Research
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Life at the Junction: where Ion channels meet
In many cell types, there are specialized sites where the plasma membrane (PM) is in close proximity to the endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) in muscle cells. Such junctions allow for a unique communication between ion channels located in different membranes. It allows for amplification of calcium signals in dendritic spines within neurons, in cardiac muscle cells, and more. One key protein is Junctophilin, which is thought to bind the plasma membrane via an ordered domain, while a transmembrane helix also anchors it in the ER or SR. Junctophilins are also able to directly interact with different ion channels, thus keeping them in proximity for optimal communication. The human genome encodes four different junctophilin isoforms (JPH1-4), expressed in different tissues. JPH2 is affected by hundreds of genetic variants that have been linked to cardiomyopathy.
Using X-ray crystallography, we solved structures of junctophilins, both alone and in complex with their binding site in L-type calcium channels (Yang et al, PNAS, 2022). Mutating the binding site leads to a redistribution of these calcium channels in skeletal muscle cells, and weakens their ability to communicate with Ryanodine Receptors in the SR membrane. We are currently investigating the architecture of neuronal Junctophilins, how Junctophilins bind lipids, how diseases lead to their proteolytic cleavage, and how they associate with other ion channels.
In many cell types, there are specialized sites where the plasma membrane (PM) is in close proximity to the endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) in muscle cells. Such junctions allow for a unique communication between ion channels located in different membranes. It allows for amplification of calcium signals in dendritic spines within neurons, in cardiac muscle cells, and more. One key protein is Junctophilin, which is thought to bind the plasma membrane via an ordered domain, while a transmembrane helix also anchors it in the ER or SR. Junctophilins are also able to directly interact with different ion channels, thus keeping them in proximity for optimal communication. The human genome encodes four different junctophilin isoforms (JPH1-4), expressed in different tissues. JPH2 is affected by hundreds of genetic variants that have been linked to cardiomyopathy.
Using X-ray crystallography, we solved structures of junctophilins, both alone and in complex with their binding site in L-type calcium channels (Yang et al, PNAS, 2022). Mutating the binding site leads to a redistribution of these calcium channels in skeletal muscle cells, and weakens their ability to communicate with Ryanodine Receptors in the SR membrane. We are currently investigating the architecture of neuronal Junctophilins, how Junctophilins bind lipids, how diseases lead to their proteolytic cleavage, and how they associate with other ion channels.
Channelopathies: investigating disease-causing mutations linked to cardiac arrhythmia, myopathies, epilepsy, and malignant hyperthermia
Sequence variants in the genes encoding ion channels often have devastating consequences. Imagine running behind the ball during a soccer match, losing consciousness, and dying on the spot. You never had any warning symptoms. Also known as 'sudden cardiac death', such events are often the result of mutations in cardiac ion channels. For example, simple point mutations in ryr2, the gene encoding Ryanodine Receptor isoform 2 (RyR2) can give rise to CPVT (catecholaminergic polymorphic ventricular tachycardia). RyR2 is located in the SR membrane, where it allows the release of calcium ions into the cytosol, driving the contraction of cardiac muscle. This calcium release needs to be precisely timed, as too much, too little, too early, or too late can have life-threatening consequences. Similarly, mutations in RyR1, primarily found in skeletal muscle, can give rise to malignant hyperthermia or central core disease (CCD), a progressive form of myopathy for which there is currently no cure. Mutations in voltage-gated calcium channels are associated with a wide range of disorders that span cardiac arrhythmia, autism, ataxias, and pain. Mutations are often also found in auxiliary proteins that regulate these channels, causing similar disorders.
We utilize cryo-electron microscopy and X-ray crystallography to understand how such mutations impact the channels, giving us much-needed insights into the disease mechanisms and providing clues for new strategies to treat the disorders. As an example, we solved cryo-EM structures of a disease mutant RyR1 carrying a malignant hyperthermia mutation (Woll et al, Nature Communications, 2021). The point mutation affects a critical domain-domain interaction that allosterically couples to the opening of the pore. However, the precise effect depends on whether auxiliary proteins are bound to the RyR1. The disease mutant RyR1 opens easier, resulting in the leak of Calcium ions from the SR into the cytosol when pharmacologically triggered. During surgery, general anesthetics like isoflurane or desflurane cause opening of such mutant RyR1, resulting in a dangerous rise in body temperature, a condition known as Malignant Hyperthermia. Unless treated immediately with a drug known as dantrolene, such events are usually fatal.
Sequence variants in the genes encoding ion channels often have devastating consequences. Imagine running behind the ball during a soccer match, losing consciousness, and dying on the spot. You never had any warning symptoms. Also known as 'sudden cardiac death', such events are often the result of mutations in cardiac ion channels. For example, simple point mutations in ryr2, the gene encoding Ryanodine Receptor isoform 2 (RyR2) can give rise to CPVT (catecholaminergic polymorphic ventricular tachycardia). RyR2 is located in the SR membrane, where it allows the release of calcium ions into the cytosol, driving the contraction of cardiac muscle. This calcium release needs to be precisely timed, as too much, too little, too early, or too late can have life-threatening consequences. Similarly, mutations in RyR1, primarily found in skeletal muscle, can give rise to malignant hyperthermia or central core disease (CCD), a progressive form of myopathy for which there is currently no cure. Mutations in voltage-gated calcium channels are associated with a wide range of disorders that span cardiac arrhythmia, autism, ataxias, and pain. Mutations are often also found in auxiliary proteins that regulate these channels, causing similar disorders.
