Jie Zheng - US grants

[unreadable] DESCRIPTION (provided by applicant): Cyclic nucleotide-gated (CNG) channels play a fundamental role in photoreceptor neurons where they mediate the final step of the signal transduction cascade triggered by absorption of photons. They are Ca2+ permeable, non-selective cation channels that are opened by binding of cyclic nucleotides. Mutations in CNG channel genes are responsible for numerous diseases in the visual system including retinitis pigmentosa (retinal degeneration) and achromatopsia (total color blindness). CNG channels are members of a large family of P-loop containing ion channels that shares considerable common structural and functional similarities. The long-term goal of the proposed experiments is to understand how conformational rearrangements in the channel protein lead to the opening of the CNG channel pore. While recent crystal structures of bacteria P-loop containing channels revealed both the closed and the open pore conformations, many fundamental questions regarding the molecular mechanism of channel gating remain unanswered. What pore structure constitutes the activation gate(s)? What are the structural movements that initiate ion permeation? To address these and related questions in CNG channels, the grant will take particular advantage of our recently developed fluorescence technique (termed Patch-Clamp Fluorometry, or PCF) that is based on site-specific fluorescence labeling of channels in isolated membrane patches. PCF will be used to simultaneously record fluorescence and current signals from the same population of channels in real time, and to directly correlate structural changes in the channel protein to their gating effects. PCF will be combined with fluorescence quenching and fluorescence resonance energy transfer (FRET) to investigate the channel pore structure as well as rearrangements in the structure during CNG channel activation. Findings from these experiments will advance our understanding of the molecular basis for CNG channel gating under physiological as well as disease states. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The heat sensor TRPV1 channel is a polymodal receptor that plays a key role in mediating neuronal pain caused by various noxious stimuli. One such stimulus is extracellular acidification caused by inflammation, tissue damage and ischemia. Low pH is thought to activate TRPV1 both directly by serving as a channel activator and indirectly by potentiating the channel's response to other stimuli. How TRPV1 activation is controlled by pH as well as its relation to activation by heat, ligands, and endogenous channel modulators remains largely unknown. Importantly, the highly unique susceptibility of TRPV1 activity to a variety of physical and chemical factors makes the channel an attractive target for clinical intervention of pain. The overarching goal of our research is to understand the cellular sensing function of TRPV1 by elucidating molecular mechanisms underlying its polymodal activation by heat, capsaicin and other stimuli. In the proposed study we aim to reveal the structural and mechanistic nature of extracellular H+ regulation of TRPV1. As H+-induced TRPV1 activity has heat-dependent and agonist-dependent components, this investigation will also shed light on how heat and agonist control TRPV1 activity. We approach our goal through a combination of optical, electrophysiological, and molecular methods. In particular, we will apply a patch fluorometry approach to directly observe structural changes in the channel protein or the binding of regulatory molecules, using fluorophores as molecular sensors. Simultaneous fluorescent and electrical recordings permit direct correlation of structural changes to their effects on channel activation. Using these methods, we will address questions on how changes in pH, temperature, and the concentration of agonists are sensed by TRPV1, what channel structures convey these stimuli, and how these stimuli converge to control TRPV1 activation. Answers to these questions should directly benefit the development of new clinical tools for treating TRPV1- mediated neuronal pain. PUBLIC HEALTH RELEVANCE: TRPV1 serves as a key molecular element in mediating low pH-induced neuronal pain due to inflammation, tissue damage and ischemia. Our research aims at understanding how TRPV1 activation is controlled by extracellular acidification and its relationship with heat- and agonist-induced channel activation, which will shed light on designing potential clinical intervention of pain.

Project Summary Capsaicin- and heat-activated TRPV1 ion channel is a primary nociceptor for both chemical and thermal stimuli, hence an attractive target for pain medication. Despite the availability of cryo-EM structures of TRPV1 at up-to-2.9 Å resolutions, molecular mechanisms underlying TRPV1 activation remains unclear. A major limitation for obtaining mechanistic information from the cryo-EM structures is the general lack of resolution to determine side-chain orientation and the associated atomic interaction. We recently demonstrated that the limitation could be overcome by combining Rosetta structural prediction with site-specific functional tests such as thermodynamic mutant cycle analysis that serve to constrain, validate, and improve structure prediction. Using this iterative approach, in combination with molecular dynamics (MD) simulation and site-specific fluorescence recordings including FRET and patch fluorometry, our proposed study aims to identify functional interactions in the capsaicin-binding domain and the outer pore and, more importantly, to reveal dynamic changes of these interactions during capsaicin- and heat-induced activation. We will take particular advantage of our newly designed fluorescent capsaicin analogs that allow us to directly monitor ligand binding, as well as the fluorescent unnatural amino acid (FUAA) incorporation method to introduce a small fluorophore to the channel's moving parts. Our goal is to elucidate key molecular interactions that mediate chemical and thermal activation, thus providing a molecular framework to guide pharmaceutical intervention.

