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.

 

Our main focus is on several types of Ca2+ selective channels, including ryanodine receptors (RyRs), and on channels whose activity is modulated by Ca2+, including voltage-gated sodium channels (NaVs). Both NaVs and RyRs are dynamic proteins that can change their state depending on various input signals. It is impossible to fully appreciate their intricate mechanisms without knowing the 3D structure. On the other hand, having a 3D structure alone is not sufficient, because they can adopt multiple conformations that affect the passage of ions. We use protein crystallography to obtain atomic models, thus generating hypotheses about their function. We use electrophysiology and biochemical techniques to test these hypotheses.

Ryanodine Receptors

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.

 

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 catecholamineric polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventrical dysplasia type 2 (ARVD2), two devastating conditions that lead to cardiac arrhythmias and 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 currently know very little about the RyR atomic structure. We have determined several crystal structures of individual domains or domain clusters, together covering ~26% of the entire protein. Together with available cryo-electron microscopy reconstructions, we show that disease mutations affect several domain-domain interactions. By weakening the contacts, it allows the RyR to open easier, leading to leakage of calcium ions into the cytoplasm.

 

One major goal is to understand the mechanisms that underlie RyR function in normal and diseased states. We aim to generate more crystal structures of important domains and their interactions with auxiliary proteins. These structures are then used to guide functional experiments in planar lipid bilayer electrophysiology experiments.

 

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

 
3pieces  

 

Voltage-gated Sodium Channels

Voltage-gated sodium channels (NaVs) are able to detect differences in voltage across the plasma membrane: when an excitable cell depolarizes to a sufficient level, they open and allow Na+ to enter. 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 NaVs work. This is due, in part, to the limited amount of high-resolution data describing their precise 3D structure. Mammalian NaVs typically contain multiple subunits. The pore-forming subunit, also known as the 'alpha' subunit, forms the permeation pathway for sodium ions and contains the voltage sensing domains. Auxiliary 'beta' subunits modulate the properties of the alpha subunit, affecting the number of channels at the plasma membrane and channel gating. Below (left) is a recent crystal structure of the NaV beta4 subunit extracellular domain, determined by Dr. Samir Das, a postdoctoral fellow in the lab. We currently try to elucidate how these beta subunits exactly engage the alpha subunits.

 

One property of NaVs is their 'inactivation', a process by which the channels are shut down. This is thought to occur via a cytoplasmic segment that acts as a plug. Many channels can already be inactivated prior to the depolarization of the membrane, which means they are not available for conducting Na+. Modulating the process of inactivation is therefore a key mechanism by which the channels can be regulated. Ca2+ ions can affect channel inactivation. This is accomplished by the binding of Calmodulin, a ubiquitous calcium sensing protein,to various cytoplasmic segments of the channell. Below (right) is a crystal structure of Calmodulin (surface) bound to the inactivation gate of the cardiac NaV (red sticks). This structure was solved by Maen Sarhan, a former PhD student co-supervised together with Dr. Chris Ahern.

 

beta4small

 

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, 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, 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/2014