Browsing by Author "Xiao, Yucheng"
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Item A-type FHFs mediate resurgent currents through TTX-resistant voltage-gated sodium channels(eLife Sciences, 2022-04-20) Xiao, Yucheng; Theile, Jonathan W.; Zybura, Agnes; Pan, Yanling; Lin, Zhixin; Cummins, Theodore R.; Biology, School of ScienceResurgent currents (INaR) produced by voltage-gated sodium channels are required for many neurons to maintain high-frequency firing and contribute to neuronal hyperexcitability and disease pathophysiology. Here, we show, for the first time, that INaR can be reconstituted in a heterologous system by coexpression of sodium channel α-subunits and A-type fibroblast growth factor homologous factors (FHFs). Specifically, A-type FHFs induces INaR from Nav1.8, Nav1.9 tetrodotoxin (TTX)-resistant neuronal channels, and, to a lesser extent, neuronal Nav1.7 and cardiac Nav1.5 channels. Moreover, we identified the N-terminus of FHF as the critical molecule responsible for A-type FHFs-mediated INaR. Among the FHFs, FHF4A is the most important isoform for mediating Nav1.8 and Nav1.9 INaR. In nociceptive sensory neurons, FHF4A knockdown significantly reduces INaR amplitude and the percentage of neurons that generate INaR, substantially suppressing excitability. Thus, our work reveals a novel molecular mechanism underlying TTX-resistant INaR generation and provides important potential targets for pain treatment.Item Advanced glycation end (AGE) product modification of laminin downregulates Kir4.1 in retinal Müller cells(PLOS, 2018-02-23) Thompson, Kayla; Chen, Jonathan; Luo, Qianyi; Xiao, Yucheng; Cummins, Theodore R.; Bhatwadekar, Ashay D.; Ophthalmology, School of MedicineDiabetic retinopathy (DR) is a major cause of adult blindness. Retinal Müller cells maintain water homeostasis and potassium concentration via inwardly rectifying Kir4.1 channels. Accumulation of advanced glycation end products (AGEs) is a major pathologic event in DR. While diabetes leads to a decrease in the Kir4.1 channels, it remains unknown whether AGEs-linked to the basement membrane (BM) affect normal Kir4.1 channels. For this study, we hypothesized that AGE-modification of laminin is detrimental to Kir4.1 channels, therefore, disrupting Müller cell function. The AGE-modified laminin-coated substrates were prepared by incubating Petri-dishes with laminin and methylglyoxal for seven days. The rat Müller cells (rMC-1) were propagated on AGE-modified laminin, and Kir4.1 expression and function were evaluated. Quantification of AGEs using ELISA revealed a dose-dependent increase in methylglyoxal-hydro-imidazolone adducts. The rMC-1 propagated on AGE-modified laminin demonstrated a decrease in Kir4.1 levels in immunofluorescence and western blot studies and a decrease in the Kir4.1 channel function. Kir4.1 decrease on AGE-modified laminin resulted in a disorganization of an actin cytoskeleton and disruption of α-dystroglycan-syntrophin-dystrophin complexes. Our studies suggest that AGE-modification of laminin is detrimental to Kir4.1 channels. By studying the role of AGEs in Kir4.1 channels we have identified a novel mechanism of Müller cell dysfunction and its subsequent involvement in DR.Item Cardiac sodium channel palmitoylation regulates channel availability and myocyte excitability with implications for arrhythmia generation(Nature Publishing Group, 2016-06-23) Pei, Zifan; Xiao, Yucheng; Meng, Jingwei; Hudmon, Andy; Cummins, Theodore R.; Department of Pharmacology and Toxicology, IU School of MedicineCardiac voltage-gated sodium channels (Nav1.5) play an essential role in regulating cardiac electric activity by initiating and propagating action potentials in the heart. Altered Nav1.5 function is associated with multiple cardiac diseases including long-QT3 and Brugada syndrome. Here, we show that Nav1.5 is subject to palmitoylation, a reversible post-translational lipid modification. Palmitoylation increases channel availability and late sodium current activity, leading to enhanced cardiac excitability and prolonged action potential duration. In contrast, blocking palmitoylation increases closed-state channel inactivation and reduces myocyte excitability. We identify four cysteines as possible Nav1.5 palmitoylation substrates. A mutation of one of these is associated with cardiac arrhythmia (C981F), induces a significant enhancement of channel closed-state inactivation and ablates sensitivity to depalmitoylation. Our data indicate that alterations in palmitoylation can substantially control Nav1.5 function and cardiac excitability and this form of post-translational modification is likely an important contributor to acquired and congenital arrhythmias.Item Characterization of Membrane Patch-Ion Channel Probes for Scanning Ion Conductance Microscopy(Wiley, 2018-05) Shi, Wenqing; Zeng, Yuhan; Zhu, Cheng; Xiao, Yucheng; Cummins, Theodore