Biology subjects

Senning, E. N.

Publications and source records attributed to Senning, E. N..

6 recordsLinked to original sources

bioRxiv2025

A single residue determines regulation of TRPV1 by phosphoinositides

The ion channel TRPV1 is expressed in the peripheral nervous system where it mediates heat sensation and pain signaling. How lipids contribute to TRPV1 regulation remains an open topic. Although an inhibitory binding site for phosphoinositides was identified at the vanilloid site in TRPV1 structures, a mechanism for phosphatidylinositol-2-bisphosphate (PI(4,5)P2) to both inhibit and potentiate TRPV1 activity is lacking. This gap in knowledge led us to examine structural and sequence overlap between TRPV1 and the PI(4,5)P2 binding site of TRPV5. In a space located adjacent to the turn from S6 to TRP helices, we identify a second, possible PI(4,5)P2 binding site in TRPV1, which we term the "front porch" site and includes residue H410. On the one hand, we obtained electrophysiological evidence with the TRPV1-H410R mutant that PI(4,5)P2 associates with TRPV1 at the front porch site through an electrostatic interaction to potentiate channel activity. On the other hand, the charge reversal in H410D conferred an increase in channel activity, which was not expected if the electrostatic interaction with PI(4,5)P2 is lost and channel function is crippled. Importantly, capsaicin activated currents from H410D experience significant current run-up, consistent with the slow displacement by capsaicin of a tighter-bound lipid in the vanilloid site, which structures suggest is phosphoinositide (PI). Lastly, we tested TRPV1-H410D sensitivity to PI(4,5)P2 manipulations by dephosphorylating the membrane pool of PI(4,5)P2 to PI with the Pseudojanin rapamycin-phosphatase (PJ) system. Depletion of PI(4,5)P2 with the PJ-system in wild type channels induces 1) a robust increase in baseline activity as channels are released from inhibition and 2) loss of current potentiation, driven by PI(4,5)P2 dissociation from the front porch, both of which were absent in TRPV1-H410D. Based on these findings we hypothesize that plasma membrane TRPV1-H410D maintains PI rather than PI(4,5)P2 in its vanilloid site and does not interact with PI(4,5)P2 in the front porch site. We integrated our discoveries that H410 interacts with PI(4,5)P2 at two distinct sites in a mutually exclusive manner and present a cohesive model for phosphoinositide inhibition and potentiation in TRPV1, addressing a maturation process for TRPV1 that relies on H410 to coordinate the exchange of PI for PI(4,5)P2. Statement of SignificanceA lingering question that hangs over TRPV1 research is how the lipid PI(4,5)P2 regulates the function of this channel. We devised a study based on structural homology between TRPV5 and TRPV1 to identify critical contact points between the lipid and a putative PI(4,5)P2 binding site in TRPV1, which we probed using mutagenesis experiments. The results we obtained lay the groundwork for how a two-site binding mechanism of PI(4,5)P2 couples to TRPV1 gating and address important questions about the channels maturation and association with phosphoinositides as it is transferred through membrane compartments with different phosphoinositide compositions. We conclude that TRPV1 expressed and purified from different compartments may preserve the phosphoinositide character of that compartment (PI in the endoplasmic reticulum, PI(4,5)P2 in the plasma membrane) and bestow on structures a state that is not necessarily relevant to the biological function of the channel in the plasma membrane.

biophysics↗

bioRxiv2025

Missense variant analysis in the TRPV1 ARD reveals the unexpected functional significance of a methionine

The Transient Receptor Potential Vanilloid sub-type 1 (TRPV1) is an ion channel that is activated by heat, extracellular protons, oxidation, and it is implicated in various aspects of inflammatory pain. In this study, we uncover that residue M308 in the TRPV1 ankyrin repeat domain (ARD) stands out from most other buried ARD residues because of the greater number of human missense variants at this position while maintaining a high degree of conservation across species and TRPV channel subtypes. We use mutagenesis and electrophysiology to examine this apparent discrepancy and show that substitutions at position M308 that preserve or reduce side-chain volume have no effect on channel function, whereas substitutions with larger or more polar residues increase channel activity in response to capsaicin or temperature. Substitution of M308 with a histidine bestows channels with pH-dependence that is different from wild type, consistent with the side-chain at position 308 exerting an influence on channel gating. We speculate that M308 is highly conserved because its side-chain could serve as a target for oxidation-dependent modification. On the other hand, we show that a previously described splice variant of TRPV1 that relies on M308 as a start codon diminishes surface expression of co-transfected full-length TRPV1 in HEK293 cells. Together, our findings reveal a functionally important conserved site within the ARD of TRPV1 that could have roles in oxidation-dependent channel regulation as well as tuning the number of active channels in the membrane by enabling expression of a shorter dominant-negative splice variant.

biophysics↗

bioRxiv2024

Fluorescence labeling strategies for the study of ion channel and receptor cell surface expression: A comprehensive toolkit for extracellular labeling of TRPV1.

