyze exchange and counterflow. In contrast to wild-type LacY, these mutants exhibit only weak electrogenic events upon addition of LacY substrates, representing only ~6% of the total charge displacement of the wild type. This activity is due either to substrate binding per se or to a conformational transition following substrate binding and is not due to sugar/H+ symport. Thus, it was proposed that turnover of LacY involves at least two electrogenic reactions: (i) a minor electrogenic step that occurs upon sugar binding and is due to a conformational transition in LacY; and (ii) a major electrogenic step due to deprotonation during downhill sugar/H+ symport, which is the limiting step for downhill lactose/H+ symport. Downhill lactose/H+ symport is inhibited by deuterium oxide (D2O), which is consistent with the interpretation of the electrophysiological findings indicating that deprotonation is the rate limiting-step for this reaction. The electrical signals generated by downhill lactose/H+ symport are inhibited 2- to 3-fold when H2O is replaced with D2O, thereby providing further evidence for the conclusion that deprotonation is rate limiting for lactose/H+ symport in the absence of the H+ electrochemical gradient. Postdoctoral fellows and undergraduate students participated in this study had a unique opportunity to study time-resolved electrophysiology using SSM technology. In addition to publication of our findings, the PI has given numerous lectures to academic personnel, as well as undergraduate and graduate students, at universities, national and international meetings.

National Science Foundation (NSF)
Division of Molecular and Cellular Biosciences (MCB)
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Gregory W. Warr
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University of California Los Angeles
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