R.; Hou, Jianghui; Baker, Lane A.; Biology, School of ScienceIntegration of dual‐barrel membrane patch‐ion channel probes (MP‐ICPs) to scanning ion conductance microscopy (SICM) holds promise of providing a revolutionized approach of spatially resolved chemical sensing. A series of experiments are performed to further the understanding of the system and to answer some fundamental questions, in preparation for future developments of this approach. First, MP‐ICPs are constructed that contain different types of ion channels including transient receptor potential vanilloid 1 and large conductance Ca2+‐activated K+ channels to establish the generalizability of the methods. Next, the capability of the MP‐ICP platforms in single ion channel activity measurements is proved. In addition, the interplay between the SICM barrel and the ICP barrel is studied. For ion channels gated by uncharged ligands, channel activity at the ICP barrel is unaffected by the SICM barrel potential; whereas for ion channels that are gated by charged ligands, enhanced channel activity can be obtained by biasing the SICM barrel at potentials with opposite polarity to the charge of the ligand molecules. Finally, a proof‐of‐principle experiment is performed and site‐specific molecular/ionic flux sensing is demonstrated at single‐ion‐channel level, which show that the MP‐ICP platform can be used to quantify local molecular/ionic concentrations.Item The Diurnal Rhythm of Insulin Receptor Substrate-1 (IRS-1) and Kir4.1 in Diabetes: Implications for a Clock Gene Bmal1(ARVO, 2019-05) Luo, Qianyi; Xiao, Yucheng; Alex, Alpha; Cummins, Theodore R.; Bhatwadekar, Ashay D.; Ophthalmology, School of MedicinePurpose: Diabetes leads to the downregulation of the retinal Kir4.1 channels and Müller cell dysfunction. The insulin receptor substrate-1 (IRS-1) is a critical regulator of insulin signaling in Müller cells. Circadian rhythms play an integral role in normal physiology; however, diabetes leads to a circadian dysrhythmia. We hypothesize that diabetes will result in a circadian dysrhythmia of IRS-1 and Kir4.1 and disturbed clock gene function will have a critical role in regulating Kir4.1 channels. Methods: We assessed a diurnal rhythm of retinal IRS-1 and Kir4.1 in db/db mice. The Kir4.1 function was evaluated using a whole-cell recording of Müller cells. The rat Müller cells (rMC-1) were used to undertake in vitro studies using a siRNA. Results: The IRS-1 exhibited a diurnal rhythm in control mice; however, with diabetes, this natural rhythm was lost. The Kir4.1 levels peaked and troughed at times similar to the IRS-1 rhythm. The IRS-1 silencing in the rMC-1 led to a decrease in Kir4.1 and BMAL1. The insulin treatment of retinal explants upregulated Kir4.1 possibly via upregulation of BMAL1 and phosphorylation of IRS-1 and Akt-1. Conclusions: Our studies highlight that IRS-1, by regulating BMAL1, is an important regulator of Kir4.1 in Müller cells and the dysfunctional signaling mediated by IRS-1 may be detrimental to Kir4.1.Item FHF2 isoforms differentially regulate Nav1.6-mediated resurgent sodium currents in dorsal root ganglion neurons(Springer Nature, 2017-02) Barbosa, Cindy; Xiao, Yucheng; Johnson, Andrew J.; Xie, Wenrui; Strong, Judith A.; Zhang, Jun-Ming; Cummins, Theodore R.; Pharmacology and Toxicology, School of MedicineNav1.6 and Nav1.6-mediated resurgent currents have been implicated in several pain pathologies. However, our knowledge of how fast resurgent currents are modulated in neurons is limited. Our study explored the potential regulation of Nav1.6-mediated resurgent currents by isoforms of fibroblast growth factor homologous factor 2 (FHF2) in an effort to address the gap in our knowledge. FHF2 isoforms colocalize with Nav1.6 in peripheral sensory neurons. Cell line studies suggest that these proteins differentially regulate inactivation. In particular, FHF2A mediates long-term inactivation, a mechanism proposed to compete with the open-channel blocker mechanism that mediates resurgent currents. On the other hand, FHF2B lacks the ability to mediate long-term inactivation and may delay inactivation favoring open-channel block. Based on these observations, we hypothesized that FHF2A limits resurgent currents, whereas FHF2B enhances resurgent currents. Overall, our results suggest that FHF2A negatively regulates fast resurgent current by enhancing long-term inactivation and delaying recovery. In contrast, FHF2B positively regulated resurgent current and did not alter long-term inactivation. Chimeric constructs of FHF2A and Navβ4 (likely the endogenous open channel blocker in sensory neurons) exhibited differential effects on resurgent currents, suggesting that specific regions within FHF2A and Navβ4 have important regulatory functions. Our data also indicate that FHFAs and FHF2B isoform expression are differentially regulated