Browsing by Subject "Ion channels"

The non-secreted fibroblast growth factor (FGF) homologous factor (FHF) FGF13 is a noncanonical FGF with identified roles in neuronal development, pain sensation, and cardiac physiology, but recent reports suggest broader roles. The in vivo functions of FGF13 have not been widely studied. In this study, we have generated a global heterozygous Fgf13 knockout mouse model. In these animals, we observed hyperactivity and accompanying reduced core body temperature in mice housed at 22 °C. In mice housed at 30 °C (thermoneutrality) we observed development of a pronounced obesity. Defects in thermogenesis and metabolism were found to be due to impaired central nervous system regulation of sympathetic activation of brown fat. Neuronal and hypothalamic specific ablation of Fgf13 recapitulated weight gain at 30 °C. In global heterozygous animals, norepinephrine turnover in brown fat was reduced at both housing temperatures, while direct activation of brown fat by a β3 agonist showed an intact response. Further, we found that FGF13 is a direct regulator of NaV1.7, a hypothalamic Na+ channel associated with regulation of body weight. Our data expand the physiologic roles for FGF13, and enhance the understanding of the multifunctional FHFs.

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.

Nociceptors are specialized primary sensory neurons that represent the first line of defense against potentially tissue damaging environmental stimuli, and are involved in pathological pain states caused by nerve damage, inflammation and many chronic diseases. In nociception, these neurons detect harmful stimuli and contribute to the reactions to avoid them. Nociceptors transduce noxious stimuli into membrane depolarization, which in turn, triggers action potentials. These action potentials are conducted to synapses in the central nervous system (CNS), resulting in release of neurotransmitters at the presynaptic terminal. The unifying factor in the progression of nociceptive signaling i.e. transduction, action potential propagation, and neurotransmitter release, is the contribution of ion channels.

In this study, I use Drosophila melanogaster larvae as a model system to study the contribution of ion channels to nociception. Larvae stimulated with a noxious thermal or mechanical stimulus perform a stereotyped and quantifiable escape behavior. Larvae exhibiting this nocifensive behavior rotate around their long body axis in a corkscrew-like manner thus escaping the damage of the noxious stimulus. This behavior is triggered by the Class IV multidendritic (md) neurons, which are the main larval nociceptors. I describe here, the results of my systematic screen for ion channels required for larval thermal nociception. To perform this screen, I utilized RNAi to knock down the expression of 98% of the predicted ion channels in the Drosophila genome. I observed the effects of ion channel knockdown in the thermal nociception behavioral assay.

In addition, I present detailed characterization of an ion channel that I found to be critical for inhibition of nociceptor excitability, the small conductance calcium-activated potassium channel, SK. This channel inhibits both thermal and mechanical nociception. Results of calcium imaging studies show enhanced excitability of larval nociceptors in SK mutant animals. My findings support a role for SK function at the sensory afferents, cell body, and axon.

Another candidate ion channel gene, shadrach, encodes a Degenerin/Epithelial Na+ channel (DEG/ENaC) that I found to be required for thermal nociception. DEG/ENaCs are conserved in flies, nematodes, and several vertebrates including humans. These channels are expressed in a variety of tissues including kidney epithelia, muscle, and neurons. Members of this superfamily play a role in a host of biological processes including salt homeostasis, neurodegeneration, proprioception, touch transduction, and nociception. RNAi knockdown of shadrach results in increased thermal nociceptive threshold. Optogenetic experiments suggest that shadrach functions downstream of transduction.

Furthermore, I identified seven ion channel genes in the thermal nociception screen, which affect nociceptor dendrite morphology. It is possible that thermal nociception behavioral phenotypes in these RNAi mutants are a consequence of the altered dendritic field. Reduction in segmental coverage by the nociceptors may influence the ability to detect noxious stimuli. Future research in our laboratory will establish the relationship between these ion channels, nociceptor development, and nociceptive behavioral output.

Drosophila melanogaster is emerging as a powerful model for the study of pain signaling. I have uncovered several candidate ion channel genes that contribute to thermal nociception; of these, SK and shadrach are required for the response to noxious heat. I have shown that dendritic field coverage is important for the detection of noxious stimuli, and I have identified many candidate genes that are required for normal dendrite morphology.

Disruption of coordinated impulse propagation in the heart as a result of fibrosis or myocardial infarction can create an asynchronous substrate with poor conduction and impaired contractility. This can ultimately lead to cardiac failure and make the heart more vulnerable to life-threatening arrhythmias and sudden cardiac death. The transplantation of exogenous cells into the diseased myocardium, "cardiac cell therapy," has been proposed as a treatment option to improve compromised cardiac function. Clinical trials of stem cell-based cardiac therapy have shown promising results, but also raised concerns about our inability to predict or control the fate of implanted cells and the electrical consequences of their interactions with host cardiomyocytes. Alternatively, genetically engineered somatic cells could be implanted to selectively and safely modify the cardiac electrical substrate, but their unexcitable nature makes them incapable of electrically repairing large conduction defects. The objective of this thesis was thus to develop a methodology to generate actively conducting excitable cells from an unexcitable somatic cell source and to demonstrate their utility for studies of basic electrophysiology and cardiac cell therapy.