Regulation of ion channel expression on the plasma membrane is a major determinant of neuronal excitability, and identifying the underlying mechanisms of this expression is critical to our understanding of neurons. A critical aspect of measuring changes in ion channel expression is uniquely identifying ion channels located on the cell surface. To accomplish this goal we demonstrate two orthogonal strategies to label extracellular sites of the ion channel TRPV1 that minimally perturb the function of the channel: 1) We use the amber codon suppression technique to introduce a non-canonical amino acid (ncAA) with tetrazine click chemistry compatible with a trans-cyclooctene coupled fluorescent dye. 2) By inserting the circularly permutated HaloTag (cpHaloTag) in an extracellular loop of TRPV1, we incorporate a click-chemistry site for a chloroalkane-linked fluorescent dye of our choosing. Optimization of ncAA insertion sites was accomplished by screening residue positions between the S1 and S2 transmembrane domains with elevated missense variants in the human population, and we identified T468 as a rapid labeling site ([~]5 minutes) based on functional as well as biochemical assays in HEK293T/17 cells. After several rounds of adapting the linker lengths and backbone placement of cpHaloTag on the extracellular side of TRPV1, our efforts led to a channel construct that robustly expressed as a fully functional TRPV1exCellHalo fusion with intact wild-type gating properties. The TRPV1exCellHalo construct was used in a single molecule experiment to track TRPV1 on the cell surface and validate studies that show decreased mobility of the channel upon activation. The success of these extracellular label TRPV1 (exCellTRPV1) constructs as tools to track surface expression of the channel will shed significant light on the mechanisms regulating expression and provide a general scheme to introduce similar modifications to other cell surface receptors.

biophysics↗

bioRxiv2023

OptoPI3K, genetic code expansion, and click chemistry reveal mechanisms underlying reciprocal regulation between TRPV1 and PI3K

ABSTRACTLigands such as insulin, epidermal growth factor, platelet derived growth factor, and nerve growth factor (NGF) initiate signals at the cell membrane by binding to receptor tyrosine kinases (RTKs). Along with G-protein coupled receptors, RTKs are the main platforms for transducing extracellular signals into intracellular signals. Studying RTK signaling has been a challenge, however, due to the multiple signaling pathways to which RTKs typically are coupled, including MAP/ERK, PLC{gamma}, and Class 1A phosphoinositide 3-kinases (PI3K). The multi-pronged RTK signaling has been a barrier to isolating the effects of any one downstream pathway. Here, we used optogenetic activation of PI3K to decouple its activation from other RTK signaling pathways. In this context, we used genetic code expansion to introduce a click chemistry noncanonical amino acid into the extracellular side of membrane proteins. Applying a cell- impermeant click chemistry fluorophore allowed us to visualize delivery of membrane proteins to the plasma membrane in real time. Using these approaches, we demonstrate that activation of PI3K, without activating other pathways downstream of RTK signaling, is sufficient to traffic the TRPV1 ion channels and insulin receptors to the plasma membrane.

physiology↗

bioRxiv2021

Phosphatidylinositol phosphate binding domains exhibit complex dissociation properties at the inner leaflet of plasma membrane sheets

The pleckstrin homology (PH) domain is a lipid targeting motif that binds with high specificity to phosphatidylinositol phosphate (PIP) lipids. Using TIRF microscopy, we followed the dissociation of GFP-tagged PH domains from the plasma membranes of rapidly unroofed cells and found that AKT-PH and PLC{delta}1-PH dissociation kinetics can be distinguished by their effective koff values determined from fitting fluorescence traces to a single exponential equation. Our measurements for the koff of AKT-PH-GFP and PLC{delta}1-PH-GFP were significantly different (p < 0.05) at 0.39 {+/-} 0.05 s-1 and 0.56 {+/-} 0.17 s-1, respectively. Furthermore, we identified substantial rebinding events in our measurements of PLC{delta}1-PH-GFP dissociation kinetics. By applying inositol triphosphate (IP3) to samples during the unroofing process, we measured a much faster koff of 1.54 {+/-} 0.42 s-1 for PLC{delta}1-PH-GFP, indicating that rebinding events are significantly depressed through competitive action by IP3 for the same PH domain binding site as phosphatidylinositol (4,5)-bisphosphate (PIP2). We discuss the complex character of our PLC{delta}1-PH-GFP fluorescence decays in the context of membrane receptor and ligand theory to address the question of how free PIP2 levels modulate the interaction between membrane associated proteins and the plasma membrane.

biophysics↗

bioRxiv2018

Reciprocal regulation among TRPV1 channels and phosphoinositide 3-kinase in response to nerve growth factor

Although it has been known for over a decade that the inflammatory mediator NGF sensitizes pain-receptor neurons through increased trafficking of TRPV1 channels to the plasma membrane, the mechanism by which this occurs remains mysterious. NGF activates phosphoinositide 3-kinase (PI3K), the enzyme that generates PIP3, and PI3K activity is required for sensitization. One tantalizing hint came from the finding that the N-terminal region of TRPV1 interacts directly with PI3K. Using 2-color total internal reflection fluorescence microscopy, we show that TRPV1 potentiates NGF-induced PI3K activity. A soluble TRPV1 fragment corresponding to the N-terminal Ankyrin repeats domain (ARD) was sufficient to produce this potentiation, indicating that allosteric regulation was involved. Further, other TRPV channels with conserved ARDs also potentiated NGF-induced PI3K activity whereas TRP channels lacking ARDs did not. Our data demonstrate a novel reciprocal regulation of PI3K signaling by the ARD of TRPV channels.

biochemistry↗