in a radicular pain model and that associated neuronal hyperexcitability is substantially attenuated by a FHFA peptide. As such, these findings suggest that FHF2A and FHF2B regulate resurgent current in sensory neurons and may contribute to hyperexcitability associated with some pain pathologies.Item Gating-pore currents demonstrate selective and specific modulation of individual sodium channel voltage-sensors by biological toxins(ASPET, 2014-08) Xiao, Yucheng; Blumenthal, Kenneth; Cummins, Theodore R.; Pharmacology and Toxicology, School of MedicineVoltage-gated sodium channels are critical determinants of nerve and muscle excitability. Although numerous toxins and small molecules target sodium channels, identifying the mechanisms of action is challenging. Here we used gating-pore currents selectively generated in each of the voltage-sensors from the four α-subunit domains (DI-DIV) to monitor the activity of individual voltage-sensors and to investigate the molecular determinants of sodium channel pharmacology. The tarantula toxin huwentoxin-IV (HWTX-IV), which inhibits sodium channel current, exclusively enhanced inward gating-pore currents through the DII voltage-sensor. By contrast, the tarantula toxin ProTx-II, which also inhibits sodium channel currents, altered the gating-pore currents in multiple voltage-sensors in a complex manner. Thus, whereas HWTX-IV inhibits central-pore currents by selectively trapping the DII voltage-sensor in the resting configuration, ProTx-II seems to inhibit central-pore currents by differentially altering the configuration of multiple voltage-sensors. The sea anemone toxin anthopleurin B, which impairs open-channel inactivation, exclusively enhanced inward gating-pore currents through the DIV voltage-sensor. This indicates that trapping the DIV voltage-sensor in the resting configuration selectively impairs open-channel inactivation. Furthermore, these data indicate that although activation of all four voltage-sensors is not required for central-pore current generation, activation of the DII voltage-sensor is crucial. Overall, our data demonstrate that gating-pore currents can determine the mechanism of action for sodium channel gating modifiers with high precision. We propose this approach could be adapted to identify the molecular mechanisms of action for gating modifiers of various voltage-gated ion channels.Item Human Nav1.6 Channels Generate Larger Resurgent Currents than Human Nav1.1 Channels, but the Navβ4 Peptide Does Not Protect Either Isoform from Use-Dependent Reduction(Public Library of Science, 2015) Patel, Reesha R.; Barbosa, Cindy; Xiao, Yucheng; Cummins, Theodore R.; Department of Pharmacology and Toxicology, IU School of MedicineVoltage-gated sodium channels are responsible for the initiation and propagation of action potentials (APs). Two brain isoforms, Nav1.1 and Nav1.6, have very distinct cellular and subcellular expression. Specifically, Nav1.1 is predominantly expressed in the soma and proximal axon initial segment of fast-spiking GABAergic neurons, while Nav1.6 is found at the distal axon initial segment and nodes of Ranvier of both fast-spiking GABAergic and excitatory neurons. Interestingly, an auxiliary voltage-gated sodium channel subunit, Navβ4, is also enriched in the axon initial segment of fast-spiking GABAergic neurons. The C-terminal tail of Navβ4 is thought to mediate resurgent sodium current, an atypical current that occurs immediately following the action potential and is predicted to enhance excitability. To better understand the contribution of Nav1.1, Nav1.6 and Navβ4 to high frequency firing, we compared the properties of these two channel isoforms in the presence and absence of a peptide corresponding to part of the C-terminal tail of Navβ4. We used whole-cell patch clamp recordings to examine the biophysical properties of these two channel isoforms in HEK293T cells and found several differences between human Nav1.1 and Nav1.6 currents. Nav1.1 channels exhibited slower closed-state inactivation but faster open-state inactivation than Nav1.6 channels. We also observed a greater propensity of Nav1.6 to generate resurgent currents, most likely due to its slower kinetics of open-state inactivation, compared to Nav1.1. These two isoforms also showed differential responses to slow and fast AP waveforms, which were altered by the Navβ4 peptide. Although the Navβ4 peptide substantially increased the rate of recovery from apparent inactivation, Navβ4 peptide did not protect either channel isoform from undergoing use-dependent reduction with 10 Hz step-pulse stimulation or trains of slow or fast AP waveforms. Overall, these two channels have distinct biophysical properties that may differentially contribute to regulating neuronal excitability.Item Identification and Characterization of Novel Proteins from Arizona Bark Scorpion Venom That Inhibit