This project aims to determine closed and opened structures of three physiologically important K+ ion channels to develop a physical model of their function as molecular devices.
The aims will be addressed through (1) development of expression and purification protocols, (2) reconstitution for functional analysis using high time resolution electrical recordings, and finally (3) application of new advances in cryo-electron microscopy for structure determination. We propose methods to control the conformational state - closed versus opened - of each channel during structural analysis. The three channels belong to the same architectural family, but each is activated in the cell by unique stimuli: Na+ in one case, membrane voltage in another, and membrane voltage plus Ca2+ in the third. To control a voltage-dependent channel's conformation while determining structure we will use a spider toxin to hold the channel closed while it is embedded in a nanodisc-mediated lipid membrane. The contrast and comparison of these channels' distinct signal responses, given that they are structurally related, should yield a deeper mechanistic understanding of each. The three channels under focus mediate smooth and skeletal muscle contraction, regulate neuronal firing rates, and propagation of action potentials along axons. Heritable defects within these channels underlie asthma and various forms of epilepsy in humans. I believe that the deeper mechanistic understanding that these experiments will yield will bring us closer to pharmacological control of these ion channels for the benefit of human health. Furthermore, the methods developed for membrane protein preparation and structure determination should be applicable to other membrane proteins, which account for 25% of proteins encoded in the human genome, and thus advance a broader field of medical science.
Heritable defects in ion channels under study in this proposal underlie asthma and various forms of epilepsy including malignant migrating partial seizures of infancy and autosomal dominant nocturnal frontal lobe epilepsy. Ion channels are fundamental to neuronal as well skeletal, smooth and cardiac muscle function. A deeper understanding of the structure and chemistry of ion channels I believe will lead to new pharmacological therapies for epilepsy, cardiac arrhythmia and skeletal and smooth muscle dysfunction.
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