News and Publications

Ion Channels

N. Unwin, B. Sheehan

Our research goal is to understand how ion channels work by analyzing their 3-dimensional structures. We use electron cryo-microscopy combined with rapid freezing to trap different conformational states. Our studies center on the nicotinic acetylcholine receptor obtained from Torpedo electric organs and on voltage-gated potassium channels obtained by overexpression in insect cells. We wish to determine how such ion channels achieve their specific ion selectivities and transport rates and to understand, in physical terms, how the channels open, close, and desensitize in response to chemical or electrical stimuli.

The nicotinic acetylcholine receptor is a pentameric complex of approximately 300 kD with large water-filled vestibules extending from the membrane that shape the acetylcholine-binding pockets and facilitate selective transport of cations across a narrow membrane-spanning pore. The recently determined structure of the receptor, at nearly 4-Å resolution, shows several new details. Specifically, the gate of the channel is made by side chains extending from a ring of a-helical segments, creating a hydrophobic girdle around the middle part of the pore. Tunnels, framed by twisted ß-sheet strands, are resolved in the extracellular wall of the channel, connecting the vestibule to the acetylcholine-binding pockets in the 2 a-subunits. Sets of narrow openings through which ions can flow are resolved between a-helical segments, forming part of the cytoplasmic wall of the channel. The extracellular tunnels appear to be access routes to the binding pockets for acetylcholine, and the cytoplasmic openings appear to act as filters to exclude anions and other impermeant species from the vicinity of the pore.

Freeze-trapping experiments showed that when acetylcholine enters the binding pockets, it triggers a concerted conformational change that opens the pore by destabilizing the gate in the middle of the membrane. The alternative "open" configuration of pore-lining a-helical segments reshapes the lumen and creates new surfaces, allowing the ions to pass through. It should soon become possible to model these different conformations at atomic resolution. Research on potassium channels is at the initial stage of exploring ways to make specimens suitable for electron crystallographic analysis.

PUBLICATIONS

Tierney, L.M., Unwin, N. Electron microscopic evidence for the assembly of soluble pentameric extracellular domains of the nicotinic acetylcholine receptor. J. Mol. Biol. 303:185, 2000.

Unwin, N. The Croonian Lecture 2000: Nicotinic acetylcholine receptor and the structural basis of fast synaptic transmission. Philos. Trans. R. Soc. Lond. B Biol. Sci. 355:1813, 2000.