Regulation of Ca-transport across membranes
Ca2+ ions are crucial in many cellular processes, including neuronal response, muscle contraction, enzyme activity, gene transcription, cell death, proliferation and differentiation. The versatile but potentially dangerous intracellular Ca2+ message requires precise spatial and temporal regulation. We study ion transport across the membrane from a structural- and biophysical perspective, with the aim to explain biological functioning. In particular, we focus on the regulatory mechanisms that govern Ca2+ fluxes across membranes.Specific systems currently involve the Na+/Ca2+-exchanger (NCX), a highly ubiquitous ion transporter that constitutes the dominant Ca2+ efflux mechanism in heart and sensory neurons and the Na+/K+-ATPase, an ion transporter functionally connected to the NCX. Using high-resolution Nuclear Magnetic Resonance spectroscopy and X-ray crystallography we solve structures at atomic detail. These structural studies are complemented by biophysical characterization of the proteins and their complexes. Together, they serve as the basis for the design and implement ion of structurally inspired modifications of function and testing of these concepts with cell-biological assays. The resulting comprehensive knowledge regarding structure and function will aid the successful development of new drugs targeting the numerous devastating diseases associated with aberrant Ca2+ signaling, such as multiple sclerosis, heart disease, stroke, osteoporosis and Alzheimer's disease.
This project is supported by a VICI grant.
The plasma membrane Na+/Ca2+ exchanger (NCX) (Mark Hilge)
Figure 1. Solution structures of the first Ca-binding domain of NCX (CBD1), the second Ca-binding domain (CBD2) and a overall model of the full exchanger.
Binding of Na+ and Ca2+ ions to
the large cytosolic loop of the NCX regulates its ion
transport. We solved the structures of the first- and
second Ca2+ binding domains (CBD1 and CBD2) of the Na+/Ca2+ exchanger.
The CBD1 and CBD2 domains constitute a novel Ca2+
binding motif and are very similar in their Ca2+-bound
state. Strikingly, in the absence of Ca2+ the upper half
of CBD1 unfolds while CBD2 maintains its structural
integrity. Together with a seven-fold higher affinity
for Ca2+ this suggests that CBD1 is the primary Ca2+
sensor.
Key publication:
■ Hilge, M., Aelen,
J.M.A., Vuister, G.W. (2006) “Ca2+-regulation in the
Na+/Ca2+ exchanger involves two Markedly Different Ca2+
Sensors”, Mol. Cell 22,
15-25.
The Na/K-ATPase (Mark Hilge)
Figure 2. 11 Å EM-map of Na,K-ATPase and structures of the N-domain in native and ATP-bound conformation. Binding of ATP induces a conformational change that is transmitted to the N- and C-terminal regions connecting the N- and P-domains.
The Na,K-ATPase hydrolyses ATP in order to drive the coupled extrusion and uptake of Na+ and K+ ions across the plasma membrane, thereby establishing the electrochemical gradient utilized by other molecules, such as the NCX.
We solved the structure of its nucleotide binding domain in native form and in complex with ATP or ADP. Binding of ATP induces a conformational change that triggers a series of events necessary for the release of K+ ions to the cytosol.
Key publication:
■ Hilge, M, Siegal, G., Vuister, G.W., Guntert, P., Gloor, S.M. & Abrahams, J.P. (2003) “ATP-induced conformational changes of the nucleotide binding domain of Na,K-ATPase”, Nature Struct. Biol. 10, 468-474.