Dynaflow® Resolve and the heat sensing receptor TRPV1
Dynaflow Resolve is a powerful platform for ion channel screening and for studying biological processes associated with ion channel activation. Here, we want to highlight some of the research done on the receptor molecule TRPV1 using Dynaflow Resolve. TRPV1 is a membrane receptor responsible for sensing heat and pain and is of great pharmacological interest for finding new ways to treat pain.
Rational antibody design for undruggable targets using kinetically controlled biomolecular probes
Carolina L. Trkulja et al., Science Advances (2021) https://doi.org/10.1126/sciadv.abe6397
Several important drug targets, e.g., ion channels and G protein–coupled receptors, are extremely difficult to approach with current antibody technologies. To address these targets classes, we explored kinetically controlled proteases as structural dynamics–sensitive druggability probes in native-state and disease-relevant proteins. By using low–Reynolds number flows, such that a single or a few protease incisions are made, we could identify antibody binding sites (epitopes) that were translated into short-sequence antigens for antibody production. We obtained molecular-level information of the epitope-paratope region and could produce high-affinity antibodies with programmed pharmacological function against difficult-to-drug targets. We demonstrate the first stimulus-selective monoclonal antibodies targeting the transient receptor potential vanilloid 1 (TRPV1) channel, a clinically validated pain target widely considered undruggable with antibodies, and apoptosis-inducing antibodies selectively mediating cytotoxicity in KRAS-mutated cells. It is our hope that this platform will widen the scope of antibody therapeutics for the benefit of patients.
Probing Structure and Function of Ion Channels Using Limited Proteolysis and Microfluidics
Carolina L. Trkulja et al., Journal of the American Chemical Society (2014). https://doi.org/10.1021/ja507285w
Even though gain, loss, or modulation of ion channel function is implicated in many diseases, both rare and common, the development of new pharmaceuticals targeting this class has been disappointing, where it has been a major problem to obtain correlated structural and functional information. Here, we present a microfluidic method in which the ion channel TRPV1, contained in proteoliposomes or in excised patches, was exposed to limited trypsin proteolysis. Cleaved-off peptides were identified by MS, and electrophysiological properties were recorded by patch clamp. Thus, the structure–function relationship was evaluated by correlating changes in function with removal of structural elements. Using this approach, we pinpointed regions of TRPV1 that affect channel properties upon their removal, causing changes in current amplitude, single-channel conductance, and EC50 value toward its agonist, capsaicin. We have provided a fast “shotgun” method for chemical truncation of a membrane protein, which allows for functional assessments of various peptide regions.
Effect of Cholesterol Depletion on the Pore Dilation of TRPV1
Erik T. Jansson et al., Molecular Pain (2013) https://doi.org/10.1186%2F1744-8069-9-1
The TRPV1 ion channel is expressed in nociceptors, where pharmacological modulation of its function may offer a means of alleviating pain and neurogenic inflammation processes in the human body. The aim of this study was to investigate the effects of cholesterol depletion of the cell on ion-permeability of the TRPV1 ion channel. The ion-permeability properties of TRPV1 were assessed using whole-cell patch-clamp and YO-PRO uptake rate studies on a Chinese hamster ovary (CHO) cell line expressing this ion channel. Prolonged capsaicin-induced activation of TRPV1 with N-methyl-D-glucamine (NMDG) as the sole extracellular cation, generated a biphasic current which included an initial outward current followed by an inward current. Similarly, prolonged proton-activation (pH 5.5) of TRPV1 under hypocalcemic conditions also generated a biphasic current including a fast initial current peak followed by a larger second one. Patch-clamp recordings of reversal potentials of TRPV1 revealed an increase of the ion-permeability for NMDG during prolonged activation of this ion channel under hypocalcemic conditions. Our findings show that cholesterol depletion inhibited both the second current, and the increase in ion-permeability of the TRPV1 channel, resulting from sustained agonist-activation with capsaicin and protons (pH 5.5). These results were confirmed with YO-PRO uptake rate studies using laser scanning confocal microscopy, where cholesterol depletion was found to decrease TRPV1 mediated uptake rates of YO-PRO. Hence, these results propose a novel mechanism by which cellular cholesterol depletion modulates the function of TRPV1, which may constitute a novel approach for treatment of neurogenic pain.