Research: Cryo-EM, X-ray crystallography, and electrophysiology to study ion channels

 

Our lab studies the structure and function of ion channels, membrane proteins that mediate electrical signaling in excitable cells. They are essential for neuronal signaling, the beating of our hearts, the contraction of muscle, and much more. Ion channel genes are targeted by hundreds of genetic variants that result in 'channelopathies', a set of severe disorders ranging from inherited cardiac arrhythmias to chronic pain and congenital epilepsy. How do the ion channels gate? How do auxiliary proteins, small molecules, and post-translational modifications change their behavior? How do they assemble into larger complexes? How do disease-associated mutation change their function? We try to answer these questions via various methods.

 

As ion channels are complex and dynamic proteins, we utilize a combination of techniques to study their behaviour. This includes X-ray crystallography and cryo-electron microscopy to study their 3D structures, and electrophysiology to measure the electrical currents generated when the channels open.

 

Recent stories

Yang et al (2022) Structures of the junctophilin/voltage-gated calcium channel interface reveal hot spot for cardiomyopathy mutations. PNAS. 119, e2120416119

 

In various cell types, there are close connections between the plasma membrane (PM) and the endoplasmic or sarcoplasmic reticulum (ER/SR). The physical proximity of these membranes allows for specialized communication between ion channels that are located in different membranes. Junctophilins are a special class of proteins that are thought to mediate ER-PM junctions. They also interact with various ion channels, further ensuring their physical proximity. For example, they allow the communication between L-type calcium channels and Ryanodine Receptors, a critical process that has mostly been studied in skeletal and cardiac muscle tissue. Junctophilins are associated with several diseases, including cardiomyopathy and huntington disease-like 2. In this study, we elucidated the peculiar structure of Junctophilin and how it interacts with L-type calcium channels. Targeted mutations at the interface strongly attenuate communication between L-type calcium channels and Ryanodine Receptors.

 

 

 

Woll et al (2021) Pathological conformations of disease mutant Ryanodine Receptors revealed by cryo-EM. Nature Communications. 12:807

 

RyR1, the main calcium release channel expressed in skeletal muscle, is the target for a genetic disease known as Malignant Hyperthermia. In patients with the disorder, volatile anaesthetics can trigger a dangerous rise in body temperature and ultimately death. Using muscle tissue from pigs homozygous for a disease mutation, we extracted mutant RyR1 and obtained cryo-EM structures in various conformational states. These show that the mutation affects critical domain-domain interactions in the closed state of the channel, thus facilitating channel opening. Below: planar lipid bilayer recordings of wild-type and disease mutant RyR1, along with a side view of the overall structures (wild-type:color; mutant: black).

 

 

Haji Ghassemi et al (2019) The Cardiac Ryanodine Receptor Phosphorylation Hot Spot Embraces PKA in a Phosphorylation-Dependent Manner.  Molecular Cell 75, 39-52

 

Stress signals allow for greater cardiac output.  The cardiac Ryanodine Receptor (RyR2) is the target for kinases that can upregulate it's activity, as part of the normal fight-or-flight response. However, excessive phosphorylation can cause or exacerbate cardiac arrhytmias. Using x-ray crystallography, we found how PKA engages RyR2 via an unusual interface, and how phosphorylation can result in changes in secondary structure.

Below: embrace between PKA (blue) and RyR2 (red) 

 

 

 

Ryanodine Receptors (RyRs): from excitation-contraction coupling to Alzheimer's disease  

RyRs are channels that release Ca²⁺ from the sarcoplasmic/endoplasmic reticulum. In cardiac muscle, the activation of voltage-gated calcium channels (Ca_Vs) 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, Ca_Vs 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. However, faulty regulation of RyRs through post-translational modifications can also lead or contribute to various pathologies, including atrial fibrillation, heart failure, and Alzheimer's disease.  

 

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 use cryo-EM to look at overall 3D structures in various bound forms. X-ray crystallography is very helpful for regions that are poorly resolved in the cryo-EM maps.  We combine these with binding assays (ITC) and planar lipid bilayer electrophysiology.    

 

 

Voltage-gated Calcium Channels: Molecular Memory

Voltage-gated calcium channels (Ca_Vs) 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 Ca_Vs work.

 

A key characteristic of Ca_Vs is their ability to 'remember' whether they've opened before. Depending on the exact timing and the exact Ca_V 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 and the factors that affect channel inactivation