Filip Van Petegem
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Affiliations to Research Centres, Institutes & Clusters
1) Muscle excitation-contraction coupling: How does an electrical signal in a muscle cell get transmitted into contraction? We investigate the membrane proteins involved in this process (L-type calcium channels, Ryanodine Receptors), as well as the various proteins that modulate these channels. Projects include solving crystal and cryo-EM structures of these channels in complex with the additional proteins. Functional experiments (e.g. electrophysiology) are used to test the hypotheses originating from these structures. 2) Channelopathies Ion channels are responsible for electrical signals in excitable cells. Mutations in the ion channel genes can lead to severe and often fatal disorders, including cardiac arrhythmias, epilepsy, ataxias, chronic pain and much more. We investigate the primary disease mechanisms by mapping disease mutations on the 3D structures, comparing structures of wild-type and disease mutant proteins, and functional experiments. Together these provide very detailed insights in the disease process. Current projects include congenital cardiac arrhythmias (CPVT, LongQT, Brugada Syndromes) and epilepsy (Dravet Syndrome)
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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - April 2022)
Kv1.2 channels are prominently expressed in neurons where they help to set the threshold of action potential firing. While we have a good understanding of the mechanism of voltage sensing and gating, we have comparatively little information on the compendium of regulatory molecules that can impact Kv1.2 expression and function. Kv1.2 channels are subject to a unique mechanism of regulation whereby a train of brief, repetitive depolarizations elicit increasing amounts of current, a phenotype we term ‘use-dependent activation’. In heterologous cells expressing Kv1.2 and primary hippocampal cultures from rats, there is remarkable diversity in this phenotype. While use-dependent activation is absent in all other Kv1 channels, it persists in heteromeric channels containing at least one Kv1.2 subunit. Exposing cells expressing Kv1.2 to reducing conditions causes a dramatic shift in use-dependent activation where there is very little or no current elicited by the first pulse, but over the course of the train there is a hundred-fold or more increase in current. Additionally, reducing conditions cause a depolarizing shift in the activation curve of Kv1.2 by +64 mV. Taken together, we postulate that use-dependence arises from an extrinsic, redox-sensitive inhibitory regulator that associates with Kv1.2 preferentially in the closed, reduced state. We have identified a new regulator of Kv1.2 function, Slc7a5, an amino acid transporter. Co-expression of these two proteins decreases Kv1.2 expression and produces a hyperpolarizing shift of the activation and inactivation curves. Together these effects result in Kv1.2 channels being caught in an ‘inactivation trap’. These effects of Slc7a5 can be rescued by co-expressing a third protein, Slc3a2, which is known to heterodimerize with the Slc7a5 channel. Using BRET we show that Slc7a5 and Kv1.2 can be within 10 nm of each other. Other Kv1 channels we have tested (Kv1.1 and Kv1.5) are insensitive to the activation shift produced by Slc7a5, however Kv1.1 channels are exquisitely sensitive to current inhibition. Overall, the work in this thesis expands our knowledge of how Kv1.2 channels are regulated and opens the door to examining how these interactions contribute to normal neuronal function.
The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.
Excitation-contraction (EC) coupling describes the process whereby the depolarizing action potential is transduced into a rapid increase of cytosolic calcium (Ca²⁺) that initiates muscle contraction. Proper execution of EC coupling relies on the coordinated communication between two calcium channels: plasma membrane-bound, L-type voltage-gated calcium channels (CaVs) and the intracellular Ryanodine Receptors (RyRs). CaVs respond to membrane depolarization by conveying an intracellular signal to the RyR. In skeletal muscle, CaV1.1 mechanically couples to the RyR; in cardiac tissue, extracellular Ca²⁺ entry via CaVs trigger RyR opening. The net effect of RyR activation is elevation of intracellular Ca²⁺ levels, activating the contractile machinery. In skeletal muscle, the nature of the physical CaV-RyR coupling has been an area of intense interest: do the channels directly interact or are auxiliary proteins required? Recently, a novel adaptor protein, STAC3, has been identified as playing a role in trafficking and maintaining components of the EC coupling machinery in a functional state. Indeed, STAC3-null mice and fish exhibit failure of skeletal muscle EC coupling. Chapter 2 presents x-ray crystallographic and isothermal titration calorimetry (ITC) data showing a direct interaction between STAC3 and CaV1.1. EC coupling assays reveal the importance of this interaction in EC coupling. The CaV1.1-STAC3 interaction is perturbed by the Native American Myopathy STAC3 mutation. L-type voltage-gated calcium channels fulfill dual roles as voltage-sensors for EC coupling and calcium ion conduits. In non-muscle cells, STAC3 facilitates CaV1.1’s functional membrane expression and alters the current properties of CaV1.2, suggesting a role of STAC proteins as a CaV regulator. Chapter 3 presents electrophysiology data illustrating the significant effect of STAC3 on modulating CaV1.2 currents. Detection of an interaction to Calmodulin (CaM), a well-known CaV regulator, suggests that STAC proteins may exert its effect on ion conduction via CaM. Genetic defects in the EC coupling machinery underlie numerous congenital myopathies and life-threatening cardiac arrhythmias. Chapter 4 explores the implications of disease-associated mutations within the cardiac Ryanodine Receptor (RyR2) using structural, spectroscopic, and thermal stability assays. An anion binding site within the N-terminal RyR2 region and maintenance of proper domain interfaces are key to RyR2 stability and normal functioning.
