|
Licensee in Biochemistry, Ghent University, 1998
PhD in Biochemistry, Ghent University, 2002
Postdoctoral research, University of California, 2007
Assistant Professor, University of British Columbia, 2007
CIHR new investigator
MSFHR career investigator
Tel: 604-827-4267
Email: petegem@interchange.ubc.ca
See the Van Petegem
Lab Website
Our thoughts and memories, heartbeats, and muscle contractions require the fast transmission of electrical signals throughout our bodies. Two steps are essential in generating these signals. Ion pumps consume energy to generate concentration gradients of ions across a membrane. Ion channels allow these ions to pass selectively down their electrochemical gradient when an appropriate signal arrives.
Of all the ions that can pass through ion channels, Ca2+ plays a crucial role: besides merely carrying charge, it is also an important intracellular second messenger. The resting concentration of Ca2+ in the cytoplasm is extremely low, and Ca2+ can enter either from the extracellular space or from intracellular stores. Our lab is interested in channels that allow the selective passage of Ca2+, how they interact with regulatory proteins, how they integrate various signals, and what their molecular architecture looks like. In particular, we are interested in 2 classes of channels: voltage-gated calcium channels (CaVs) and ryanodine receptors (RyRs).
Both CaVs and RyRs are dynamic proteins that can change their state depending on various input signals. It is impossible to fully appreciate their intricate functioning without knowing their 3D structure. On the other hand, having a 3D structure alone is not sufficient, because they can adopt multiple conformations in vivo. We use protein crystallography to obtain atomic models, and use these to generate hypotheses about their function. We use electrophysiology and biochemical techniques to test these hypotheses.
Prospective students or postdocs would benefit from learning cloning, expression, and purification of proteins, crystallogenesis and X-ray diffraction to determine high-resolution structures of protein complexes, biophysical methods such as isothermal titration calorimetry to study binding interactions, and two-electrode voltage clamp to measure ion channel currents.
CaVs are able to detect differences in voltage across the plasma membrane: when an excitable cell depolarizes to a sufficient level, they open and allow Ca2+ to enter. CaVs thus convert an electrical signal into an intracellular chemical signal. Because Ca2+ signalling is important in virtually every cell, CaVs have been recruited for very diverse tasks. These include excitation-contraction (EC) coupling in heart and skeletal muscle, hormone and neurotransmitter release, regulation of gene expression, neuronal migration, and generation and control of the cardiac action potential. Their dysfunction leads to various severe and often lethal genetic diseases, and they form the targets for many drugs that are being used to treat cardiovascular diseases, hypertension, epilepsy, and chronic pain. Despite their importance, we still lack a profound insight into how CaVs work. This is due, in part, by the limited amount of high-resolution data describing their precise architecture.
CaVs can be built up by multiple subunits (Figure). The CaV a 1 subunit allows passage of the ions. The other subunits ( b , g , a 2 d ) help in trafficking of the channel and regulate its kinetic properties. In addition, a myriad of auxiliary proteins (kinases, phosphatases, calmodulin, scaffolding molecules, and components of the synaptic vesiscle release machinery) can interact with CaVs in a dynamic manner, regulating the channel properties. CaVs are sensitive integrators: the output (the time and amount of Ca2+ entering the cell) is dependent on several input signals (Ca2+ concentration, phosphorylation status, various protein-protein interactions). Our primary focus lies with dissecting the mechanisms whereby auxiliary proteins regulate the channels.
Figure: Typical subunit arrangement of a skeletal muscle voltage-gated calcium channel
RyRs are channels that release Ca2+ from the sarcoplasmic/endoplasmic reticulum. In cardiac muscle, the activation of CaVs evokes an increase in cytoplasmic Ca2+ concentration. The RyR detects the increase and releases more Ca2+ into the cytoplasm, thus ‘enforcing' the signal. In skeletal muscle, CaVs and RyRs are thought to communicate directly through protein-protein interactions.
Mutations in RyRs are linked to several genetic diseases, including cardiac arrhythmias, central core disease, and malignant hyperthermia, one of the major causes of death due to anaesthesia. RyRs are regulated by a wide array of small molecules (such as caffeine) and auxiliary proteins (calmodulin, FKBP, kinases, phosphatases,…). RyRs are huge ion channels (up to 2 MDa), built up by 4 identical subunits. We currently know next to nothing about their atomic structure. Our major focus is to generate crystal structures of important domains and their interactions with auxiliary proteins. This work will lay the basis for novel strategies to interfere with RyR function in diseased states.
Sarhan MS, Van Petegem F , Ahern CA (2009) A double tyrosine motif in the cardiac sodium channel Domain
III-IV linker couples calcium dependent calmodulin binding to inactivation gating. J. Biol. Chem . In Press.
Lobo PA, Van Petegem F (2009) Crystal structures of the N-terminal domains of cardiac and skeletal muscle ryanodine receptors: insights into disease mutations. Structure. In Press.
The structural biology of voltage-gated calcium channel function and regulation.
F. Van Petegem and D.L. Minor
Biochem Soc Trans 34, 887-893 (2006).
Insights into voltage-gated calcium channel regulation from the structure of the CaV1.2 IQ domain-Ca2+/calmodulin complex.
F. Van Petegem, FC Chatelain , D.L. Minor Nat Struct Mol Biol 12, 1108-1115 (2005)
Structure of a complex between a voltage-gated calcium channel beta-subunit and an alpha-subunit domain.
F. Van Petegem, K.A Clark, F.C. Chatelain, D.L. Minor Nature 429, 671-675 (2004)
Alanine-scanning mutagenesis defines a conserved energetic hotspot in the Ca V a AID-Ca V ß interaction site that is critical for channel modulation.
Van Petegem F, Duderstadt KE, Clark KA, Wang M, Minor DL
Structure 16 ( 2 ), 280-294
. (2007)
Kim EY, Rumpf CH, Fujiwara Y, Cooley ES, Van Petegem F,
Minor DL Structures of Ca(V)2 Ca(2+)/CaM-IQ Domain Complexes Reveal Binding Modes that Underlie Calcium-Dependent Inactivation and Facilitation.
Structure 16 ( 10 ), 1455-1467 (2008)
back to top |
