Inaugural lecture H.R. Kaback

Bioenergetics in energy-transducing membranes represents the bridge between biochemistry and physiology. While ATP is the currency of energy exchange in the cytosol, electrochemical ion gradients across energy-transducing membranes are involved in a large number of seemingly unrelated processes such as oxidative phosphorylation, active transport of many different metabolites, signal transduction, maintenance of internal pH, intracellular organelle function, protein secretion, bacterial motility, and other cellular processes.

THE “BIG PICTURE”

Epithelial transport. Transport across the epithelium of the intestine will be discussed briefly merely to demonstrate the similarity of the basic principles. The topic will be discussed in more detail by Professor Wright. At the lumenal or brush-border side of this polarized intestinal epithelial cell (top), sodium and glucose are symported via a sodium-coupled symport protein (SGLT1), leading to accumulation of glucose against a large concentration gradient in the cytosol. The accumulated glucose is then allowed to flow down its chemical gradient via a glucose uniporter (a.k.a., facilitated diffusion carrier) on the basolateral surface (bottom) into the serosal fluid, and sodium is pumped out on the basolateral surface by the Na+/K+-ATPase. The tight junctions keep the membrane proteins on the brush border from mixing with the proteins on the basolateral membrane and vice versa.

Synaptic function in the CNS Synaptic function in the CNS. At neuromuscular junction, acetylcholine is released from pre-synaptic cells and binds to the nicotinic acetylcholine receptor on the muscle cell membrane, thereby opening a sodium channel which leads to depolarization of the post-synaptic muscle cell. Subsequently, the signal is terminated by acetylcholine esterase which hydrolyzes the neurotransmitter, and in addition, the acetylcholine receptor becomes desensitized. In contrast, at synapses in the CNS, the primary means of terminating the signal is by re-uptake of neurotransmitters (e.g., dopamine, serotonin, glutamate, glycine) into the pre-synaptic cell and subsequent repackaging into synaptic vesicles.

Starting with the synapse at rest, synaptic vesicles loaded with neurotransmitter (l, Tr) are concentrated in the axon terminal, and an action potential travels down the nerve fiber to the axon terminal. Upon depolarization of the axon terminal, calcium enters and triggers fusion of synaptic vesicles with the synaptic membrane of the pre-synaptic cell, a process that involves specific interactions between a number of proteins. At this point, the contents of the synaptic vesicles (i.e., neurotransmitters) are released into the synaptic cleft (1) where they diffuse to the post-synaptic cell membrane, bind specifically to a receptor (2) and open a channel for sodium (with depolarization of the post-synaptic cell) or chloride (with hyperpolarization of the post-synaptic cell). In order to terminate the signal, the neurotransmitter is transported back into the pre-synaptic cells via neurotransmitter re-uptake proteins that are specific for different neurotransmitters (3). Transport is usually sodium-coupled, but frequently, translocation of other ions (K+ or Cl-) is also required for accumulation. Once inside of the axon terminal, the neurotransmitters are actively transported and re-packaged in synaptic vesicles (4). There is a so-called vacuolar ATPase on the outer surface of the synaptic vesicle membrane that utilizes ATP hydrolysis to pump protons into the synaptic vesicles, thereby generating a (interior positive and acid, as in ISO E. coli membrane vesicles). The neurotransmitter is then actively transported into the synaptic vesicles by means of specific antiporters in the synaptic vesicle membrane (i.e., protons move out of the synaptic vesicles down their electrochemical gradient, and the energy released by this process is used to accumulate neurotransmitter against a concentration gradient). The synaptic vesicles in the pre-synaptic axon terminal are now reloaded and ready to release neurotransmitter again.

The next three lectures The next three lectures* will focus primarily upon one aspect of bioenergetics, active transport of metabolites in a specific experimental model system, bacterial cytoplasmic membrane vesicles, which revolutionized the field by leading to the development of similar systems from epithelia and the nervous system. The intent is to use this highly defined system to give you a feel for what an electrochemical ion gradient is, how the components are measured in microscopic systems that are not readily amenable to electrophysiology and how one can demonstrate convincingly that an electrochemical ion gradient is the immediate driving force for the accumulation of metabolites. The general relevance of this relatively simple experimental system to more complex systems that have direct relevance to human physiology will be illustrated. * Lecture 1: THE MODEL SYSTEM: PREPARATION AND CHARACTERIZATION OF BACTERIAL MEMBRANE VESICLES Lecture 2: MEMBRANE POTENTIALS AND pH GRADIENTS IN MICROSCOPIC SYSTEMS: THE CHEMIOSMOTIC PARADIGM Lecture 3: BIOENERGETICS OF ION-COUPLED ACTIVE TRANSPORT

Reading: Lectures from Professors Bezanilla and Wright contain related information that will help you understand the concepts under consideration. For those of you who would like more in-depth reading, the following are suggested: Kaback, H.R. 1971. Bacterial membranes. Methods in Enzymology 22, 99-130; Nichols, D. G. and Ferguson, S. 1992. Bioenergetics 2 (Academic Press, London).