Professor Nicholas Keep, Dean of the Faculty of Science (and director of the MSc in Structural Molecular Biology and the second-year option TSMB) introduced Professor Wallace. She has been at Birkbeck for about twenty years and now directs the department’s impressive research work on the structural biology of membrane ion channels. You will learn a lot about membrane proteins in general in section 11 of PPS; they are ubiquitous, are responsible for the transport of both chemicals and signals into and out of cells, and form some of the most important drug targets. They are also, as Wallace made very clear in her talk, some of the most challenging of all proteins for structural biologists to work with.
All cell membranes are semi-permeable, which means that some substances can pass across them easily while others are excluded. Ions, which are charged, are generally excluded by the hydrophobic (“water hating”) membranes. This could be something of a problem, as ion transport into and out of cells is an essential physiological process. Ion channels are evolution’s solution to this problem: proteins embedded in membranes that allow ions to selectively enter and leave cells.
Much of Wallace’ work over the last ten years has focused on the structures of voltage gated sodium channels. These open to allow sodium ions to enter cells, and close to prevent them from doing so, in response to changes in potential across the membrane, and they are found throughout nature. Small molecules can bind to these channels, holding them either open or closed; some of these are severely toxic, but others are important drugs for cardiac arrhythmias, epilepsy, and pain.
Human voltage gated sodium channels are composed of a single protein chain, divided into four similar domains. Each of these domains has six transmembrane helices, four of which (labelled S1-S4) act as a voltage sensor while the other two (S5 and S6) fold together to form an eight-helix pore. This protein has so far proved impossible to crystallise, and the breakthrough involved a bacterial protein. Similar proteins are found in the membranes of some species of bacteria, enabling them to live in “extreme” environments that are rich in salt. Their structures are similar to those of the human protein, but in this case the channel is built up from a complex of four identical proteins, each of which is homologous to a single domain of the human channel.
Although this simpler bacterial protein proved easier to work with than the human protein, it was still not at all easy. It took over ten years for Professor Wallace and her group to isolate the gene, clone and purify the protein, obtain crystals and finally solve the structure of the pore. The structure was finally solved using the powerful X-rays generated at Diamond, the UK’s only synchrotron radiation source located near Harwell in Oxfordshire.
These channels exist in three different structural forms: “open”, “closed” and “inactivated”. Many years before the detailed structures were solved Wallace and her group had used a biophysical technique, circular dichroism (CD) spectroscopy, to examine the conformational changes that occurred when mammalian and bacterial channels switched from one state to the other. As always, however, the full atomic-crystal structures yielded very much more information.
The first of these structures to be solved was a slightly strange one: the pore was held in the “closed” conformation that prevents sodium ions from entering the cell, although the voltage sensor was in the structure associated with the “active” state (PDB code 3RVY). The “top” part of this structure, towards the extracellular membrane surface, has a hydrophobic surface, and the pore in this part of the membrane acts as a selectivity filter to allow sodium ions in while keeping others, including potassium and calcium ions, out. Wallace and her group were the first to solve the structure of a fully open channel and showed that the upper portion of the channel containing the selectivity filter was virtually unchanged. The conformational change associated with opening and closing the channel occurs at the internal or cytoplasmic side of the protein (PDB 4F4L). When the pore closes, a small turning motion of the “bottom” part of the helical bundle causes the diameter of the pore to shrink, in a motion rather like the closure of a camera lens; the resulting channel is too small for sodium ions to pass through, so any inside the pore become trapped there.
Two subunits of the bacterial sodium channel pore in the “open” conformation, shown as a ribbon structure
All voltage gated sodium channels have a domain at the C-terminal end of the molecule that is necessary for channel activity but that was not visible in any of the crystal structures. Wallace and her group looked at this part of the molecule in the bacterial protein using a particularly powerful form of CD spectroscopy called synchrotron radiation CD spectroscopy that she had pioneered, and showed that each subunit had an extremely flexible protein chain separating the pore from a C-terminal helix. Using this information, the group have proposed a novel mechanism for channel opening in which the conformational change in the pore is enabled by these helices oscillating up and down.
Two subunits of the bacterial sodium channel pore in the “open” conformation, shown as a ribbon structure
The final part of Wallace’ talk was devoted to the role of sodium channels in health and disease, and as a drug target. A few unfortunate individuals have mutations in a type of channel that is involved in the response to painful stimuli. If this channel is jammed open, patients experience a constant, burning pain termed erythromelalgia, most commonly in their hands and feet. Wallace showed that an equivalent mutation from phenylalanine to valine at the base of one of the bacterial protein subunits caused the channel to open just enough for ions to pass through. There are also people in whom these channels are jammed in the closed position, and they feel no pain, even if they walk on hot coals. It may one day be possible for drugs based on our knowledge of these structures to be designed to ease both these conditions.
