Biomedical Frontiers: Winter 1994, Vol.1, No.2
Center for Molecular Recognition

Thousands of integral membrane proteins exist within mammalian membranes, where they mediate vital functions, including intracellular communication, ion and substrate transport, electrical excitability, and cell-to-cell adhesion.

To understand the functions of these proteins in molecular terms, investigators have used affinity labeling, peptide mapping, site- directed mutagenesis, heterologous expression--even crystallography and X-ray diffraction analysis.

All these approaches are being taken by director Dr. Arthur Karlin, Eugene Higgins Professor of Neurology, professor of biochemistry & molecular biophysics, and professor of physiology & cellular biophysics, and his colleagues at the Center for Molecular Recognition. Crystallization of membrane proteins, however, is notoriously difficult and progress has been slow. A new technique used at the center combining site-directed mutagenesis and protein labelling has yielded exciting information about acetylcholine receptors, GABAA-receptors, the cystic fibrosis transmembrane transport regulator, and dopamine receptors.

What is the technique? For the ion channels, the researchers sequentially mutagenize each of the amino acids known to span a membrane to cysteine. They then determine whether the cysteine is lining the channel by using chemical probes that specifically react with cysteine. If the cysteine faces the channel, the chemical probe will react with it, and ion flow through the channel will be blocked. In this way, the researchers can obtain information about the channel structure.

This cysteine-replacement approach is also used with the dopamine receptors, which are not ion channels. Cysteine replaces the amino acids near the presumed binding site. Cysteine replacements that do not affect agonist or antagonist binding--indicating that the receptor function is still active--will be studied further. Next, the ability of the probe to affect the binding of the agonist or antagonist will be determined. If binding is affected, the cysteine is replacing an amino acid involved in the binding site.

Because of these novel strategies, center researchers are uncovering new details about channel structures and how ions flow through them. With these methods, they also hope to delineate the differences in neurotransmitter binding to the distinct brain dopamine receptor types.