[Show abstract][Hide abstract] ABSTRACT: The capsaicin receptor TRPV1 ion channel is a polymodal nociceptor that responds to heat with exquisite sensitivity through an unknown mechanism. Here we report the identification of a novel toxin, RhTx, from the venom of the Chinese red-headed centipede that potently activates TRPV1 to produce excruciating pain. RhTx is a 27-amino-acid small peptide that forms a compact polarized molecule with very rapid binding kinetics and high affinity for TRPV1. We show that RhTx targets the channel's heat activation machinery to cause powerful heat activation at body temperature. The RhTx–TRPV1 interaction is mediated by the toxin's highly charged C terminus, which associates tightly to the charge-rich outer pore region of the channel where it can directly interact with the pore helix and turret. These findings demonstrate that RhTx binding to the outer pore can induce TRPV1 heat activation, therefore providing crucial new structural information on the heat activation machinery.
[Show abstract][Hide abstract] ABSTRACT: Lung cancer or pulmonary carcinoma is primarily derived from epithelial cells that are thin and line on the alveolar surfaces of the lung for gas exchange. ANO1/TMEM16A, initially identified from airway epithelial cells, is a member of Ca2+-activated Cl- channels (CaCCs) that function to regulate epithelial secretion and cell volume for maintenance of ion and tissue homeostasis. ANO1/TMEM16A has recently been shown to be highly expressed in several epithelium originated carcinomas. However, the role of ANO1 in lung cancer remains unknown. In this study, we show that inhibition of calcium-activated chloride channel ANO1/TMEM16A suppresses tumor growth and invasion in human lung cancer. ANO1 is upregulated in different human lung cancer cell lines. Knocking-down ANO1 by small hairpin RNAs inhibited proliferation, migration and invasion of GLC82 and NCI-H520 cancel cells evaluated by CCK-8, would-healing, transwell and 3D soft agar assays. ANO1 protein is overexpressed in 77.3% cases of human lung adenocarcinoma tissues detected by immunohistochemistry. Furthermore, the tumor growth in nude mice implanted with GLC82 cells was significantly suppressed by ANO1 silencing. Taken together, our findings provide evidence that ANO1 overexpression contributes to tumor growth and invasion of lung cancer; and suppressing ANO1 overexpression may have therapeutic potential in lung cancer therapy.
PLoS ONE 08/2015; 10(8):e0136584. DOI:10.1371/journal.pone.0136584 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mexiletine and lidocaine are widely used class IB anti-arrhythmic drugs that are considered to act by blocking voltage-gated open sodium currents for treatment of ventricular arrhythmias and relief of pain. To gain mechanistic insights into action of anti-arrhythmics, we characterized biophysical properties of Nav1.5 and Nav1.7 channels stably expressed in HEK293 cells and compared their use-dependent block in response to mexiletine and lidocaine using whole-cell patch clamp recordings. While the voltage-dependent activation of Nav1.5 or Nav1.7 was not affected by mexiletine and lidocaine, the steady-state fast and slow inactivation of Nav1.5 and Nav1.7 were significantly shifted to hyperpolarized direction by either mexiletine or lidocaine in dose-dependent manner. Both mexiletine and lidocaine enhanced the slow component of closed-state inactivation, with mexiletine exerting stronger inhibition on either Nav1.5 or Nav1.7. The recovery from inactivation of Nav1.5 or Nav1.7 was significantly prolonged by mexiletine compared to lidocaine. Furthermore, mexiletine displayed a pronounced and prominent use-dependent inhibition of Nav1.5 than lidocaine, but not Nav1.7 channels. Taken together, our findings demonstrate differential responses to blockade by mexiletine and lidocaine that preferentially affect the gating of Nav1.5, as compared to Nav1.7; and mexiletine exhibits stronger use-dependent block of Nav1.5. The differential gating properties of Nav1.5 and Nav1.7 in response to mexiletine and lidocaine may help explain the drug effectiveness and advance in new designs of safe and specific sodium channel blockers for treatment of cardiac arrhythmia or pain.
PLoS ONE 06/2015; 10(6):e0128653. DOI:10.1371/journal.pone.0128653 · 3.23 Impact Factor
[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: Mtype potassium current (IM) was initially isolated from sympathetic neurons in 1980 and named as it was inhibited by muscarine. In 1998, the molecular identity of Mcurrent was revealed to be heterotetramers of KCNQ2 and KCNQ3 subunits, whose mutations cause neonatal epilepsy. Reduction of voltage-gated KCNQ2/3 K+ channel (M-channel) activity leads to neuronal hyperexcitability that defines the fundamental mechanism of neurological disorders such as epilepsy and pain. Thus, suppression of neuronal hyperexcitability by activation of KCNQ2/3 channels serves the basis for development of the channel openers for treatment of epilepsy and pain. The well-known KCNQ opener is retigabine (Potiga) that was approved by FDA as an antiepileptic drug in 2011. Recent studies also provide evidence that KCNQ2/3 channel openers are effective in animal models of bipolar disorder, anxiety and schizophrenia, whereas KCNQ2/3 inhibitors, on the other hand, are indicated for improvement of learning and memory in animal models. We recently designed and validated a novel series of pyrazolo [1,5a] pyrimidin7(4H)-ones (PPOs) that selectively activate KCNQ2/3 and show antiepileptic and analgesic activity in vivo. Up to date, all the progress made enforces the view that targeting voltage-gated KCNQ/M-channel may provide therapeutic potential for treatment of neuropsychiatric disorders
Journal of Chinese Pharmaceutical Sciences 01/2014; 23(1). DOI:10.5246/jcps.2014.01.001
[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 · 4.79 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 · 4.79 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 · 3.25 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 · 2.30 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.
[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.