Discovery at the VICB
Solving the Mysteries of Potassium Transport
By: Carol A. Rouzer, VICB Communications
Published: September 22, 2016
A structure of the potassium channel modulating protein KCNE3 revealed by NMR provides new insight into the pathogenesis of cystic fibrosis.
Cystic fibrosis is a genetic disorder associated with recurrent lung infections, poor digestion, stunted growth, and a shortened life expectancy. It is caused by mutation of the gene that encodes the cystic fibrosis transmembrane conductance regulator protein (CFTR), which transports chloride ion across the apical membranes of epithelial cells, particularly in the respiratory and digestive tracts. A well-established but poorly understood clinical observation about cystic fibrosis is that female patients usually suffer more severe disease than males. Now, Vanderbilt Institute of Chemical Biology members Chuck Sanders and Jens Meiler along with their collaborators Carlos Vanoye (Northwestern University) and Wade Van Horn (Arizona State University) provide new insight into the structure and function of KCNE3, a potassium channel modulating protein that is required for normal chloride ion transport (Figure 1A). Their findings offer new insight into the pathogenesis of cystic fibrosis and the basis for the differences in clinical severity between the sexes. [B. M. Kroncke, et al. (2016) Sci. Adv., published online September 9, DOI:10.1126/sciadv.1501228].
FIGURE 1. (A) Role of the KCNQ1/KCNE3 potassium channel in Cl- transport. In epithelial cells of the intestines and trachea, the Na+/K+ pump, found in the basolateral membrane, uses the energy from ATP hydrolysis to pump K+into and Na+ out of the cell against their concentration gradients. The K+/Cl-/Na+cotransporter then transports two Cl- ions along with one Na+ and one K+ ion into the cell. These Cl- ions can then be transported across the apical membrane of the epithelial cell by the cystic fibrosis transmembrane conductance regulator protein (CFTR). The concentrations of Na+ and Cl- are high in the extracellular environment, so they are readily available to the K+/Cl-/Na+ cotransporter. However, to provide enough K+ ions for Cl- cotransport, a flow of those ions from inside to outside the cell is required. This flow is provided by the KCNQ1/KCNE3 potassium channel. (B) Diagram of KCNE3 topography, showing the transmembrane domain, two amphipathic helices, and connecting loops. Sites of mutations associated with known disease states (red) and sites used for site-directed spin labeling (yellow) are also indicated. Figure reproduced under the Creative Commons Attribution-NonCommercial 4.0 International License from B. M. Kroncke, et al. (2016) Sci. Adv., published online September 9, DOI:10.1126/sciadv.1501228.
KCNE3 is a transmembrane protein that binds to and moderates the activities of a number of potassium channel proteins, including KCNQ1. In the absence of KCNE3, KCNQ1 acts as a voltage-gated channel, meaning that it opens and closes in response to changes in membrane potential. Upon binding to KCNE3, however, KCNQ1 becomes permanently open under physiologically relevant conditions, serving as a "leak" channel through which potassium ions continually exit the cell. KCNQ1/KCNE3 complexes are found in the basolateral membranes of intestinal and tracheal epithelial cells, where they facilitate potassium ion efflux that is coupled to the transport of chloride ions into the cell (Figure 1A). These chloride ions are subsequently transported back out of the cell across its apical membrane by CFTR. The KCNE3/KCNQ1 complex-dependent leakage of potassium is required for normal chloride ion homeostasis. To better understand how KCNE3 modulates KCNQ1 and the role of the complex in chloride transport, the investigators determined the structure of KCNE3 and developed a model of its complex with KCNQ1.
The researchers first used solution nuclear magnetic resonance (NMR) supplemented with computational molecular dynamics (MD) simulations to determine the structure of KCNE3 in lipid bicelles. They also evaluated the effects of paramagnetic probes (one lipophilic and one hydrophilic) on the NMR signals of the protein to delineate its conformational dynamics. These studies revealed the presence of an extracellular N-terminal amphipathic helix that is loosely associated with the membrane surface. A flexible loop connects this helix to the protein's single helical transmembrane domain (TMD). At the opposite end of the TMD, a six amino-acid loop precedes a short juxtamembrane helix that is then followed by a disordered C-terminus (Figure 1B). A curvature at the C-terminal (intracellular) end of the TMD was an interesting structural feature of the protein (Figure 2). The investigators confirmed the presence of this curvature by generating a site-directed KCNE3 mutant protein bearing cysteine residues at both ends of the TMD. They then coupled a nitroxide spin label to these cysteines, enabling them to use double electron-electron spin resonance to measure the end-to-end length of the TMD helix. The results were consistent with the presence of the curvature, which was also detected in KCNE3 proteins embedded in detergent micelles and lipid bilayers. Thus, the researchers concluded that the curvature was an intrinsic property of the protein.
FIGURE 2. Structure of KCNE3 as determined by solution NMR spectroscopy and molecular dynamics refinement. (A) Close up of the transmembrane helical domain, showing the curvature at the C-terminal (intracellular) end, and highlighting the three amino acids on the internal face of the curve. (B) Model of the complete protein. (C) Same as (B), but with amino acid side chains also shown. Figure reproduced under the Creative Commons Attribution-NonCommercial 4.0 International License from B. M. Kroncke, et al. (2016) Sci. Adv., published online September 9, DOI:10.1126/sciadv.1501228.
