Browsing by Subject "Ion channel"

Pain is defined by the international association of pain as an "unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage". Most people have experienced one form of pain or another and although such experiences can be unsavory, pain serves the basic need for the detection of dangerous stimuli that can cause bodily harm. Because pain serves such an essential need, it is important to understand how the nervous system processes and encodes noxious or potentially tissue damaging stimuli. This neural processing is called nociception.

In this study, I use Drosophila larvae as a genetic model organism to study nociception. In response to noxious thermal and mechanical stimuli, Drosophila larvae perform a nociceptive defensive behavior (termed nocifensive) where larvae rotate in a corkscrew like fashion along the long axis causing them to move in a lateral direction. Using this behavior and genetic tools which can manipulate neuronal output, we have identified the sensory neurons which serve as larval nociceptors as class IV multidendritic sensory neurons. Further characterization of these larval nociceptors, has also shown that they are both cholinergic and peptidergic.

After the identifying the larval nociceptors, I next identified several molecular components which are required for larval mechanical nociception. I have found that the degenerin epithelial sodium channel (DEG/ENaC) called pickpocket is required for larval mechanical nociception by using genetic mutants and RNAi knockdwon. In addition, after performing a screen using RNAi to knockdown ion channel transcripts in larval nociceptors, I have identified two other DEG/ENaC channels which are required for larval mechanical nociception. DEG/ENaCs are particularly interesting because they have been identified as candidate mechanotransducers in C. elegans for the gentle touch behavior. I propose that DEG/ENaCs may serve as candidate mechanotransducers in larval mechanical nociception because they are not generally required for neuronal excitability. However, future research will be required to establish their true role in mechanical nociceptive signaling.

In addition to DEG/ENaCs, transient receptor potential (TRP) channels also play a role in nociception. painless, a channel that was first identified in a thermal nociception screen on Drosophila larvae, is required for both thermal and mechanical nociception. The last section shows that multiple isoforms of painless exist and that these different isoforms may play different roles in thermal and mechanical nociception.

Taken together, these results have begun to establish Drosophila larva as a model for studying nociception. I have identified the sensory neurons used as larval nociceptors and shown that DEG/ENaC channels play an important role in larval mechanical nociception.

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.

Fibroblast growth factor homologous factors (FHFs) are non-canonical members of the fibroblast growth factor family (FGF11-14) that were initially discovered to bind and regulate neuronal and cardiac voltage-gated Na+ channels. Loss-of-function mutations that disrupt interaction between FHFs and Na+ channels cause spinocerebellar ataxias and cardiac arrhythmias such as Brugada syndrome. Although recent studies in brain of FHF knockout mice suggested novel functions for FHFs beyond ion channel modulation, it is unclear whether FHFs in the heart serve additional roles beyond regulating cardiac excitability. In this study, we performed a proteomic screen to identify novel interacting proteins for FGF13 in mouse heart. Mass spectrometry analysis revealed an interaction between FGF13 and a complex of cavin proteins that regulate caveolae, membrane invaginations that organize protective signaling pathways and provide a reservoir to buffer membrane stress. FGF13 controls the relative distribution of cavin 1 between the plasma membrane and cytosol and thereby acts as a negative regulator of caveolae. In inducible, cardiac-specific Fgf13 knockout mice, cavin 1 redistributed to the plasma membrane and stabilized the caveolar structural protein caveolin 3, leading to an increased density of caveolae. In a transverse aortic constriction model of pressure overload, this increased caveolar abundance enhanced cardioprotective signaling through the caveolar-organized PI3 kinase pathway, preserving cardiac function and reducing fibrosis. Additionally, the increased caveolar reserve provided mechanoprotection, as indicated by reduced membrane rupture in response to hypo-osmotic stress. Thus, our results establish FGF13 as a novel regulator of caveolae-mediated mechanoprotection and adaptive hypertrophic signaling, and suggest that inhibition of FHFs in the adult heart may have cardioprotective benefits in the setting of maladaptive hypertrophy.

Nucleoside transport is required for the salvage pathway of DNA and RNA, provides a transport route for nucleoside-like drugs and regulates adenosine signaling. The eukaryotic cell possesses two evolutionarily unrelated protein families of nucleoside transporters, the concentrative nucleoside transporters and the equilibrative nucleoside transporters. We aimed to elucidate their transport mechanisms using X-ray crystallography and biochemical assays. We solved multiple crystal structures of CNT along its transport cycle at 3.45-4.2 Å, which provide novel insights into the elevator-type transport mechanism of secondary active transporters.