I had the pleasure of attending several excellent seminars over the weekend describing recent studies of gating mechanisms of ion channels, beginning with the the Cryo-EM subgroup session on Saturday. What I appreciate about these studies is how the researchers utilized cutting-edge biophysical and structural methods to address important questions and find answers that were elusive by more conventional means.
On Saturday, Doreen Matthies presented cryo-EM studies of the magnesium channel, CorA, that she and a group of researchers from the NIH and the University of Chicago had recently published in Cell. The study provided a key missing link in the transport mechanism that could not be derived from crystal structures. CorA is a bell-shaped homopentameric channel for magnesium uptake, and previously reported X-ray structures revealed little conformational changes either in the presence of absence of magnesium. (Magnesium inactivates the channel by binding on the cytoplasmic side.) EPR studies of CorA had suggested large conformational changes between the subunits, and it was suspected the lack of change was an artifact of crystallization.
Enter the Cryo-EM revolution. With the availability of advanced imaging technology, the groups determined closed- and open-structures of the 200 kDa channel that revealed an unexpected mechanism of magnesium transport. The five-fold symmetry was broken by asymmetric conformational changes in the cytoplasmic gating region, with one subunit displaced away from the central axis while another towards it. The asymmetric opening was demonstrated both in detergent and in nanodiscs, and revealed the difficulties of crystallizing the channel in this state. These open, active structures provided the physical basis for a magnesium conductance pathway supported by an ensemble of asymmetric open states that allowed ion uptake, with a closed, magnesium-bound inactive state.
Cryo-EM is far from the only cutting-edge biophysical technique being utilized for these pentameric ion channels. On Sunday, Claudio Grosman presented a variety of approaches to studying gating mechanisms of muscle nicotinic acetylcholine receptors. Using a combination of single-channel electrophysiology and all-atom molecular dynamics simulations, he showed that rotamers of pore-lining glutamate side-chains provided the rate-limiting step in conductance of these neurotransmitter-gated channels. As the average distance of the side-chains from the pore center increased, so too did the conductance. These studies showed the importance of side-chain conformations on protein function, details which may be missing in static crystal structures.
Finally, Cynthia Czajkowski and colleagues showed the utility of a more sensitive EPR spectroscopy technique, known as Double Electron Electron Resonance, or DEER. This method allows the probing of intermolecular distances up to 60 angstroms while using smaller amounts of sample at lower concentrations than those typically required for conventional EPR experiments. This is of particular importance for studies of membrane proteins by these methods, allowing for measurements in natural membrane environments such as liposomes and nanodiscs. Czajkowski and colleagues showed the importance of lipids for the function of prokaryotic ligand-gated ion channels. The DEER measurements revealed large-scale structural changes in the interfacial region between the transmembrane and extracellular regions leading to channel activation. These conformational changes could not be observed in detergent reconstituted channels.
The combination of these methods, and many more, have us poised for a revolution in membrane protein structural biology, and provides researchers with the ability to gain a more sophisticated and detailed understanding of membrane protein structure-function than ever before, while at the same time breaking the most significant logjams for their study. (Mainly, expressing these proteins in milligram quantities and crystallizing them.) It’s certainly an exciting time to be in the field, and I look forward to hearing much more on this topic at this meeting.