KCNE3 interacts with the KCNQ4 potassium channel as well as KCNQ1. However, whereas KCNE3 binding promotes opening of KCNQ1, it inhibits potassium conductance through KCNQ4. This led the investigators to search for the structural basis for these differences. Both potassium channel proteins are homotetramers, constructed of monomers that have six transmembrane domains, designated S1 through S6. The researchers constructed six chimeric KCNQ1 proteins, each of which contained a transmembrane domain from KCNQ4 in place of the native domain. After testing each of these proteins for potassium conductance and response to KCNE3 binding, the investigators discovered that the chimera bearing the S4 domain of KCNQ4 exhibited activity similar to that of KCNQ4 rather than KCNQ1. Further work, in which individual amino acids were substituted, revealed that either of two double mutations of KCNQ1, H240R/Q244R or H241M/Q244R were sufficient to convert the protein's response upon binding KCNE3 to that of KCNQ4. In both of these double mutants, amino acids in the S4 domain of KCNQ1 were substituted with the corresponding amino acids of KCNQ4, Clearly, these amino acids must be critical to the way in which KCNE3 promotes KCNQ1 channel opening and potassium leakage.
Preliminary modeling studies suggested that the N-terminal end of the KCNE3 TMD interacts with the extracellular end of KCNQ1's S1 domain, and that the C-terminal end of the KCNE3 TMD interacts with the intracellular end of the KCNQ1's S4 segment. The investigators used site-directed mutagenesis to introduce cysteine residues into each of the predicted interaction sites in both proteins. They then combined the mutant proteins and subjected them to oxidizing conditions. Their hypothesis was that, if the predicted sites indeed interact, they should be permanently joined by a disulfide bridge, and therefore activated, under these conditions. The somewhat unexpected results demonstrated that the current amplitude of the combined proteins was actually lower under oxidizing than under reducing conditions. The investigators explained their findings by postulating that a disulfide bond is, in fact, formed between the mutant KCNE3 and KCNQ1 proteins upon oxidation, but that the residues involved are not ideally aligned for bonding in this way. Hence, the resulting joined protein does not retain the ideal conformation for potassium conductance.
Using all of the clues they had gathered concerning interaction sites between KCNE3 and KCNQ1 and all available structural data, the investigators generated an integrative model of the complex between the two proteins (Figure 3). The model predicted that the N-terminus of the KCNE3 TMD interacts with a cleft between the extracellular end of the S1 helix of one KCNQ1 subunit and a "turret" structure comprising S5 and the P-loop of another subunit. This site in KCNQ1 is the location of a number of gain-of-function mutations that are associated with atrial fibrillation. The cytosolic end of the KCNE3 TMD binds in a cleft that enables it to make contact with the S6 domain of one subunit, the S4-S5 linker of a second subunit, and the S1 and S4 helices of a third. As predicted from the mutagenesis studies, His240, Val241, and Gln244 of KCNQ1 are all present in the KCNE3 binding cleft. A particularly important interaction predicted by the model was a hydrogen bond between Arg83 of KCNE3 and Gln244 of KCNQ1. The investigators noted that, in KCNQ4, residue 244 is Arg rather than Gln, so this hydrogen bond cannot form. Instead, Arg83 of KCNE3 is more likely to interact with Asp218 of KCNQ4, an interaction that would displace the S4 helix of the channel toward the cytosolic face of the membrane, a conformation that is consistent with channel closing rather than opening. This observation could provide the foundation for the opposite effects of KCNE3 binding to KCNQ1 (opening) versus KCNQ4 (closing).
FIGURE 3. Integrative model of the KCNQ1/KCNE3 complex. (A) Model viewed looking down on the extracellular face of the membrane. KCNQ1 is a homotetramer. Blue color indicates the voltage-sensor domain, green the S4-S5 linker, and red the channel pore. KCNE3 is shown in gold. (B) View from the side within the membrane. Key residues and structural features are labeled. (C) View looking at an angle from the extracellular to the intracellular surface. The direction of K+ flow is indicated by the arrow. Figure reproduced under the Creative Commons Attribution-NonCommercial 4.0 International License from from B. M. Kroncke, et al. (2016) Sci. Adv., published online September 9, DOI:10.1126/sciadv.1501228.
Estrogen activates protein kinase Cδ (PKCδ), which phosphorylates Ser82 of KCNE3. Phosphorylation at this site blocks the interaction of KCNE3 with KCNQ1, leading to a reduction in potassium transport out of the cell. Under normal circumstances, this reduction is not deleterious to health, but in a cystic fibrosis patient, the loss of potassium transport would likely exacerbate the already compromised ability of the cell to transport chloride ions. The model of the KCNE3/KCNQ1 complex offers important insight into the effects of Ser82 phosphorylation on complex formation. The model places Ser82 of KCNE3 close to Gln244 of KCNQ1. The investigators hypothesized that phosphorylation of Ser82 would disrupt the normal interactions of KCNE3 with this residue. They tested this hypothesis by constructing a site-directed mutant KCNQ1 protein in which Gln244 was replaced with Arg, expecting that this substitution would reduce any repulsive effects of KCNE3 Ser82 phosphorylation. Expression of this mutant in CHO-K1 cells followed by treatment of the cells with estrogen revealed that the effects of estrogen were abrogated for the Q244R mutant. Thus, the model provides a structural explanation for the increased severity of cystic fibrosis in women.
Together these studies provide important new insights into the functioning of a key potassium channel modulating protein. These findings are applicable to developing a better understanding of other members of both the KCNQ and KCNE protein families, and may lead to new therapeutic approaches to help patients, particularly women, suffering from cystic fibrosis.
View Science Advances article: "Structural basis for KCNE3 modulation of potassium recycling in epithelia"