Rockefeller University

The Rockefeller University
1230 York Avenue
New York, NY 10021

Laboratory of Cardiac/Membrane Physiology

D.W. Bronk Building, Room 307
Tel: (212)327-8617 Fax: (212)327-7589





Introduction/Overview

Cells are studded with millions of special proteins that control the passage of ions across the surface membrane. Ion flow into or out of cells underlies vital functions like electrical signaling in neurons and in heart and muscle cells, secretion of hormones and neurotransmitters, cell volume regulation, kidney function, and fertilization. Two principal classes of membrane proteins regulate ion movements: ion-motive pumps and ion channels. Though both provide a favorable environment for shepherding ions across the otherwise impenetrable phospholipid bilayer, ion pumps and channels are traditionally viewed as different entities. But our work has led us to believe that pumps and channels are far more closely related than generally assumed. Our two subjects are the Na+,K+-ATPase (or Na+/K+ exchange pump) and the cystic fibrosis Cl- ion channel, CFTR (Cystic Fibrosis Transmembrane conductance Regulator), both of biomedical importance. The Na+/K+ pump extrudes 3 Na+ from a cell and recovers 2 K+ at the expense of a single molecule of ATP, so establishing the transmembrane gradients of Na+ and K+ ions that are crucial to cell life. Mutations in the gene that encodes CFTR cause cystic fibrosis, the most prevalent lethal genetic disease in the USA. Our goal is to learn how these two molecular machines work.

Background

Why are ion pumps and ion channels perceived as being so different? A major reason is that the ion-selective pore in a channel conducts dissipative ion flow, down the electrochemical gradient, at a high rate (e.g. 107 to 108 s-1), whereas an ATPase pump moves ions against their electrochemical gradient, and far more slowly (e.g. ~102 s-1). But the high rates apply only to open ion channels, and all channels contain a gate that allows the ion flow to be turned on and off as needed. Opening and closing of the gate involve substantial conformational changes that are relatively slow (~102 to 103 s-1) and require input of energy. Energy sources include movement within the membrane's electrical field of charged residues in voltage-gated channels, or binding of an extra- or intracellular messenger in ligand-gated channels, or physical distortion in mechanosensitive channels. Nevertheless, the conformational changes that accompany opening and closing of an ion channel might not differ greatly from those associated with the ion movements effected by a pump. In fact, we suggest that an ion pump be viewed (Figure 1) as a modified ion channel with at least two gates that should never be open simultaneously: i.e., one gate must close before the other can open, implying tight coupling between them.

Current research

Because the Na+/K+ pump and CFTR Cl- channel both move charged ions across the cell membrane, they generate tiny electrical signals that we can measure to monitor their function. Using the sensitive "patch-clamp" recording technique we can readily see the ~107 Cl- ions s-1 that flow across the membrane when a single CFTR Cl- channel opens. Intriguingly, each CFTR molecule includes two highly conserved, cytoplasmic, nucleotide binding domains that place it in the large family of ABC (ATP Binding Cassette) proteins, and that are believed to sequentially bind and hydrolyze ATP. By analyzing the timing of opening and closing of individual wild-type or mutated CFTR channels during exposure to ATP and/or nucleotide analogs, we are learning how the free energy released during ATP binding and hydrolysis is harnessed to drive the conformational changes that open and close the ion pore (Figure 2). We find that both nucleotide binding domains must bind ATP before the channel opens, that the two domains interact to regulate each other's function, and that one acts like a G protein in that nucleotide bound there stabilizes the active (open) state until hydrolysis prompts channel closure. The orders-of-magnitude smaller ion movements generated by pumps preclude similar electrical assays of individual Na+/K+ pump function, but we can monitor ensemble activity of thousands of Na+/K+ pumps in small patches of membrane, or of the millions of Na+/K+ pumps in entire cells. Our high-speed electrical measurements suggest that, at least in the conformation that releases the 3 Na+ to the exterior, the Na+/K+ pump resembles an ion-channel pore closed at its cytoplasmic end. That the Na+/K+ pump does contain an ion channel gated at either end is supported by our latest work using the marine toxin, palytoxin (PTX). PTX effectively disrupts the coupling between the Na+/K+ pump's two gates, so permitting dissipative cation flow fast enough (~107 ions s-1) to be measured in a single Na+/K+ pump. This ion flow is interrupted by occasional closures of one or the other gate. Binding of external K+ tends to close, and external Na+ to open, the extracellular gate, whereas nucleotides binding from the cytoplasmic solution tend to open the intracellular gate. Thus, PTX transforms the Na+/K+ pump into a channel, in which gating seems to reflect the normal ion occlusion/deocclusion reactions that alternately entrap first 3 Na+, and then 2 K+, within the interior of the pump, between the two gates.

Future Goals

We are presently using site-specific mutagenesis combined with biochemical, electrical, and fluorescence measurements to probe the relationships between molecular structure and mechanism in these two ion transport proteins, often one molecule at a time.


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