Browsing by Author "Grandl, Jorg"

Olfaction is one of the most primitive of our senses, and the olfactory receptors that mediate this very important chemical sense comprise the largest family of genes in the mammalian genome. It is therefore surprising that we understand so little of how olfactory receptors work. In particular we have a poor idea of what odorous chemicals are detected by most of the olfactory receptors in the genome, and for those receptors which we have paired with ligands, we know relatively little about how the structure of these ligands can either activate or inhibit the activation of these receptors. Furthermore the large repertoire of olfactory receptors, which belong to the G protein coupled receptor (GPCR) superfamily, can serve as a model to contribute to our broader understanding of GPCR- ligand binding, especially since GPCRs are important pharmaceutical targets.

In this dissertation, I explore the relationship between olfactory receptors and their ligands, both by manipulating the ligands presented to the olfactory receptors, as well as by altering the structure of the receptor itself by mutagenesis. Here we report the probable requirement of a hydrated germinal-diol form of octanal for activation of the rodent OR-I7 receptor by ligand manipulation, and the successful in vitro modeling and manipulation of ketamine binding to MOR136-1. We also report the results of a large-scale screen of 1190 human and mouse olfactory receptors for receptors activated by volatile general anesthetics, which has lead to the identification of 32 olfactory receptor-volatile general anesthetic pairs.

Mechanosensation, or the ability to sense mechanical forces, is critical for the survival of all organisms. For vertebrates, the ability to sense and respond to environmental stimuli, or somatosensation, is not well understood at the neural circuit level, and the molecular underpinnings for somatosensation, especially in regards to mechanosensation, remain elusive and unresolved. In recent years, Piezo ion channels were discovered as the first mammalian excitatory bona fide mechanosensitive ion channel to be the initial molecules in sensing gentle touch in the somatosensory neural circuitry. Although physiological roles of Piezo ion channels for both somatosensation and other non-neuronal processes have been identified, the mechanisms for how these ion channels directly sense mechanical forces and transduce electrical signals remains unknown.

With the use of biochemical, molecular, and electrophysiological methods, I first developed a novel technique in which electrophysiology could be performed on microsomes, or vesicles formed from ER fractions containing proteins that are not trafficked to the plasma membrane. This experiment revealed that wild-type Piezo1 ion channels retain stretch activation in microsomes, and therefore this technique could be utilized to characterize mutants channels that lack trafficking to the plasma membrane in order to identify which domains are involved in activation and inactivation of Piezo ion channels. I next generated two separate constructs that removed domains suggested to be involved in activation and inactivation of Piezo1. Removal of the proposed inactivation domain rendered a non-functional channel that could still trimerize, suggesting that this domain not only plays a role in inactivation but also is critical for activation of Piezo1 ion channels. Partial removal of the proposed membrane-spanning mechanosensor domains produced a channel that lacked the ability to conduct large macroscopic currents but formed a conducting pore with single channel openings. This finding suggests that membrane tension is not sensed and transduced to the pore by a single domain, but rather multiple domains in concert. Together the findings provide evidence that the mechanisms for both activation and inactivation require multiple domains moving in collaboration together, and will broadly be informative for continued studies of the molecular mechanisms of mechanosensitive ion channels.

An organism’s ability to sense mechanical forces is critical for the detection of environmental stimuli as well as the regulation of internal processes necessary for survival. In vertebrates, the molecular mechanisms of somatosensation has remained an important and unresolved neurobiological question. In the past decade, Piezo ion channels have emerged as the first vertebrate ion channels identified responsible for transforming somatosensory stimuli into excitatory signals in the nervous system, generating our sense of touch. However, little still is known about the precise mechanisms for how Piezo channels activate and inactivate in the presence of stimuli and how they could potentially be modulated.

By using a combination of engineered and biomolecular methods combined with electrophysiology, I identify distinct structural domains within Piezo ion channels that are necessary for specific aspects of channel inactivation. First, I engineered a method by which magnetic nanoparticles were used to mechanically pull on individual domains of the Piezo channel to screen for mechanically sensitive structures. This experiment revealed a particularly striking effect for one domain, manifested as a profound slowing of channel inactivation kinetics. Next, I generated chimeric constructs to exchange this domain with a homologous structure and demonstrated its sufficiency for mediating the kinetics for inactivation. Finally, I introduced point mutations at key residues within the immediately adjacent pore domain and identified a structural correlate for the modulation of inactivation by voltage. These findings together provide a foundation for understanding the mechanism for inactivation in Piezo channels, and more broadly, for further studying the complexities in transducing mechanical force that create our sense of touch.