First, based on the principles of cardiac action potential propagation, we applied genetic engineering techniques to convert human unexcitable cells (HEK-293) into an autonomous source of excitable and conducting cells by the stable forced expression of only three genes encoding an inward rectifier potassium (Kir2.1), a fast sodium (Na_v1.5), and a gap junction (Cx43) channel. Systematic pharmacological and electrical pacing studies in these cells revealed the individual contributions of each expressed channel to action potential shape and propagation speed. Conduction slowing and instability of induced arrhythmic activity was shown to be governed by specific mechanisms of I_Na inhibition by TTX, lidocaine, or flecainide. Furthermore, expression of the Na_v1.5 A1924T mutant sodium channel or Ca_v3.3 T-type calcium channel was utilized to study the specific roles of these channels in action potential conduction and demonstrate that genetic modifications of the engineered excitable cells in this platform allow quantitative correlations between single-cell patch clamp data and tissue-level function.

We further performed proof-of-concept experiments to show that networks of biosynthetic excitable cells can successfully repair large conduction defects within primary excitable tissue cultures. Specifically, genetically engineered excitable cells supported active action potential propagation between neonatal rat ventricular myocytes (NRVMs) separated by at least 2.5 cm in 2-dimensional and 1.3 cm in 3-dimensional cocultures. Using elastic films with micropatterned zig-zag NRVM networks that mimicked the tortuous conduction patterns observed in cardiac fibrosis, we showed that electrical resynchronization of cardiomyocyte activation by application of engineered excitable cells improved transverse conduction by 370% and increased cardiac twitch force amplitude by 64%. This demonstrated that despite being noncontractile, engineered excitable cells could potentially improve both the electrical and mechanical function of diseased myocardial tissue.

Lastly, we investigated how activation and repolarization gradients at the interface between cardiomyocytes and other excitable cells influence the vulnerability to conduction block. Microscopic optical mapping of action potential propagation was used to quantify dispersion of repolarization (DOR) in micropatterned heterocellular strands in which either well-coupled or poorly-coupled engineered excitable cells with a short action potential duration (APD), seamlessly interfaced with NRVMs that had a significantly longer APD. The resulting electrical gradients originating from the underlying heterogeneity in intercellular coupling and APD dispersion were further manipulated by the application of barium chloride (BaCl2) to selectively prolong APD in the engineered cells. We measured how the parameters of DOR affected the vulnerable time window (VW) of conduction block and found a strong linear correlation between the size of the repolarization gradient and VW. Reduction of DOR by BaCl2 significantly reduced VW and showed that VW correlated directly with dispersion height but not width. Conversely, at larger DOR, VW was inversely correlated with the dispersion width but independent of the dispersion height. In addition, despite their similar APDs, poorly-coupled excitable cells were found to significantly increase the maximum repolarization gradient and VW compared to well-coupled excitable cells, but only at larger DOR.

In summary, this thesis presents the novel concept of genetically engineering membrane excitability and impulse conduction in previously unexcitable somatic cells. This biosynthetic excitable cell platform is expected to enable studies of ion channel function in a reproducible tissue-level setting, promote the integration of theoretical and experimental studies of action potential propagation, and stimulate the development of novel gene and cell-based therapies for myocardial infarction and cardiac arrhythmias.

Transmembrane protein 16 (TMEM16) is a novel family of transmembrane proteins that function either as ion channels, lipid scramblases or both. In mammals, the majority of TMEM16 members are Ca2+-dependent phospholipid scramblases (CaPLSases) that catalyze bidirectional movement of phospholipids across the membrane bilayer. Interestingly, some of these TMEM16 CaPLSases can also conduct ions, making them multifunctional (moonlighting) transporters. These moonlighting TMEM16 members have been linked to various physiological and pathological conditions, such as blood coagulation, ataxia, muscle dystrophy, cell-cell fusion and viral infection.To further understand their biology and design therapeutics to treat the related diseases, it is urgent to unveil the structures, machineries as well as pharmacological profiles of the multifunctional TMEM16 proteins. However, studying TMEM16 proteins has been challenging due to their unique structural topologies and biophysical properties. Despite the recent progress in the structure and function understanding of the TMEM16 family, how the moonlighting TMEM16s gate and distinguish different permeating substrates remain open questions. To resolve these unknowns and contribute to a more comprehensive understanding of the multifunctional TMEM16 proteins, this dissertation focuses on investigating the molecular mechanisms of TMEM16F – the first identified moonlighting member of the TMEM16 family. We first developed a sensitive and reliable fluorescence microscopy-based scrambling assay that can be either used alone to assess TMEM16F CaPLSase activity or combined with electrophysiology to simultaneously examine TMEM16F CaPLSase and ion channel components (Chapter 2). Next, by applying our optimized scrambling assay together with computational simulation, mutagenesis screening and electrophysiology approaches, we uncovered the gating mechanism of TMEM16F and revealed the differences in protein conformation between TMEM16 -CaPLSases and -ion channels (Chapter 3). Furthermore, during our drug screening to identify antagonists for TMEM16F CaPLSase, we made a surprising discovery about the potential pitfalls of using fluorescence-based assay that could cause false positive results and challenge the identification of bona fide inhibitors for the CaPLSases (Chapter 4). Finally, our discovery of Subdued – a TMEM16 fly homolog – as a new moonlighting protein with similar biophysical properties to those of TMEM16F further expands our knowledge about the diversity and relationship among TMEM16 members (Chapter 5). In summary, this dissertation advances the current understanding of the molecular underpinning and diverse functions of the TMEM16 family in general, and TMEM16F in particular.