We utilize cryo-electron microscopy and X-ray crystallography to understand how such mutations impact the channels, giving us much-needed insights into the disease mechanisms and providing clues for new strategies to treat the disorders. As an example, we solved cryo-EM structures of a disease mutant RyR1 carrying a malignant hyperthermia mutation (Woll et al, Nature Communications, 2021). The point mutation affects a critical domain-domain interaction that allosterically couples to the opening of the pore. However, the precise effect depends on whether auxiliary proteins are bound to the RyR1. The disease mutant RyR1 opens easier, resulting in the leak of Calcium ions from the SR into the cytosol when pharmacologically triggered. During surgery, general anesthetics like isoflurane or desflurane cause opening of such mutant RyR1, resulting in a dangerous rise in body temperature, a condition known as Malignant Hyperthermia. Unless treated immediately with a drug known as dantrolene, such events are usually fatal.
Effect of the R615C mutation, linked to MH, on the structure of RyR1. Shown is a detail around the mutation site, with a morph between the cryo-EM structures of wild-type and mutant RyR1. Arg615 (black sticks) is located at the interface between two separate domains of RyR1 (blue and green), where it forms favorable interactions. Mutation to a Cys triggers a conformational change, whereby the green domain (a.k.a. the 'bridging solenoid') tilts relative to the blue domain ('N-terminal solenoid'). This tilt translates into movements ~10 Angstrom at the end of the green domain, and results in facilitated opening of the channel pore.
Ion channel pharmacology and toxicology
Ion channels are prime drug targets to treat disorders ranging from arrhythmia, epilepsy, pain, Alzheimer's disease, and many more. Cryo-EM enables us to visualize drug binding sites and understand their mode of action, providing critical clues on how to optimize them. In addition, nature has already evolved various toxins that can bind ion channels and either activate or block them. This can paralyze prey or otherwise inflict extreme pain as a defense mechanism. Studying how such toxins associate with channels gives us clues on how to modify them into useful molecules with therapeutic potential. Cryo-EM, coupled with functional electrophysiological assays, has allowed us to understand ion channel pharmacology.
A few examples:
RyR1 in complex with diamide insecticides: Ma et al (2020) Nature Chemical Biology
KCNQ1 (cardiac potassium channel) in complex with the activator ML277: Willegems et al (2022) Nature Communications
RyR1 in complex with the scorpion toxin imperacalcin: Haji-Ghassemi et al (2023) Science Advances
Muscle excitation-contraction coupling
In skeletal muscle, there is a highly-specialized communication between L-type calcium channels (CaV1.1), located in the plasma membrane, and Ryanodine Receptors (RyR1) in the SR membrane: upon depolarization of the plasma membrane, conformational changes in CaV1.1 are transmitted mechanically to RyR1, which is then triggered to open. The communication goes both ways, as mutations in RyR1 also affect the behavior of CaV1.1. Although this process has been known to exist for decades, the precise mechanism of action has remained a long-standing mystery in the field. In addition to these 2 channels, it also involves Junctophilins and STAC3. Using X-ray crystallography, we have been able to figure out how exactly these bind to CaV1.1. We also found that several disease-causing mutations link to the interface and thus directly interfere with the mechanical coupling. We hypothesize that junctophilins and STAC3 also associate with RyR1, thus linking it with CaV1.1 to enable the mechanical coupling.
STAC3 interactions with CaV1.1: Wong King Yuen et al (2017) PNAS
STAC3 disease-causing mutations: Rufenach et al (2020) Structure
Junctophilin interactions with CaV1.1: Yang et al (2022) PNAS
In skeletal muscle, there is a highly-specialized communication between L-type calcium channels (CaV1.1), located in the plasma membrane, and Ryanodine Receptors (RyR1) in the SR membrane: upon depolarization of the plasma membrane, conformational changes in CaV1.1 are transmitted mechanically to RyR1, which is then triggered to open. The communication goes both ways, as mutations in RyR1 also affect the behavior of CaV1.1. Although this process has been known to exist for decades, the precise mechanism of action has remained a long-standing mystery in the field. In addition to these 2 channels, it also involves Junctophilins and STAC3. Using X-ray crystallography, we have been able to figure out how exactly these bind to CaV1.1. We also found that several disease-causing mutations link to the interface and thus directly interfere with the mechanical coupling. We hypothesize that junctophilins and STAC3 also associate with RyR1, thus linking it with CaV1.1 to enable the mechanical coupling.
STAC3 interactions with CaV1.1: Wong King Yuen et al (2017) PNAS
STAC3 disease-causing mutations: Rufenach et al (2020) Structure
Junctophilin interactions with CaV1.1: Yang et al (2022) PNAS
Older videos, heavily outdated but kept around for nostalgic reasons: