[Show abstract][Hide abstract] ABSTRACT: A-type Kv4 potassium channels undergo a conformational change toward a nonconductive state at negative membrane potentials, a dynamic process known as pre-open closed states or closed-state inactivation (CSI). CSI causes inhibition of channel activity without the prerequisite of channel opening, thus providing a dynamic regulation of neuronal excitability, dendritic signal integration, and synaptic plasticity at resting. However, the structural determinants underlying Kv4 CSI remain largely unknown. We recently showed that the auxiliary KChIP4a subunit contains an N-terminal Kv4 inhibitory domain (KID) that directly interacts with Kv4.3 channels to enhance CSI. In this study, we utilized the KChIP4a KID to probe key structural elements underlying Kv4 CSI. Using fluorescence resonance energy transfer two-hybrid mapping and bimolecular fluorescence complementation-based screening combined with electrophysiology, we identified the intracellular tetramerization (T1) domain that functions to suppress CSI and serves as a receptor for the binding of KID. Disrupting the Kv4.3 T1-T1 interaction interface by mutating C110A within the C3H1 motif of T1 domain facilitated CSI and ablated the KID-mediated enhancement of CSI. Furthermore, replacing the Kv4.3 T1 domain with the T1 domain from Kv1.4 (without the C3H1 motif) or Kv2.1 (with the C3H1 motif) resulted in channels functioning with enhanced or suppressed CSI, respectively. Taken together, our findings reveal a novel (to our knowledge) role of the T1 domain in suppressing Kv4 CSI, and that KChIP4a KID directly interacts with the T1 domain to facilitate Kv4.3 CSI, thus leading to inhibition of channel function.
[Show abstract][Hide abstract] ABSTRACT: The specific binding of auxiliary Kv channel-interacting proteins (KChIPs) to the N terminus of Kv4 pore-forming α-subunits results in modulation of gating properties, surface expression, and subunit assembly of Kv4 channels. However, the interactions between KChIPs and Kv4 remain elusive. Thus, affinity capillary electrophoresis (ACE) was employed to quantitatively evaluate the interactions between KChIPs and Kv4.3 N terminus (KvN) and between KChIP4a/related mutants and Ca2+ for the first time. The mobility ratio, derivatives calculated from the mobility shift method, was used to deduce the binding constants (Kb). As a result, the binding constants for KChIP4a/KvN and KChIP1/KvN complexes were (8.32 ± 1.66) × 106 L mol–1 and (5.26 ± 0.71) × 106 L mol–1, respectively. In addition, in the presence of calcium (10 μmol L–1), the binding constant of KChIP4a/KvN increased to (6.72 ± 1.66) × 107 L mol–1. In addition, the binding constant of KChIP4a with Ca2+ was (7.1 ± 1.5) × 107 L mol–1. Besides, studies on the effect of truncated mutants revealed that the third EF hand of KChIP4a was related to high-affinity binding with Ca2+, and the integrity of the molecular structure of KChIP4a was important for Ca2+ binding. This method profits from small samples, rapid analysis, and simple operation without being time-consuming.
[Show abstract][Hide abstract] ABSTRACT: Pain in masticatory muscles is among the most prominent symptoms of temperomandibular disorders (TMDs) that have diverse and complex etiology. A common complaint of TMD is that unilateral pain of craniofacial muscle can cause a widespread of bilateral pain sensation, although the underlying mechanism remains unknown. To investigate whether unilateral inflammation of masseter muscle can cause a bilateral allodynia, we generated masseter muscle inflammation induced by unilateral injection of complete Freund's adjuvant (CFA) in rats, and measured the bilateral head withdrawal threshold at different time points using a von Frey anesthesiometer. After behavioral assessment, both right and left trigeminal ganglia (TRG) were dissected and examined for histopathology and transient receptor potential vanilloid 1 (TRPV1) mRNA expression using quantitative real-time PCR analysis. A significant increase in TRPV1 mRNA expression occurred in TRG ipsilateral to CFA injected masseter muscle, whereas no significant alteration in TRPV1 occurred in the contralateral TRG. Interestingly, central injection of TRPV1 antagonist 5-iodoresiniferatoxin into the hippocampus significantly attenuated the head withdrawal response of both CFA injected and non-CFA injected contralateral masseter muscle. Our findings show that unilateral inflammation of masseter muscle is capable of inducing bilateral allodynia in rats. Upregulation of TRPV1 at the TRG level is due to nociception caused by inflammation, whereas contralateral nocifensive behavior in masticatory muscle nociception is likely mediated by central TRPV1, pointing to the involvement of altered information processing in higher centers.