Organisms need to sense temperature to avoid detrimental damage to cells and tissues. In mammals, this is thought to be mediated, at least in part, by several members of the transient receptor potential (TRP) superfamily of ion channels. TRP channels are outwardly rectifying channels with 6 transmembrane segments that assemble as a tetramer. Some TRP channels are activated by cold or heat, chemicals, and depolarizing voltages. Temperature sensitive TRP channels are expressed in a variety of cell types including keratinocytes and medium- to small-diameter nociceptors where they are involved in the detection of noxious chemicals, inflammatory mediators, and temperature. In response to noxious stimuli, TRP channels mediate the depolarization of nociceptors ultimately leading to the perception of pain. Due to their critical role in nociception they are excellent candidates for the development of analgesic drugs that can be used as treatments for different pain modalities. However, drugs that target these ion channels have many unwanted side effects that include hypo or hyperthermia. Clearly, a thorough understanding of the structures and mechanism that mediate temperature activation of TRP channels is needed for the clever development of novel drugs that do not evoked these side effects.

Although countless studies have tried to identify the structures and mechanisms that confer temperature sensitivity to TRP channels, no consensus about these have been attained. One recent proposed mechanism assumes that temperature activation is driven by the exposure of hydrophobic residues to solvent. This mechanism further predicts that residues are exposed to solvent in a coordinated way, but without necessarily being near each other. However, there is little experimental evidence supporting this mechanism in TRP channels. Here I tackle these questions using a variety of approaches: First, I tested the sufficiency of the pore domain of TRPV1 towards temperature sensitivity using minimal ‘pore-only’ channels, but found that my minimalistic approach does not yield functional channels. I then tested the sufficiency of the entire ankyrin repeat 6, or single-point mutation on the same repeat of drosophila TRPA1 for inverting the temperature directionality of the channel, but found that the structure was not sufficient to make heat-activated drosophila TRPA1 cold sensitive. Lastly, I took a combinatorial approach and used random mutagenesis, coupled to high-throughput screening and massive parallel sequencing to identify and characterize mutations from ~7,300 randomly mutated TRPV1 clones. I found that residues important for temperature activation are randomly spread throughout the entire sequence of the channel indicating that temperature does not activate the channel by acting on a single coherent domain, but rather in the entire protein. This implies that it will be very complicated to develop analgesic drugs that do not affect the temperature activation mechanism since residues throughout the protein are involved in temperature sensitivity. Additionally, I found that large decreases in hydrophobicity of amino acids are better tolerated for activation by capsaicin than for activation by hot temperature, suggesting that strong hydrophobicity might be specifically required for temperature activation. This provides initial support for a previously hypothesized temperature activation mechanism involving amino acid hydrophobicity in TRP ion channels.

Cells throughout the human body detect mechanical forces. While it is known that the rapid (millisecond) detection of mechanical forces is mediated by force-gated ion channels, technological limitations have precluded a detailed quantitative understanding of cells as sensors of mechanical energy. Here, I describe the development and validation of a system combining atomic force microscopy with patch-clamp electrophysiology. Using this system, I develop a quantitative framework for describing cells as sensors of mechanical energy and determine the physical limits of detection and resolution of mechanical energy for cells expressing the force-gated ion channels Piezo1, Piezo2, TREK1, and TRAAK. I find that, depending on the identity of the channel, cells can function either as proportional or nonlinear transducers of mechanical energy, detect mechanical energies as little as ~70 fJ, and with a resolution of up to ~1 fJ. I also make the surprising discovery that cells can transduce forces either nearly instantaneously (< 1ms), or with substantial time delay (~10 ms) dependent on the identity of the channel. Using a chimeric experimental approach and simulations we show how such delays can emerge from channel-intrinsic properties and the slow diffusion of tension in cellular membranes. Further, I explore the role that cellular properties such as cell size, channel density, and actin cytoarchitecture play in tuning the biophysical limits of rapid cellular mechanotransduction. Overall, our experiments reveal the capabilities and limits of cellular mechanosensing and provide insights into molecular mechanisms that different cell types may employ to specialize for their distinct physiological roles.