Project Summary Capsaicin- and heat-activated TRPV1 ion channel is a primary nociceptor for both chemical and thermal stimuli, hence an attractive target for pain medication. Despite the availability of cryo-EM structures of TRPV1 at up-to-2.9 Å resolutions, molecular mechanisms underlying TRPV1 activation remains unclear. A major limitation for obtaining mechanistic information from the cryo-EM structures is the general lack of resolution to determine side-chain orientation and the associated atomic interaction. We recently demonstrated that the limitation could be overcome by combining Rosetta structural prediction with site-specific functional tests such as thermodynamic mutant cycle analysis that serve to constrain, validate, and improve structure prediction. Using this iterative approach, in combination with site-specific fluorescence recordings including FRET and patch fluorometry, our study aims to identify functional interactions in the capsaicin-binding domain and the outer pore and, more importantly, to reveal dynamic changes of these interactions during capsaicin- and heat-induced activation. We will take particular advantage of the fluorescent unnatural amino acid (FUAA) incorporation method to introduce a small fluorophore to the channel?s moving parts. Our goal is to elucidate structural mechanisms for key molecular interactions that mediate chemical and thermal activation, thus providing a molecular framework to guide pharmaceutical intervention.

Abstract Limbal stem cell deficiency (LSCD) is a major cause, either primary or secondary, of significant visual loss and blindness in many common corneal disorders. Transplantation of autologous limbal stem cells (LSCs) expanded in tissue culture has successfully restored vision and revolutionized the treatment of LSCD. A higher expansion efficiency of the stem/progenitor cell population in culture corresponds to a greater probability of long-term graft survival. The long-term goal of our study is to develop small molecules that can govern LSC self-renewal and differentiation and be used as therapeutic reagents for stem cell-based treatments of LSCD? related disorders. The most efficient expansion method requires feeder cells that provide a proper microenvironment to support the growth of LSCs. Among the external signaling that the feeder cells provide to the LSCs is the Wnt signaling. The central hypothesis of this project is that small molecules can be developed to mimic the Wnt proteins and activate Wnt signaling in the cells. We further propose that these small molecules will increase the efficiency of ex vivo expansion of functional human LSCs. The goal of this application is to use a structure-based drug discovery approach to develop potent Wnt mimics small molecule and to test their ability to increase the efficiency of ex vivo LSC expansion. Because the maintenance of stem cell characteristics in the process of culture expansion is essential for the success of ocular surface reconstruction, the small molecules generated in this project will serve as a platform for the development of novel pharmaceutical reagents for treating other corneal epithelial disorders.

Project Summary Heat- and capsaicin-activated ion channel TRPV1 is an important cellular sensor for maintaining normal body temperature and detecting noxious ambient environment, hence an important drug target. TRPV1 also serves as a prototypical model for understanding polymodal activation of diverse TRP channels. Structures of TRPV1 have been solved by cryo-electron microscopy (cyro-EM) at up-to-2.9- Å resolutions, yet mechanistic understanding of channel activation, in particular by heat, remains preliminary. A major limitation has been the lack of effective tools for probing structural dynamics in functional channels. We have recently identified two structurally related peptide toxins from the venom of Chinese red-headed centipede, termed RhTx1 and RhTx2, that exhibit highly specific and sub-?M affinity binding to the outer pore region of TRPV1. Interestingly, while RhTx1 strongly activates the channel, RhTx2, having just four additional amino acids at its N-terminus, is a potent inhibitor. Our preliminary study further suggested that RhTx1 specifically promotes heat activation. Because these small, compact peptide toxins can be readily synthesized and modified, they offer unique opportunities to probe the molecular mechanism underlying TRPV1 activation, with their rapid ON and OFF kinetics being particularly favorable for biophysical investigation. The proposed study will take advantage of these novel toxins to gain a better understanding of TRPV1 activation mechanism by revealing its structural dynamics using a multidisciplinary approach combines structural, electrophysiological, optical, and computational modeling methods.