[Show abstract][Hide abstract] ABSTRACT: The specific binding of auxiliary Kv channel-interacting proteins (KChIPs) to the N-teriminus of Kv4 pore-forming -subunits results in modulation of gating properties, surface expression and subunit assembly of Kv4 channels. However, the interactions between KChIPs and Kv4 remain elusive. Thus, ACE was employed to quantitatively evaluate the interactions between KChIPs and Kv4.3 N-teriminus (KvN) and between KChIP4a/related mutants and Ca(2+)for the first time. The mobility ratio, derivatives calculated from mobility shift method, was used to deduce the binding constants (Kb). As a result, the binding constants for KChIP4a/KvN and KChIP1/KvN complexes were (8.32 ± 1.66) × 10(6) L mol(-1) and (5.26 ± 0.71) × 10(6) L mol(-1), respectively. And in the presence of calcium (10 μmol L(-1)), the binding constant of KChIP4a/KvN increased to (6.72 ± 1.66) × 10(7) L mol(-1). In addition, the binding constant of KChIP4a with Ca(2+) was (7.1 ± 1.5) × 10(7) L mol(-1). Besides, studies on the effect of structure's truncated mutants revealed that the third EF-hand of KChIP4a was related to high affinity binding with Ca(2+), and the integrity of molecular structure of KChIP4a was important for Ca(2+) binding. This method profits from small samples, rapid analysis and simple operation without time consuming.
[Show abstract][Hide abstract] ABSTRACT: Divalent cations Mg(2+) and Ba(2+) selectively and directly potentiate transient receptor potential vanilloid type 1 heat activation by lowering the activation threshold into the room temperature range. We found that Mg(2+) potentiates channel activation only from the extracellular side; on the intracellular side, Mg(2+) inhibits channel current. By dividing the extracellularly accessible region of the channel protein into small segments and perturbing the structure of each segment with sequence replacement mutations, we observed that the S1-S2 linker, the S3-S4 linker, and the pore turret are all required for Mg(2+) potentiation. Sequence replacements at these regions substantially reduced or eliminated Mg(2+)-induced activation at room temperature while sparing capsaicin activation. Heat activation was affected by many, but not all, of these structural alternations. These observations indicate that extracellular linkers and the turret may interact with each other. Site-directed fluorescence resonance energy transfer measurements further revealed that, like heat, Mg(2+) also induces structural changes in the pore turret. Interestingly, turret movement induced by Mg(2+) precedes channel activation, suggesting that Mg(2+)-induced conformational change in the extracellular region most likely serves as the cause of channel activation instead of a coincidental or accommodating structural adjustment.
