Current Research Interests: Mechanisms of Active Electrogenic Ion Transport Research projects in my laboratory are directed at answering fundamental physiological questions that have broad implications for cell function. Using biophysical and molecular biological techniques, we are currently studying mechanisms of active electrogenic ion transport.

Electrochemical gradients for ions are maintained across biological membranes by specialized active transport proteins, ion pumps. In spite of their central role in cell function, the molecular mechanisms by which these proteins transport ions across biological membranes are not known. Using the Na,K-ATPase as a model system, we have been studying a nearly universal feature of ion pumps, the involvement of voltage-dependent reaction steps in ion transport. In these experiments, steady state and transient ion transport kinetics are measured with patch-clamp techniques using cells expressing Na,K-ATPase enzymes that contain point mutations in membrane spanning regions of the protein. Our work has yielded a direct demonstration that extracellular K+ binding reactions are voltage-dependent and the identification of amino acid residues critical in determining the observed voltage dependence of extracellular ion binding. With this combination of molecular biological and electrophysiological techniques, our goal is to determine which amino acid residues are responsible for ion coordination and voltage-dependent ion binding reactions and, in doing so, identify the changes in enzyme structure that underlie ion transport reaction steps. This project, which represents the fastest growing area of my laboratory, should allow us to pinpoint the molecular mechanism of ion transport by the Na,K-ATPase and will also serve as a guide for studying transport by other ion pumps.

Representative Publications:
Argüello JM, Peluffo RD , Feng J, Lingrel JB , Berlin JR: Substitution of glutamic 779 with alanine in the Na,K-ATPase subunit removes voltage dependence of ion transport. J Biol Chem 271:24610-24616, 1996.

Peluffo RD , Berlin JR: Ion channel-like properties of the Na,K-pump demonstrated by electrogenic K+transport. J Physiol (Lond) 501:33-40, 1997.

Peluffo RD , Lingrel JB, Argüello JM , Berlin JR: Changes to Na,K-ATPase ?-subunit E779 separate the structural basis for VM and ion dependence of Na,K-pump current. Ann NY Acad Sci 834:339-342,1997.

Peluffo RD, Argüello JM , Berlin JR: The role of Na,K-ATPase ?-subunit serine 775 and glutamate 779 in determining the extracellular K+ and membrane potential dependent properties of the Na,K-pump. J Gen Physiol 116, 47-59, 2000.

Peluffo RD, Argüello JM, Lingrel JB , Berlin JR: Electrogenic sodium-sodium exchange carried out by Na,K-ATPase containing the amino acid substitution Glu779Ala. J Gen Physiol 116, 61-73, 2000.