Membrane proteins are the conduits for communication between the outside world and the interior of the cell and are central to processes that are essential for human life such as neurotransmission, cardiovascular regulation, and hormonal signaling. Despite their importance, we still know very little regarding the basic mechanisms by which they work due to a lack of three-dimensional structural information. The largest impediment to the routine biochemical and biophysical study of eukaryotic membrane proteins is the limited availability of pure protein from natural or recombinant sources. This problem is particularly acute for ion channels, which are often found in very low abundance in native tissues. Furthermore, many membrane proteins have limited stability in the detergent solutions that are required for their purification adding additional complications to their handling in biochemical and biophysical experiments. To overcome both of these limitations, we seek to develop a general strategy for the selection, overexpression, and purification of functional, stability-enhanced eukaryotic ion channels using yeast. As a model system we are investigating the prototypic inwardly rectifying potassium channel Kir2.1. Kir channels form a large family of potassium channels with central roles in the regulation of heartbeat, hormone release, sensory transduction, and cognition. Their dysfunction has been linked to a variety of human diseases including diabetes, cardiac arrhythmias, and epilepsy. Developing a means to produce large quantities of pure, functional, rare membrane proteins such as eukaryotic ion channels will have a major impact on our efforts to understand the basic biochemistry behind the membrane signaling and transport mechanisms that underlie processes like neurotransmission, cardiovascular regulation, and hormonal signaling, as well as the development of new drugs to treat dysfunctions in these systems. The methods developed here are general and, once established, should permit the overproduction of a wide range of ion channels as well as other eukaryotic membrane proteins for biochemical and structural study.