The Journal of General Physiology 12/2013; 143(1). DOI:10.1085/jgp.201311024 · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transient receptor potential vanilloid type 1 (TRPV1) channel responds to a wide spectrum of physical and chemical stimuli. In doing so, it serves as a polymodal cellular sensor for temperature change and pain. Many chemicals are known to strongly potentiate TRPV1 activation, though how this is achieved remains unclear. In this study we investigated the molecular mechanism underlying the gating effects of divalent cations Mg(2+) and Ba(2+). Using a combination of fluorescence imaging and patch-clamp analysis, we found that these cations potentiate TRPV1 gating by most likely promoting the heat activation process. Mg(2+) substantially lowers the activation threshold temperature; as a result, a significant fraction of channels are heat-activated at room temperature. Although Mg(2+) also potentiates capsaicin- and voltage-dependent activation, these processes were found either to be not required (in the case of capsaicin) or insufficient (in the case of voltage) to mediate the activating effect. In support of a selective effect on heat activation, Mg(2+) and Ba(2+) cause a Ca(2+)-independent desensitization that specifically prevents heat-induced channel activation but does not prevent capsaicin-induced activation. These results can be satisfactorily explained within an allosteric gating framework in which divalent cations strongly promote the heat-dependent conformational change or its coupling to channel activation, which is further coupled to the voltage- and capsaicin-dependent processes.
The Journal of General Physiology 12/2013; 143(1). DOI:10.1085/jgp.201311025 · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In all six members of TRPV channel subfamily, there is an ankyrin repeat domain (ARD) in their intracellular Ntermini. Ankyrin (ANK) repeat, a common motif with typically 33 residues in each repeat, is primarily involved in protein-protein interactions. Despite the sequence similarity among the ARDs of TRPV channels, the structure of TRPV3-ARD, however, remains unknown. Here, we report the crystal structure of TRPV3-ARD solved at 1.95 Å resolution, which reveals six-ankyrin repeats. While overall structure of TRPV3-ARD is similar to ARDs from other members of TRPV subfamily; it, however, features a noticeable finger 3 loop that bends over and is stabilized by a network of hydrogen bonds and hydrophobic packing, instead of being flexible as seen in known TRPV-ARD structures. Electrophysiological recordings demonstrated that mutating key residues R225, R226, Q255, and F249 of finger 3 loop altered the channel activities and pharmacology. Taken all together, our findings show that TRPV3-ARD with characteristic finger 3 loop likely plays an important role in channel function and pharmacology.
Protein & Cell 11/2013; 4(12). DOI:10.1007/s13238-013-3091-0 · 2.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aberrant expression of microRNAs is associated with development and progression of cancers. Down-regulation of miR-124 has been demonstrated in the hepatocellular carcinoma (HCC), but the underlying mechanism by which miR-124 suppresses tumorigenesis in HCC remains elusive. In this study, we found that miR-124 suppresses the tumor growth of HCC through targeting the signal transducers and activators of transcription 3 (STAT3). Overexpression of miR-124 suppressed proliferation and induced apoptosis in HepG-2 cells. Luciferase assay confirmed that miR-124 binding to the 3'-UTR region of STAT3 inhibited the expression of STAT3 and phosphorylated STAT3 proteins in HepG-2 cells. Knockdown of STAT3 by siRNA in HepG-2 cells mimicked the effect induced by miR-124. Overexpression of STAT3 in miR-124-transfected HepG-2 cells effectively rescued the inhibition of cell proliferation caused by miR-124. Furthermore, miR-124 suppressed xenograft tumor growth in nude mice implanted with HepG-2 cells by reducing STAT3 expression. Taken together, our findings show that miR-124 functions as tumor suppressor in HCC by targeting STAT3, and miR-124 may therefore serve as a biomarker for diagnosis and therapeutics in HCC.
Biochemical and Biophysical Research Communications 11/2013; 441(4). DOI:10.1016/j.bbrc.2013.10.157 · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the brain and heart, auxiliary KChIPs coassemble with pore-forming Kv4 α- subunits to form a native channel complex and regulate the expression and gating properties of Kv4 currents. Among the KChIP1-4, KChIP4a exhibits a unique N-terminus that is known to suppress Kv4 function, but the underlying mechanism for Kv4 inhibition remains unknown. Here, using confocal imaging, biochemistry and electrophysiology, we identified an ER retention motif, consisting of six hydrophobic and aliphatic residues 12-17 (LIVIVL) within the KChIP4a N-terminus, that functions to reduce surface expression of Kv4-KChIP channel complex. The ability of LIVIVL motif to retain Kv4 proteins is transferable and depends on its flanking location, but not buried in the middle of sequence. Interestingly, adjacent to the ER retention motif, the residues 19-21 (VKL motif) directly interact with Kv4.3 to enhance closed-state inactivation (CSI) and lead to Kv4.3 current inhibition. Taken together, our findings reveal two distinct mechanisms by which KChIP4a suppresses Kv4 function through its N-terminus-mediated ER retention and promoting CSI.
