Voltage-dependent ion channels are instrumental in the generation of membrane potential, receptor potential, and action potential. They are the molecular building blocks for what is an essential characteristic of many cells in neuroscience: excitability. These proteins are implicated in the physiology and pathophysiology of all excitable tissues, underlie many disease processes including epilepsies and arrhythmias, and are major targets of essential drugs used in clinical medicine. Despite the obvious biological and clinical importance of these proteins, we still know relatively little about their structure. Although important progress has been made in understanding ion permeation in potassium channels, large parts of these essential proteins remain structurally unresolved. This proposal explores a novel technique for addressing structural questions involving ion channels and other membrane proteins. We will use a combination of cysteine scanning mutagenesis, alkylation, and mass spectrometry to study structural aspects of two potassium channels, KcsA and Shaker. We refer to this novel technique as site-directed mass tagging. The long-term goal of this proposal is to provide low-resolution structural information about functional ion channels. The proposal is high risk in the sense that we do not yet know whether the technique will fulfill its potential and prove to be a useful structural tool. If successful, however, site-directed mass tagging may be able to provide important insight into the gross structure of many rare membrane proteins. It is anticipated that results from this study will provide new insight into the general structural layout of Shaker.
