Publications

Phosphatidylinositides constitute only 1%-3% of plasma membranes but play vital roles in cellular signaling. In particular, phosphatidylinositol 4,5-bisphosphate (PIP₂) is involved in processes such as cytoskeleton organization and ion-channel regulation. Pleckstrin homology (PH) domains are modular domains found in many proteins and are known for their strong affinity for PIP₂ headgroups. The role of lipid composition in PH domain binding to PIP₂, particularly the inclusion of phosphatidylserine (PS), is not well understood. This study explores the mechanisms of PH domain binding to PIP₂ using fluorescence spectroscopy, Fourier transform infrared spectroscopy, two-dimensional infrared spectroscopy, and molecular dynamics simulations. We find that anionic PIP₂ and PS alter the interfacial environment compared to phosphatidylcholines. Additionally, the PH domain promotes the localization of anionic lipid domains upon binding. Our results highlight the role of PS in lipid domain formation within membranes and its potential influence on protein binding affinities and lipid geometries. Specifically, we discovered a strong interaction between PIP₂ and PS whereby hydrogen bonding within these anionic lipids drives localization in the membrane. This interaction also regulates protein binding at the membrane interface. Our findings suggest that cooperativity between PIP₂ and PS is key to the formation of localized lipid domains and the recruitment of proteins such as the PH domain of phospholipase C-δ1.

The Transient Receptor Potential Vanilloid sub-type 1 (TRPV1) is an ion channel implicated in various aspects of inflammatory pain. Whereas ion conduction and the binding of vanilloids and many other important ligands occur within the transmembrane domain, the role of the large cytoplasmic N-terminal ankyrin repeat domain in channel function is unclear. We have previously described that the number of distinct amino acid variants observed in the human population at each TRPV1 N-terminal ankyrin repeat domain position is positively correlated with their solvent accessible surface area (SASA). Our prior study led us to hypothesize that sites with strong deviations from the correlation are likely to have functional relevance. In this study, we isolated two buried residues (M308/A256) in the ARD that have low SASA scores and yet at least 3 or 4 alternate types of amino acids are found within the human population at these two positions, setting them apart from most other sites with similarly low SASA for which no human missense variants have been found. Side chains from both residues extend into a buried pocket and are within 5 Å of each other in a closed channel structure configuration. Moreover, despite its variability among the human population, M308 is highly conserved across species as well as across TRPV subfamily members. To address these apparently contradictory observations, we used mutagenesis to examine the functional role of M308. We determined using electrophysiology that substitutions at position M308 which preserve or reduce side-chain volume had no effect on channel function, whereas substitutions with the larger residues resulted in either complete loss of channel activity or increased activity in response to both capsaicin and temperature. We confirmed that M308 is a partially buried site since M308C did not show any evidence of reactivity to various cysteine-modifying reagents. We observed that converting the methionine to histidine bestows a pH-dependent effect on the channel that is different from wild type, consistent with the side-chain at position 308 exerting a functional effect on the channel either sterically or electrostatically by action of protonation. These observations establish that steric or electrostatic perturbations at position M308 influence channel activity, but that a methionine is not required for normal channel function, raising the question of why M308 is so highly conserved. We speculate that given the functional effect of perturbations introduced at position 308, M308 could have a conserved role in redox signaling as a target for oxidation-dependent side-chain covalent modification. On the other hand, M308 could be used as an alternative ‘start’ codon, which is consistent with a short 5’ splice variant of TRPV1 that has been reported in the literature. We show that the truncated splice variant significantly diminishes surface expression of full-length TRPV1 when co-expressed in HEK293 cells. Together, our findings reveal a functionally important conserved site within the ARD of TRPV1 that could have physiologically relevant 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.

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. Here, we present two orthogonal strategies to label extracellular sites of the ion channel TRPV1 that minimally perturb its function. 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. Additionally, by inserting the circularly permutated HaloTag (cpHaloTag) in an extracellular loop of TRPV1, we can incorporate a 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. We identified T468 as a rapid labeling site (∼5 min) based on functional and biochemical assays in HEK293T/17 cells. Through adapting linker lengths and backbone placement of cpHaloTag on the extracellular side of TRPV1, we generated a fully functional channel construct, TRPV1exCellHalo, with intact wild-type gating properties. We used TRPV1exCellHalo 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 application of these extracellular label TRPV1 (exCellTRPV1) constructs to track surface localization of the channel will shed significant light on the mechanisms regulating its expression and provide a general scheme to introduce similar modifications to other cell surface receptors.

Ligands 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, PLCγ, 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.

Transient receptor potential (TRP) channels encompass a diverse class of non-selective
cation channels that are primarily grouped by sequence homology to an ion channel
discovered in the light transduction pathway of fly photoreceptors. Emphasis on gene
sequence and protein domains rather than a functional classification scheme is necessary
given the broad array of physiological roles for these channels as well as theirmodes of
activation. To date, approximately 30 mammalian orthologues of the original fly TRP
have been identified, and these have been further classified into six sub-groups: TRPC,
TRPV, TRPA, TRPM, TRPP and TRPML. Functional characterization by electrophysiology
andmutational or chimeric analysis have been the cornerstone of advancing
our knowledge about TRP channels. A recent wave of TRP channel structures has
confirmed many of the keenest observations from electrophysiology experiments and
provided fertile starting ground for a newwave of functional analysis.This chapter aims
to consolidate an understanding of TRP channel function with the growing number of
TRP channel structures that are being solved at an incredible pace. In these structures,
4TRPsubunits assemble into a pore-forming ion channel, andeachsubunit is defined by
six transmembrane segments separated into a pore domain and voltage sensor-like
domain, common to the voltage-gated ion channel super-family. A high degree of
structural similarity in the transmembrane region will be the backdrop to a discussion on
general principles of gating, lipid interactions, Ca2+ dependent modulation and cell
signaling in the TRP channel family.

Protein structures and mutagenesis studies have been instrumental in elucidating molecular mechanisms of ion channel function, but making informed choices about which residues to target for mutagenesis can be challenging. Therefore, we investigated the potential for using human population genomic data to further refine our selection of mutagenesis sites in TRPV1. Single nucleotide polymorphism data of TRPV1 from gnomAD 2.1.1 revealed a lower number of missense variants within buried residues of the ankyrin repeat domain and an increased number of variants between secondary structure elements of the transmembrane segments. We hypothesized that residues critical to interactions at interfaces between subunits or domains in the channel would exhibit a similar reduction in variants. We identified in the structure of ground squirrel TRPV1 (PDB: 7LQY) a possible electrostatic network between K155 and K160 in the N-terminal ankyrin repeat domain and E761 and D762 in the C-terminus (K-KED). Consistent with our hypothesis for residues at key interface sites, none of the four residues have any variants reported in gnomAD 2.1.1. Ca²⁺ imaging of TRPV1 K-KED mutants confirmed significant roles for these residues, but we found that the electrostatic interaction is not essential since channel function is still observed in total charge reversals on the C-terminal side of the interface (E761K/D762K). Interestingly, Ca²⁺ imaging responses for a charge swap experiment with K155D/D762K showed partially restored wild-type responses. Using electrophysiology, we found that charge reversals on either K155 or D762 increased the baseline currents of TRPV1, and the charge swapped double mutant, K155D/D762K, partially restored baseline currents to wild-type levels. We interpret these results to mean that contacts across residues in the K-KED interface shift the equilibria of conformations to closed pore states. Our study demonstrates the utility and applicability of a combined missense variant and structure targeted investigation of residues at TRPV1 subunit interfaces.