Membrane Transport: Riding elevators into and out of cells
The mechanisms responsible for the trafficking of carboxylate ions across cell membranes are becoming clearer.
- Department of Integrative Biology, University of Colorado Denver, United States
- Chemistry Department, University of Colorado Denver, United States
Small carboxylate ions such as citrate and succinate are intermediates in the citric acid cycle, which is a crucial metabolic pathway in aerobic organisms. However, small carboxylate ions also have many other roles: for example, they function as signaling molecules in processes ranging from DNA transcription and replication (Wellen et al., 2009) to heat generation (Mills et al., 2018), and they have also been linked to obesity (Birkenfeld et al., 2011) and seizures (Thevenon et al., 2014).
Cells rely on transmembrane proteins belonging to the DASS family (short for divalent anion sodium symporter) to move small carboxylate ions into and out of cells. There are two clades in the DASS family: cotransporters that import carboxylate ions from the bloodstream into cells (Prakash et al., 2003), and antiporters/exchangers that move some carboxylate ions into cells while moving others out (Pos et al., 1998).
Previously the structure of just one member of the DASS family – a cotransporter called VcINDY, which is found in the bacterium Vibrio cholerae – had been determined (Mancusso et al., 2012; Mulligan et al., 2016; Nie et al., 2017). VcINDY contains two subunits, and each of these contains two domains: (i) a scaffold domain, which is anchored in the plasma membrane of the cell and is not, therefore, free to move; (ii) a transport domain, which is more mobile.
It has been predicted that DASS proteins operate with an 'elevator mechanism' that involves the transport domain (to which the carboxylate ion is attached) sliding up and down the scaffold domain between an inward-facing state and an outward-facing state (Figure 1; Mulligan et al., 2016). However, since the structure of VcINDY has only ever been determined for the inward-facing state, evidence in support of this mechanism has remained inconclusive. Now, in eLife, Da-Neng Wang (New York University School of Medicine), Emad Tajkhorshid (University of Illinois at Urbana-Champaign) and co-workers – including David Sauer as first author – report the results of a combined experimental and computational study that helps to shed light on this mystery (Sauer et al., 2020).
Figure 1
Schematics of the elevator mechanism.
Each of the two sub-units in a DASS transporter contains a transport domain (yellow) that is mobile, and a scaffold domain (blue) that is anchored within the plasma membrane of the cell (green). When the transporter is in an outward-facing state (left) two carboxylate ions outside the cell (such as the succinate ion shown here) can each bind to one transport domain and be dragged across the membrane and into the cell by the transport domain as it slides along the scaffold domain. This leaves the transporter is in an inward-facing state (right). In cotransporters the positive charges of two sodium ions in the transport domain have an important role in trafficking carboxylate ions through the membrane; in antiporters/exchangers two positively charged residues have a similar role in the trafficking process. DASS: divalent anion/sodium symporter.
The researchers used a combination of X-ray crystallography and cryo-electron microscopy to determine structures for VcINDY and also for LaINDY, an antiporter that is found in the bacterium Lactobacillus acidophilus. Remarkably, they were able to obtain structures for the previously elusive outward-facing state for both. Moreover, they determined the structures when a carboxylate ion was bound to the transport domain and also for the substrate-free case. Relative to the inward-facing state, the transport domain in the outward-facing state is rotated by an angle of 37° and is about 13 Å further away from the inside of the cell (Figure 1).
Sauer et al. then used computer simulations to model the transition from an initial state in which two succinate ions were bound to the outward-facing state in the LaINDY antiporter (based on their experimental structures) to a final state in which the succinate ions were inside the cell and the antiporter was in an inward-facing state: the researchers used an approximate structure for the final state as the actual structure for the inward-facing state in LaINDY has not been determined yet. Jointly, the experiments and simulations lend strong support to the elevator mechanism.
One may ask: how does the cotransporter or antiporter make sure that the carboxylate ion has been loaded into the elevator before the button is pressed? The experimental structures suggest that a 'passport check' is enforced via electrostatic effects. In the absence of the carboxylate ion, the binding site on the transport domain has a positive net charge, which makes it difficult for this domain to pass through the membrane, because charged particles prefer polar environments (such as aqueous solutions) to the nonpolar environments found in the membrane. Loading the negative carboxylate ion onto the binding site neutralizes the postive charge, allowing the transport domain to cruise through. This mechanism limits a form of unproductive transport called slippage (that is, the passage of substrate-free transport domains) in both cotransporters and antiporters.