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.

Action potential firing in various excitable cells (cardiomyocytes, neurons, smooth and skeletal muscle cells) is enabled by the presence of inwardly rectifying potassium channels, such as Kir2.1, which set the resting membrane potential to a negative value allowing the activation of depolarizing voltage-gated currents. In the heart, Kir2.1-mediated IK1 current is upregulated during development, but downregulated in certain pathologies, including myocardial infarction, heart failure, and the Anderson-Tawil syndrome, a congenital disease characterized by periodic paralysis and polymorphic tachycardias. Such electrophysiological changes in the heart are often associated with profound alterations in cardiomyocyte size and shape, extracellular matrix (ECM), as well as cytoskeletal tension. Cardiomyocytes sense physical changes in their environment through integrins, heterodimeric receptors for ECM proteins that relay information to focal adhesions (FAs), force-sensitive sub-cellular structures connected to actin cytoskeleton. In different systems, dynamic changes in FA proteins as well as current flow through ion channels, including Kir2.1, are known to govern important cellular processes, including survival, proliferation, differentiation, migration, and matrix remodeling. Still, relationships between ion channels, FAs, and mechanosensitive cell electrophysiology are not well-understood. The objective of this thesis has been to explore how changes in integrin engagement and FA assembly within excitable cells affect Kir2.1 membrane localization, IK1 amplitude, and action potential characteristics. We hypothesized that integrin engagement will increase the number of active Kir2.1 channels to the membrane, thus increasing IK1 amplitude and changing action potential characteristics.

To accomplish this objective, we utilized a monoclonal line of HEK293 cells engineered to express fluorescently tagged Kir2.1 to visualize channels, a monoclonal line of HEK293 cells (“Ex293”) engineered to express Kir2.1, cardiac sodium channel Nav1.5, and gap junctional channel Connexin-43 as a well-defined excitable cell source, and neonatal rat cardiomyocytes as native excitable cells. Using microfabrication techniques, we created a platform for robustly and precisely controlling cell shape and size. Combining this technique with single cell electrophysiology and quantitative image analysis, we characterized local and global membrane distributions of Kir2.1, IK1 amplitude, and FAs. We established that membrane-bound Kir2.1 localizes in proximity to FAs, giving a non-uniform distribution of IK1 in the cell membrane.

Next we applied micropatterning of ECM proteins, pharmacological, and environmental manipulations to alter FA size and distribution in individual cells. Combining these techniques with single cell electrophysiology, confocal and total internal reflection fluorescence (TIRF) microscopy, and quantitative image analysis, we provide evidence that FAs and active integrins play a critical role in regulating Kir2.1 membrane localization, IK1 amplitude, and action potential morphology in excitable cells.

Furthermore, by studying Kir2.1 turnover dynamics using fluorescence recovery after photobleaching (FRAP), we show that the channels are uniformly transported to the membrane, where they preferentially accumulate near FA-rich sites via the local inhibition of dynamin-mediated endocytosis. We conclude this thesis with a modeling summary of the Kir2.1 trafficking and localization near FAs at the membrane.

Overall, this thesis shows links between the electrophysiology of ion channels and FA biology, coupling action potential dynamics to changes in the cellular environment and cytoskeletal mechanics. Our results propose a novel mechanism whereby engaged integrins are an important regulator of the membrane localization of Kir2.1 channels via the local inhibition of dynamin-dependent endocytosis. This mechanism renders the IK1 and cellular electrophysiology indirectly mechanosensitive to various intra- and extracellular signals affecting integrin engagement and FA dynamics. The work in this thesis warrants future in-depth studies of how cell-matrix interactions modulate the function of various ion channels across diverse cell types and pathophysiological conditions.