Research

 

Calcium ions play crucial roles in our bodies: they shape part of the cardiac action potential, mediate contraction of both cardiac and skeletal muscle, allow for the release of neurotransmitter and hormones, and regulate gene transcription, cell motility and much more. Ca2+ enters the cytosol through specialized channels, selective for Ca2+. Mutations in these channels can result in severe genetic diseases that are often fatal. Our lab is dedicated to understanding how these channels work in both native and diseased states, and how Ca2+ regulates various ion channels.

 

Our main focus is on several types of Ca2+ selective channels, including ryanodine receptors (RyRs) and voltage-gated calcium channels (CaVs), and on the Ca2+-dependent regulation of voltage-gated sodium channels (NaVs). We use cryoEM and X-ray crystallography to obtain atomic models, thus generating hypotheses about their function. We use electrophysiology and biochemical techniques to test these hypotheses.

Ryanodine Receptors (RyRs) and muscle contraction

RyRs are channels that release Ca2+ from the sarcoplasmic/endoplasmic reticulum. In cardiac muscle, the activation of voltage-gated calcium channels (CaVs) evokes an increase in cytosolic Ca2+ concentration. The RyR detects the increase and releases more Ca2+ into the cytoplasm, thus amplifying the signal. In skeletal muscle, CaVs and RyRs are thought to communicate directly through protein-protein interactions. Understanding this coupling is major project within the laboratory.

 

Mutations in RyRs are linked to several genetic diseases. Mutations in the skeletal muscle isoform (RyR1) can lead to central core disease (CCD) and malignant hyperthermia (MH), one of the major causes of death due to use of halogenated anaesthetics. Mutations in the cardiac isoform (RyR2) may result in severe inherited arrhythmia, including catecholamineric polymorphic ventricular tachycardia (CPVT) a devastating conditions that may result in sudden cardiac death.

 

RyRs are the largest ion channels currently known (with sizes up to 2.2MDa), built up by 4 identical subunits. They are regulated by a wide array of small molecules (such as caffeine) and auxiliary proteins (calmodulin, FKBP, kinases, phosphatases,…).We have determined several crystal structures of individual domains or domain clusters, together covering ~26% of the entire protein. These allow us to map and analyze the positions of disease-associated mutations. Structures of mutant forms reveal the conformational changes induced by the sequence variants. Cryo-electron microscopy reconstructions are utilized to map the binding sites of various auxiliary proteins and small molecules. The structures are used to guide functional experiments using planar lipid bilayer electrophysiology.

 

The Ryanodine Receptor: Fitting crystal structures in cryo-EM maps.

 
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Voltage-gated Calcium Channels: Molecular Memory

Voltage-gated calcium channels (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. This then turns on a Ca2+-dependent process, ranging from the release of neurotransmitters or hormones, muscle contraction (by activating RyRs), transcription, etc.

 

Their dysfunction leads to various severe and often lethal genetic diseases, and they form the targets for many drugs to treat cardiovascular diseases and pain. Despite their importance, we still lack a profound insight into how CaVs work.

 

A key characteristic of CaVs is their ability to 'remember' whether they've opened before. Depending on the exact timing and the exact CaV isoform , they respond with either higher or lower electrical currents. This intriguing feat is mediated by the same ion that permeates these channels: Ca2+. Upon entering the cytosol, Ca2+ binds to Calmodulin, which results either in inactivation (decreased currents) or facilitation (faster opening of the channels). The exact mechanisms still remain a mystery, but it is clear that disease-associated mutations, either in Calmodulin or in the channel itself, can interfere with the process.

 

The related voltage-gated sodium channel (NaV) also shows a Ca2+-dependent feedback mechanism similar to CaVs. We're studying the interaction between Calmodulin and NaVs, as well as the structure of auxiliary NaVbeta subunits.

 

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Techniques

We use a variety of complementary biochemical and biophysical techniques to investigate ion channels. Examples include x-ray crystallography to elucidate the 3D structure of individual subunits, domains, and their complexes with ligands, cryo-electron microscopy (cryo-EM) to look at intact channels and their complexes. We have frequent access to an in-house Titan Krios microscope with Volta phase plate and Falcon III direct electron detector. A Tecnai Spirit and Tecnai F20 are available for screening.

 

Other techniques: isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) to detect and quantify protein-ligand and protein-protein interactions, circular dichroism (CD) and differential scanning calorimetry (DSC) to analyze secondary structure and protein stability, and electrophysiology (two-electrode voltage clamp and Planar lipid bilayer electrophysiology) to measure the ionic conductance and gating properties.

 

X-ray crystallography

Isothermal titration calorimetry

 

 

Two-electrode voltage clamp

keywords: cardiac arrhythmia, epilepsy, ion channel, CPVT, Dravet syndrome, malignant hyperthermia, central core disease, excitation-contraction coupling, ryanodine receptor, sodium channel, calcium channel, voltage-gated sodium channel, calmodulin, X-ray crystallography, ryanodine, RyR, RyR1, RyR2, RyR3, high resolution, structure, arrhythmia, genetic disorder, ITC, isothermal titration calorimetry, Brugada syndrome, Long QT syndrome, arrhythmia, channel, cryo-EM, electrophysiology, GEFS+, Vancouver, UBC, Biochemistry, Canada
tel:604.827.4267 | fax:604.822.5227 | email:filip.vanpetegem'at'gmail.com

Van Petegem Lab © 2014, updated: Oct/2017