Journal of Biological Chemistry 04/2013; DOI:10.1074/jbc.M113.466052 · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Ca2+-permeable transient receptor potential vanilloid subtype 4 (TRPV4) channel mediates crucial physiological functions such as calcium signaling, temperature sensing, and maintaining cell volume and energy homeostasis. Noticeably, most disease-causing genetic mutations are concentrated in the cytoplasmic domains. In the present study, we focused on the role of TRPV4 C-terminus in modulating protein folding, trafficking and activity. By examining a series of C-terminal deletions, we identified a 20-amino acid distal region covering residues 838-857 that is critical for channel folding, maturation and trafficking. Surface biotinylation, confocal imaging and fluorescence-based calcium influx assay demonstrated that mutant proteins missing this regionwere trapped in the ER and unglycosylated, leading to accelerated degradation and loss of channel activity. Rosetta de novo structural modeling indicated that residues 838-857 assume a defined conformation, withGly849 and Pro851 located at critical positions. Patch clamp recordings confirmed that lowering temperature from 37°C to 30°C rescued channel activity of folding-defective mutants. Further dissection of the segment revealed two key residues, Gly849 and Pro851, to be essential for correct folding. Moreover, biochemical tests demonstrated that, in addition to participating in C-C interaction, the C-terminus also interacts with the N-terminus. Taken together, our findings indicate that a novel segment comprising 20 amino acids in C-terminal region of TRPV4 is critical for channel protein folding and maturation, and the short distal region plays an essential role in this process. Therefore, selectively disrupting the folding-sensitive region may present therapeutic potential for treating overactive TRPV4-mediated diseases such as pain and skeletal dysplasias.
Journal of Biological Chemistry 03/2013; DOI:10.1074/jbc.M113.457291 · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The gut-derived orexigenic peptide hormone ghrelin enhances neuronal firing in the substantia nigra pars compacta, where dopaminergic neurons modulate the function of the nigrostriatal system for motor coordination. Here we describe a novel mechanism by which ghrelin enhances firing of nigral dopaminergic neurons by inhibiting voltage-gated potassium Kv7/KCNQ/M-channels through its receptor GHS-R1a and activation of the PLC-PKC pathway. Brain slice recordings of substantia nigra pars compacta neurons reveal that ghrelin inhibits native Kv7/KCNQ/M-currents. This effect is abolished by selective inhibitors of GHS-R1a, PLC and PKC. Transgenic suppression of native Kv7/KCNQ/M-channels in mice or channel blockade with XE991 abolishes ghrelin-induced hyperexcitability. In vivo, intracerebroventricular ghrelin administration causes increased dopamine release and turnover in the striatum. Microinjection of ghrelin or XE991 into substantia nigra pars compacta results in contralateral dystonic posturing, and attenuation of catalepsy elicited by systemic administration of the D2 receptor antagonist haloperidol. Our findings indicate that the ghrelin/KCNQ signalling is likely a common pathway utilized by the nervous system.
