An estimated 30 % of the proteins encoded in the human genome code for transmembrane proteins, highlighting the crucial importance of these macromolecules to human physiology. Our lab focuses on a family of transmembrane proteins known as ion channels. These molecular switches regulate the flux of ions across the otherwise ion-impermeable hydrophobic core of biological membranes. They proteins play crucial roles in numerous physiological processes, such as neuronal and cardiac excitability. Genetic or acquired dysfunctions in ion channels are the known cause of pain, epilepsy, and cardiac arrhythmias, making them front-line therapeutic targets.
Left: Top down view of a P2X receptor with ATP shown in blue
IN A NUTSHELL
Why do we need these receptors?! Cells, too, need to talk: the cell that has something to say sends out a signal (for example a small molecule), which then needs to be somehow recognized/sensed by a cell nearby. This often happens through a specialized protein in the cell membrane, called a receptor – which is exactly the kind of molecule we work on. Basic communication, really.
Trimeric ion channels
A major focus of our lab are acid-sensing ion channels (ASICs) and P2X receptors. They trimeric ligand-gated ion channels that open cation-selective pores in response to binding of extracellular protons and ATP, respectively. They play important roles in synaptic transmission throughout the body and their dysfunction has been implicated in numerous diseases, such as hearing loss, pain and stroke (Heusser & Pless, in preparation; Illes et al., Br J Pharm, 2020). We are interested in all molecular aspects of these ion channels, including the basic mechanism of activation, the origin of their ion selectivity, their pharmacology, as well as functional modulation by protein-protein interactions.
Left: Sodium ions binding to the ASIC selectivity filter
NALCN channel complex
Another key area of interest is the sodium leak channel NALCN. This large protein is required for survival in mammals and gain- or loss-of-function mutations can result in devastating diseases. Although its sequence resembles that of well-known voltage-gated sodium and calcium channels, NALCN itself is nonfunctional. We have recently shown that NALCN is only functional when it forms a complex with three other proteins (Chua et al, Science Advances, 2020). This breakthrough enabled the first detailed functional characterisation of NALCN and led our collaborators to obtain a structure of NALCN together with its auxiliary factor FAM155A (Kschonsak et al, Nature, 2020). We continue to study the function and pharmacology, as well as disease-causing mutations of the sodium leak channel complex.
Left: Close up view of ATP bound to a P2X receptor
WHY ALL THOSE FANCY METHODS?
The receptors we work on are incredibly small, making them essentially invisible, even for powerful microscopes –
which is why we constantly try to come up with new (and often indirect) ways to study their function.
Recent progress in structure determination of trimeric ion channels and the sodium leak channel (Kschonsak et al., Nature, 2020; Yoder & Gouaux, eLife, 2020; McCarthy et al., Cell, 2019) provides an excellent framework to shed new light on these fascinating macromolecules. In particular, our lab employs a combination of electrophysiology, chemical biology, protein engineering and fluorescence spectroscopy to address crucial questions regarding the molecular function and pharmacology of these proteins:
- ElectrophysiologyWhen ions flow across a membrane they generate electrical currents. Although minute, they can be measured with great precision, down to the level of ions flowing through a single ion channel. In our lab we employ a variety of electrophysiological recording techniques, e.g. two-electrode voltage-clamp and patch-clamp methodologies, including single molecule and high throughput recordinds (Lynagh et al., eLife, 2017; Braun et al., 2021, bioRxiv).
- Protein engineeringEmerging chemical biology techniques allow the site-directed incorporation of ncAAs or post-translational modifications via either non-sense suppression approaches (Braun et al, J Physiol, 2020) or protein semi-synthesis (Khoo & Galleano et al., Nature Comms, 2020). These modifications offer the ability to either incorporate subtle derivatives of naturally occurring amino acids or to confer entirely new properties to a given protein.
- FluorometryPatch-clamp fluorometry (PCF) and its cousin voltage-clamp fluorometry (VCF) allow labeling an ion channel with an organic dye. When the protein changes its conformation, the dye reports a change in environment by changing its brightness (Borg & Braun et al, PNAS, 2020). We recently increased the sensitivity of this approach, allowing to track protein conformational changes independent of ionic currents with greater temporal resolution (Wulf & Pless, Cell Rep, 2018).