Structural studies of human potassium channels and lipid scramblases in genetic disease

Project Overview

Ion channels are critical for the function of almost every aspect of the human body, playing essential roles in the brain, nervous system and the heart. Defects in these channels lead to a host of medical conditions such as heart disease, cystic fibrosis and depression. We aim to study these complex, membrane embedded proteins using a range of structure determination techniques (X-ray crystallography, serial femtosecond crystallography, Electron microscopy)  to solve their three dimensional structures and a range of biophysical and biochemical techniqes to understand their function. Many commercial small molecule drugs bind to ion channels and we also aim to throw light on how these drugs bind to channels by comparing the structures with and without bound drugs. At the SGC we have developed a system for producing human membrane proteins. We have solved the structures of two potassium channels, TREK-1 and TREK-2 in multiple conformations and in complex with antagonists.  This has allowed us to start to understand how these channels are gated by a range of stimuli, including stretch, temperature, fatty acids and drugs. We have also obtained crystals and electron microscopy data for several other ion channels and we have solved three ion channel structures by electron microscopy.  In addition we have many well expressed proteins ready for crystallisation and electron microscopy studies.

Two projects that are available next year:

A collaboration between Liz Carpenter and Stephen Tucker . K2P channels are involved in the perception of pain, in migraine with aura and pulmonary hypertension. We have structures of two of the 15 members of this family, but key questions remain about how activators turn on these channels, how the channels involved in pain and migraine (TRESK) and pulmonary hypertension (TASK-1) differ from the TREK channels we have already solved.

We have solved the structure of an intracellular scramblase, TMEM16K, by cryo-EM and X-ray crystallography, revealing a range of conformations. This scramblase/channel protein is associated with the cerebellar ataxia SCAR10 and we are keen to investigate how disease associated mutations affect the function of the protein. We are also interested in studying structures of a range of other TMEM16 family members and using MD simulations to study conformational changes associated with opening and closing of lipid scramblase groove. This work is a collaboration with Alessio Accardi (New York), Phill Stansfield (Biochemistry, Oxford), Rebecca Sitsapecan and Paolo Tammaro (Pharmacology, Oxford).

I have a number of other ion channel related projects, so feel free to contact me if you would like to discuss further.

For these projects we would welcome enthusiastic and determined students who are interested in structural and functional studies of medically important proteins. The student would work both in the Carpenter lab for the structural work, and either at the SGC or in collaborators labs for functional studies.  Initially the project would involve extensive screening, purification and crystallization/cryo-EM work, leading to structures of human ion channels. In this challenging area it is helpful to start with a variety of different proteins, identify those that can be produced on a 500 ug scale and those that crystallise. Once a suitable target has been identified the student would learn crystallography and/or cryo-EM, and would be involved in solving the structure. Functional and small molecule probe studies would also be possible, depending on the progress of the project. This is an excellent opportunity for a student to be involved in cutting edge research and to contribute to our understanding of a family of medically important proteins.

Training Opportunities

The student will gain a solid grounding in the emerging area of membrane protein structural biology, combined with a knowledge of ion channel structure and function.

They would work both in the Carpenter lab for structural studies and in the collaborators lab for the channel projects.  They will learn how to product proteins in the insect cells/baculovirus and mammalian expression systems, methods for screening for high levels of expression and purification, membrane protein purification, crystallisation and cryoEM grid preparation. The student will learn how to screen crystals and collect X-ray crystallographic data at the synchrotron. They will be involved in solving structures of complexes and novel ion channels. There will also be opportunities to learn electrophysiology, including single channel techniques in Oxford and in our the labs of our Pharmaceutical company collaborators. The student will learn how to work in a team, manage experiments, present data and how to write research papers and a thesis.


Protein Science & Structural Biology and Physiology, Cellular & Molecular Biology


Project reference number: 328

Funding and admissions information


Name Department Institution Country Email
Professor Liz Carpenter Structural Genomics Consortium Oxford University, Old Road Campus Research Building GBR
Prof Stephen Tucker Physics Oxford University,
Professor Rebecca Sitsapesan Pharmacology University of Oxford GBR

Quigley A, Dong YY, Pike AC, Dong L, Shrestha L, Berridge G, Stansfeld PJ, Sansom MS, Edwards AM, Bountra C, von Delft F, Bullock AN, Burgess-Brown NA, Carpenter EP. 2013. The structural basis of ZMPSTE24-dependent laminopathies. Science, 339 (6127), pp. 1604-7. Read abstract | Read more

Mutations in the nuclear membrane zinc metalloprotease ZMPSTE24 lead to diseases of lamin processing (laminopathies), such as the premature aging disease progeria and metabolic disorders. ZMPSTE24 processes prelamin A, a component of the nuclear lamina intermediate filaments, by cleaving it at two sites. Failure of this processing results in accumulation of farnesylated, membrane-associated prelamin A. The 3.4 angstrom crystal structure of human ZMPSTE24 has a seven transmembrane α-helical barrel structure, surrounding a large, water-filled, intramembrane chamber, capped by a zinc metalloprotease domain with the catalytic site facing into the chamber. The 3.8 angstrom structure of a complex with a CSIM tetrapeptide showed that the mode of binding of the substrate resembles that of an insect metalloprotease inhibitor in thermolysin. Laminopathy-associated mutations predicted to reduce ZMPSTE24 activity map to the zinc metalloprotease peptide-binding site and to the bottom of the chamber. Hide abstract

Shintre CA, Pike AC, Li Q, Kim JI, Barr AJ, Goubin S, Shrestha L, Yang J, Berridge G, Ross J, Stansfeld PJ, Sansom MS, Edwards AM, Bountra C, Marsden BD, von Delft F, Bullock AN, Gileadi O, Burgess-Brown NA, Carpenter EP. 2013. Structures of ABCB10, a human ATP-binding cassette transporter in apo- and nucleotide-bound states. Proc. Natl. Acad. Sci. U.S.A., 110 (24), pp. 9710-5. Read abstract | Read more

ABCB10 is one of the three ATP-binding cassette (ABC) transporters found in the inner membrane of mitochondria. In mammals ABCB10 is essential for erythropoiesis, and for protection of mitochondria against oxidative stress. ABCB10 is therefore a potential therapeutic target for diseases in which increased mitochondrial reactive oxygen species production and oxidative stress play a major role. The crystal structure of apo-ABCB10 shows a classic exporter fold ABC transporter structure, in an open-inwards conformation, ready to bind the substrate or nucleotide from the inner mitochondrial matrix or membrane. Unexpectedly, however, ABCB10 adopts an open-inwards conformation when complexed with nonhydrolysable ATP analogs, in contrast to other transporter structures which adopt an open-outwards conformation in complex with ATP. The three complexes of ABCB10/ATP analogs reported here showed varying degrees of opening of the transport substrate binding site, indicating that in this conformation there is some flexibility between the two halves of the protein. These structures suggest that the observed plasticity, together with a portal between two helices in the transmembrane region of ABCB10, assist transport substrate entry into the substrate binding cavity. These structures indicate that ABC transporters may exist in an open-inwards conformation when nucleotide is bound. We discuss ways in which this observation can be aligned with the current views on mechanisms of ABC transporters. Hide abstract

Carpenter EP, Beis K, Cameron AD, Iwata S. 2008. Overcoming the challenges of membrane protein crystallography. Curr. Opin. Struct. Biol., 18 (5), pp. 581-6. Read abstract | Read more

Membrane protein structural biology is still a largely unconquered area, given that approximately 25% of all proteins are membrane proteins and yet less than 150 unique structures are available. Membrane proteins have proven to be difficult to study owing to their partially hydrophobic surfaces, flexibility and lack of stability. The field is now taking advantage of the high-throughput revolution in structural biology and methods are emerging for effective expression, solubilisation, purification and crystallisation of membrane proteins. These technical advances will lead to a rapid increase in the rate at which membrane protein structures are solved in the near future. Hide abstract

Weyand S, Shimamura T, Yajima S, Suzuki S, Mirza O, Krusong K, Carpenter EP, Rutherford NG, Hadden JM, O'Reilly J, Ma P, Saidijam M, Patching SG, Hope RJ, Norbertczak HT, Roach PC, Iwata S, Henderson PJ, Cameron AD. 2008. Structure and molecular mechanism of a nucleobase-cation-symport-1 family transporter. Science, 322 (5902), pp. 709-13. Read abstract | Read more

The nucleobase-cation-symport-1 (NCS1) transporters are essential components of salvage pathways for nucleobases and related metabolites. Here, we report the 2.85-angstrom resolution structure of the NCS1 benzyl-hydantoin transporter, Mhp1, from Microbacterium liquefaciens. Mhp1 contains 12 transmembrane helices, 10 of which are arranged in two inverted repeats of five helices. The structures of the outward-facing open and substrate-bound occluded conformations were solved, showing how the outward-facing cavity closes upon binding of substrate. Comparisons with the leucine transporter LeuT(Aa) and the galactose transporter vSGLT reveal that the outward- and inward-facing cavities are symmetrically arranged on opposite sides of the membrane. The reciprocal opening and closing of these cavities is synchronized by the inverted repeat helices 3 and 8, providing the structural basis of the alternating access model for membrane transport. Hide abstract

Newstead S, Fowler PW, Bilton P, Carpenter EP, Sadler PJ, Campopiano DJ, Sansom MS, Iwata S. 2009. Insights into how nucleotide-binding domains power ABC transport. Structure, 17 (9), pp. 1213-22. Read abstract | Read more

The mechanism by which nucleotide-binding domains (NBDs) of ABC transporters power the transport of substrates across cell membranes is currently unclear. Here we report the crystal structure of an NBD, FbpC, from the Neisseria gonorrhoeae ferric iron uptake transporter with an unusual and substantial domain swap in the C-terminal regulatory domain. This entanglement suggests that FbpC is unable to open to the same extent as the homologous protein MalK. Using molecular dynamics we demonstrate that this is not the case: both NBDs open rapidly once ATP is removed. We conclude from this result that the closed structures of FbpC and MalK have higher free energies than their respective open states. This result has important implications for our understanding of the mechanism of power generation in ABC transporters, because the unwinding of this free energy ensures that the opening of these two NBDs is also powered. Hide abstract

Berridge G, Chalk R, D'Avanzo N, Dong L, Doyle D, Kim JI, Xia X, Burgess-Brown N, Deriso A, Carpenter EP, Gileadi O. 2011. High-performance liquid chromatography separation and intact mass analysis of detergent-solubilized integral membrane proteins. Anal. Biochem., 410 (2), pp. 272-80. Read abstract | Read more

We have developed a method for intact mass analysis of detergent-solubilized and purified integral membrane proteins using liquid chromatography-mass spectrometry (LC-MS) with methanol as the organic mobile phase. Membrane proteins and detergents are separated chromatographically during the isocratic stage of the gradient profile from a 150-mm C3 reversed-phase column. The mass accuracy is comparable to standard methods employed for soluble proteins; the sensitivity is 10-fold lower, requiring 0.2-5 μg of protein. The method is also compatible with our standard LC-MS method used for intact mass analysis of soluble proteins and may therefore be applied on a multiuser instrument or in a high-throughput environment. Hide abstract

Newstead S, Drew D, Cameron AD, Postis VL, Xia X, Fowler PW, Ingram JC, Carpenter EP, Sansom MS, McPherson MJ, Baldwin SA, Iwata S. 2011. Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2. EMBO J., 30 (2), pp. 417-26. Read abstract | Read more

PepT1 and PepT2 are major facilitator superfamily (MFS) transporters that utilize a proton gradient to drive the uptake of di- and tri-peptides in the small intestine and kidney, respectively. They are the major routes by which we absorb dietary nitrogen and many orally administered drugs. Here, we present the crystal structure of PepT(So), a functionally similar prokaryotic homologue of the mammalian peptide transporters from Shewanella oneidensis. This structure, refined using data up to 3.6 Å resolution, reveals a ligand-bound occluded state for the MFS and provides new insights into a general transport mechanism. We have located the peptide-binding site in a central hydrophilic cavity, which occludes a bound ligand from both sides of the membrane. Residues thought to be involved in proton coupling have also been identified near the extracellular gate of the cavity. Based on these findings and associated kinetic data, we propose that PepT(So) represents a sound model system for understanding mammalian peptide transport as catalysed by PepT1 and PepT2. Hide abstract