Nav1.8, a Voltage-Gated Sodium Channel Regulator of Pain Signaling(MDPI, 2021-07-18) El-Aziz, Tarek Mohamed Abd; Xiao, Yucheng; Kline, Jake; Gridley, Harold; Heaston, Alyse; Linse, Klaus D.; Ward, Micaiah J.; Rokyta, Darin R.; Stockand, James D.; Cummins, Theodore R.; Fornelli, Luca; Rowe, Ashlee H.; Biology, School of ScienceThe voltage-gated sodium channel Nav1.8 is linked to neuropathic and inflammatory pain, highlighting the potential to serve as a drug target. However, the biophysical mechanisms that regulate Nav1.8 activation and inactivation gating are not completely understood. Progress has been hindered by a lack of biochemical tools for examining Nav1.8 gating mechanisms. Arizona bark scorpion (Centruroides sculpturatus) venom proteins inhibit Nav1.8 and block pain in grasshopper mice (Onychomys torridus). These proteins provide tools for examining Nav1.8 structure-activity relationships. To identify proteins that inhibit Nav1.8 activity, venom samples were fractioned using liquid chromatography (reversed-phase and ion exchange). A recombinant Nav1.8 clone expressed in ND7/23 cells was used to identify subfractions that inhibited Nav1.8 Na+ current. Mass-spectrometry-based bottom-up proteomic analyses identified unique peptides from inhibitory subfractions. A search of the peptides against the AZ bark scorpion venom gland transcriptome revealed four novel proteins between 40 and 60% conserved with venom proteins from scorpions in four genera (Centruroides, Parabuthus, Androctonus, and Tityus). Ranging from 63 to 82 amino acids, each primary structure includes eight cysteines and a "CXCE" motif, where X = an aromatic residue (tryptophan, tyrosine, or phenylalanine). Electrophysiology data demonstrated that the inhibitory effects of bioactive subfractions can be removed by hyperpolarizing the channels, suggesting that proteins may function as gating modifiers as opposed to pore blockers.Item Increased Resurgent Sodium Currents in Nav1.8 Contribute to Nociceptive Sensory Neuron Hyperexcitability Associated with Peripheral Neuropathies(Society for Neuroscience, 2019-02-20) Xiao, Yucheng; Barbosa, Cindy; Pei, Zifan; Xie, Wenrui; Strong, Judith A.; Zhang, Jun-Ming; Cummins, Theodore R.; Biology, School of ScienceNeuropathic pain is a significant public health challenge, yet the underlying mechanisms remain poorly understood. Painful small fiber neuropathy (SFN) may be caused by gain-of-function mutations in Nav1.8, a sodium channel subtype predominantly expressed in peripheral nociceptive neurons. However, it is not clear how Nav1.8 disease mutations induce sensory neuron hyperexcitability. Here we studied two mutations in Nav1.8 associated with hypersensitive sensory neurons: G1662S reported in painful SFN; and T790A, which underlies increased pain behaviors in the Possum transgenic mouse strain. We show that, in male DRG neurons, these mutations, which impair inactivation, significantly increase TTX-resistant resurgent sodium currents mediated by Nav1.8. The G1662S mutation doubled resurgent currents, and the T790A mutation increased them fourfold. These unusual currents are typically evoked during the repolarization phase of action potentials. We show that the T790A mutation greatly enhances DRG neuron excitability by reducing current threshold and increasing firing frequency. Interestingly, the mutation endows DRG neurons with multiple early afterdepolarizations and leads to substantial prolongation of action potential duration. In DRG neurons, siRNA knockdown of sodium channel β4 subunits fails to significantly alter T790A current density but reduces TTX-resistant resurgent currents by 56%. Furthermore, DRG neurons expressing T790A channels exhibited reduced excitability with fewer early afterdepolarizations and narrower action potentials after β4 knockdown. Together, our data demonstrate that open-channel block of TTX-resistant currents, enhanced by gain-of-function mutations in Nav1.8, can make major contributions to the hyperexcitability of nociceptive neurons, likely leading to altered sensory phenotypes including neuropathic pain in SFN.SIGNIFICANCE STATEMENT This work demonstrates that two disease mutations in the voltage-gated sodium channel Nav1.8 that induce nociceptor hyperexcitability increase resurgent currents. Nav1.8 is crucial for pain sensations. Because resurgent currents are evoked during action potential repolarization, they can be crucial regulators of action potential activity. Our data indicate that increased Nav1.8 resurgent currents in DRG neurons greatly prolong action potential duration and enhance repetitive firing. We propose that Nav1.8 open-channel block is a major factor in Nav1.8-associated pain mechanisms and that targeting the molecular mechanism underlying these unique resurgent currents represents a novel therapeutic target for the treatment of aberrant pain sensations.