Ryanodine Receptors (RyR) are large ion channels that are responsible for the release of Ca²⁺ from the sarco/endoplasmic reticulum. The channel consists of a large cytosolic cap which functions as a giant allosteric protein, capable of being modulated by an assortment of binding partners and small molecules. To understand its function and mechanisms one needs to dissect the channel to its smallest parts. Using a combination of isothermal titration calorimetry and x-ray crystallography, two areas have been analyzed: binding by calmodulin (CaM) and the structure of a RyR domain, SPRY2.Calmodulin (CaM) is a Ca²⁺ binding protein that can regulate RyR under conditions of both high and low Ca²⁺ by tuning their Ca²⁺ sensitivity to channel opening and closing in an isoform-specific manner. I analyze the binding of CaM and its individual domains to three different RyR CaM binding regions using isothermal titration calorimetry. I compared binding to skeletal muscle (RyR1) and cardiac (RyR2) isoforms, under both Ca²⁺-loaded and Ca²⁺ free conditions. I find that CaM is able to bind all three regions, but with different binding modes, between the isoforms. Disease mutations target one of the three sites and affect CaM binding and energetics.The SPRY2 domain is one of three repeats of the same fold that are present within the RyR. It has been suggested as a key protein interaction site with dihydropyridine receptors to mediate excitation-contraction coupling in skeletal muscle tissue. RyR1 and RyR2 SPRY2 domains were crystallized and reveal differences with several other known SPRY domain structures. Docking of the RyR1 SPRY2 structure places it in between the central rim and the clamp region. The structure of a disease mutant causing cardiomyopathy is also determined and shows local misfolding. Finally, RyR1 SPRY2 binding to the DHPR II-III loops is undetectable by isothermal titration calorimetry.
Ryanodine receptors (RyRs) are calcium release channels located in the endo/sarcoplasmic reticulum that play a crucial role in the excitation-contraction coupling. Over 500 mutations have been found in the skeletal muscle (RyR1) and cardiac (RyR2) isoforms that cause severe muscle disorders or life-threatening arrhythmias. Mechanisms of these mutations have remained elusive largely due to the lack of high-resolution structures. Here, we compare pseudo-atomic models of the N-terminal region of RyR1 in the open and closed states together with crystal structures and thermal melts of multiple disease-associated mutants. We describe a model in which the intersubunit interface at the N-terminal region acts as a brake in channel opening. Next, we depict crystal structures of mutants at the intersubunit interface of RyR2 N-terminal region that perturb the structure of a loop targeted by multiple mutations. Furthermore, the crystal structure of the N-terminal domains of RyR2 reveals a unique, central anion-binding site. This anion binding is ablated in a disease-associated mutant that targets one of the anion-coordinating arginine residues, resulting in domain reorientations. Several other disease-causing mutations destabilize the protein. Taken together, the results illustrate a common theme across the RyR isoforms and their homologous IP₃ receptors that conformational changes at the N-terminal region caused by the destabilization of the interfaces are allosterically coupled to channel opening.
No abstract available.
Master's Student Supervision (2010 - 2021)
The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.
Voltage-gated calcium channels (Cay) have functions ranging from regulatingrelease of hormones and neurotransmitters, generating cardiac action potentials, andexcitation-contraction coupling. At nerve terminals, N- and P/Q- type Cavs convert theaction potential into aC²⁺ signal that in turn triggers neurotransmitter release.Neurotransmitter release requires several components, such as SNARE proteins.SNAREs, as well as many other presynaptic proteins, can interact with Cavs and inhibitthem by increasing their inactivation. The interaction is localized in the intracellular loopbetween domains II and III of the CL 1 subunit, in a domain termed ‘synprint’ (synapticprotein interaction site). In this study, we tried to solve the structure of the synprint siteby crystallography. To date, long needle-shape crystals were obtained; however, thequality of these crystals was not good enough for X-ray diffraction. in addition,isothermal titration calorimetry (ITC) was used to determine the interaction betweenSNARE protein syntaxinlA and the synprint site. It turned out that not any binding wasdetected, suggesting that the interaction between SNARE proteins and the presynapticCas, if at all present, is weak.