[Show abstract][Hide abstract] ABSTRACT: Honokiol, a major bioactive constituent of the bark of Magnolia officinalis has been confirmed to have the neuroprotective effect on ischemic stroke in rats. This study was designed to observe the therapeutic time window of honokiol microemulsion on cerebral ischemia-reperfusion injury to support its potential for future clinical trials and further explore the underlying mechanisms. Honokiol microemulsion (50μg/kg, i.v. at 0, 1 or 3h after reperfusion) significantly reduced neurological deficit, infarct volume and brain water content in rats subjected to cerebral ischemia-reperfusion, and honokiol (0.1-10μM) significantly attenuated oxygen-glucose deprivation- or glutamate- induced injury of fetal rat cortical neurons. In co-immunoprecipitation and western blot test, honokiol decreased the intensity of nNOS related to PSD95 but failed to affect that of PSD95 related to NR2B in NR2B-PSD95-nNOS complex, and it also inhibited the translocation of nNOS from cytosol to membrane without affecting total nNOS expression, and then markedly decreased NO production in cortical neurons. Besides, the results of whole-cell patch-clamp recordings showed that honokiol reversibly inhibited the NMDA current by about 64%. In conclusion, honokiol has a therapeutic window of at least 5h after the onset of cerebral ischemia or 3h after reperfusion in rats, which may be in part ascribed to the disruption of the PSD95-nNOS interaction leading to the inhibition of neurotoxic NO production.
Brain research 11/2012; 1491. DOI:10.1016/j.brainres.2012.11.004 · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The etiology of prostatic adenocarcinoma remains unclear. Prostate cancer cells of varying metastatic potential and apoptotic resistance show altered expression of plasma membrane ion channels and unbalanced Ca(2+) homeostasis. Ca(2+)-activated Cl(-) channels (CaCCs) are robustly expressed in epithelial cells and function to regulate epithelial secretion and cell volume for maintenance of ion and tissue homeostasis in proliferation, differentiation and apoptosis. ANO1/TMEM16A was recently identified as a CaCC, and it is of interest to determine whether ANO1 plays a role in development and metastasis of prostate carcinoma. Here we show that ANO1 mRNA and protein are highly expressed in human metastatic prostate cancer LNCaP and PC-3 cells by quantitative analysis of real-time PCR and Western blot. These findings were confirmed by whole-cell patch clamp recording of LNCaP and PC-3 cells with increased current density of ANO1 channels. Immunohistochemistry staining further revealed overexpression of ANO1 in human prostate cancer tissues, which correlated with the clinical TNM stage and Gleason score. Experiments with small hairpin RNAs (shRNAs) targeting human ANO1 resulted in a significant reduction of proliferation, metastasis and invasion of PC-3 cells using WST-8, colony formation, wound-healing and transwell assays. Moreover, intratumoral injection of ANO1 shRNA completely inhibited established tumor growth and survival in orthotopic nude mice implanted with PC-3 cells. Our findings provide compelling evidence that upregulation of CaCC ANO1 is involved in the proliferation, progression and pathogenesis of metastatic prostate cancer. Membrane ANO1 protein may therefore serve as a biomarker, and inhibition of overexpressed ANO1 has potential for use in prostate cancer therapy.
Cancer letters 07/2012; 326(1):41-51. DOI:10.1016/j.canlet.2012.07.015 · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: α-Hydroxyl acids (AHAs) from natural sources act as proton donors and topical compounds that penetrate skin and are well known in the cosmetic industry for their use in chemical peels and improvement of the skin. However, little is known about how AHAs cause exfoliation to expose fresh skin cells. Here we report that the transient receptor potential vanilloid 3 (TRPV3) channel in keratinocytes is potently activated by intracellular acidification induced by glycolic acid. Patch clamp recordings and cell death assay of both human keratinocyte HaCaT cells and TRPV3-expressing HEK-293 cells confirmed that intracellular acidification led to direct activation of TRPV3 and promoted cell death. Site-directed mutagenesis revealed that an N-terminal histidine residue, His-426, known to be involved in 2-aminoethyl diphenylborinate-mediated TRPV3 activation, is critical for sensing intracellular proton levels. Taken together, our findings suggest that intracellular protons can strongly activate TRPV3, and TRPV3-mediated proton sensing and cell death in keratinocytes may serve as a molecular basis for the cosmetic use of AHAs and their therapeutic potential in acidic pH-related skin disorders.