The work of Sauer et al. also highlights interesting differences between the cotransporter and the antiporter. For the VcINDY cotransporter the positive charge on the transport domain is provided by sodium ions, and the binding of the carboxylate ion to the transport domain leads to large changes in conformation. It also appears that the relatively weak initial binding of sodium ions in the cotransporter is strengthened by the arrival of the carboxylate ion. In contrast, the positive charges in the LaINDY antiporter are provided by amino acid residues rather than sodium ions, and the conformational changes caused by binding appeared to be small and local.
It would be interesting to explore the effects of introducing mutations to the residues at the sites of the sodium ions in VcINDY cotransporters or the equivalent sites in LaINDY antiporters. Would it be possible to engineer a sodium-independent cotransporter? And could a cotransporter be converted into an antiporter, and vice versa? Answers to these questions will lead to a deeper understanding of the structure-function relationships for proteins belonging to the DASS family.
References
-
The bacterial dicarboxylate transporter VcINDY uses a two-domain elevator-type mechanismNature Structural & Molecular Biology 23:256–263.https://doi.org/10.1038/nsmb.3166
-
The ion transporter superfamilyBiochimica et Biophysica Acta (BBA) - Biomembranes 1618:79–92.https://doi.org/10.1016/j.bbamem.2003.10.010
-
Mutations in SLC13A5 cause autosomal-recessive epileptic encephalopathy with seizure onset in the first days of lifeAmerican Journal of Human Genetics 95:113–120.https://doi.org/10.1016/j.ajhg.2014.06.006
Article and author information
Author details
Publication history
Copyright
© 2020, Duster and Lin
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 691
- views
-
- 80
- downloads
-
- 1
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Membrane Transport: Riding elevators into and out of cells
eLife 9:e62925.
https://doi.org/10.7554/eLife.62925
Further reading
-
- Structural Biology and Molecular Biophysics
The KCNH family of potassium channels serves relevant physiological functions in both excitable and non-excitable cells, reflected in the massive consequences of mutations or pharmacological manipulation of their function. This group of channels shares structural homology with other voltage-gated K+ channels, but the mechanisms of gating in this family show significant differences with respect to the canonical electromechanical coupling in these molecules. In particular, the large intracellular domains of KCNH channels play a crucial role in gating that is still only partly understood. Using KCNH1(KV10.1) as a model, we have characterized the behavior of a series of modified channels that could not be explained by the current models. With electrophysiological and biochemical methods combined with mathematical modeling, we show that the uncovering of an open state can explain the behavior of the mutants. This open state, which is not detectable in wild-type channels, appears to lack the rapid flicker block of the conventional open state. Because it is accessed from deep closed states, it elucidates intermediate gating events well ahead of channel opening in the wild type. This allowed us to study gating steps prior to opening, which, for example, explain the mechanism of gating inhibition by Ca2+-Calmodulin and generate a model that describes the characteristic features of KCNH channels gating.
-
- Structural Biology and Molecular Biophysics
The M2 proton channel aids in the exit of mature influenza viral particles from the host plasma membrane through its ability to stabilize regions of high negative Gaussian curvature (NGC) that occur at the neck of budding virions. The channels are homo-tetramers that contain a cytoplasm-facing amphipathic helix (AH) that is necessary and sufficient for NGC generation; however, constructs containing the transmembrane spanning helix, which facilitates tetramerization, exhibit enhanced curvature generation. Here, we used all-atom molecular dynamics (MD) simulations to explore the conformational dynamics of M2 channels in lipid bilayers revealing that the AH is dynamic, quickly breaking the fourfold symmetry observed in most structures. Next, we carried out MD simulations with the protein restrained in four- and twofold symmetric conformations to determine the impact on the membrane shape. While each pattern was distinct, all configurations induced pronounced curvature in the outer leaflet, while conversely, the inner leaflets showed minimal curvature and significant lipid tilt around the AHs. The MD-generated profiles at the protein–membrane interface were then extracted and used as boundary conditions in a continuum elastic membrane model to calculate the membrane-bending energy of each conformation embedded in different membrane surfaces characteristic of a budding virus. The calculations show that all three M2 conformations are stabilized in inward-budding, concave spherical caps and destabilized in outward-budding, convex spherical caps, the latter reminiscent of a budding virus. One of the C2-broken symmetry conformations is stabilized by 4 kT in NGC surfaces with the minimum energy conformation occurring at a curvature corresponding to 33 nm radii. In total, our work provides atomistic insight into the curvature sensing capabilities of M2 channels and how enrichment in the nascent viral particle depends on protein shape and membrane geometry.
