Phosphoproteome Analysis Identifies a Synaptotagmin-1-Associated Complex Involved in Ischemic Neuron Injury.

Cerebral stroke is one of the leading causes of death in adults worldwide. However, the molecular mechanisms of stroke-induced neuron injury are not fully understood. Here, we obtained phosphoproteomic and proteomic profiles of the acute ischemic hippocampus by LC-MS/MS analysis. Quantitative phosphoproteomic analyses revealed that the dysregulated phosphoproteins were involved in synaptic components and neurotransmission. We further demonstrated that phosphorylation of Synaptotagmin-1 (Syt1) at the Thr112 site in cultured hippocampal neurons aggravated oxygen-glucose deprivation-induced neuronal injury. Immature neurons with low expression of Syt1 exhibit slight neuronal injury in a cerebral ischemia model. Administration of the Tat-Syt1T112A peptide protects neurons against cerebral ischemia-induced injury in vitro and in vivo. Surprisingly, potassium voltage-gated channel subfamily KQT member 2 (Kcnq2) interacted with Syt1 and Annexin A6 (Anxa6) and alleviated Syt1-mediated neuronal injury upon oxygen-glucose deprivation treatment. These results reveal a mechanism underlying neuronal injury and may provide new targets for neuroprotection after acute cerebral ischemia onset.

Impaired dendritic spike generation in the Fragile X prefrontal cortex is due to loss of dendritic sodium channels.

Patients with Fragile X syndrome, the leading monogenetic cause of autism, suffer from impairments related to the prefrontal cortex, including working memory and attention. Synaptic inputs to the distal dendrites of layer 5 pyramidal neurons in the prefrontal cortex have a weak influence on the somatic membrane potential. To overcome this filtering, distal inputs are transformed into local dendritic Na+ spikes, which propagate to the soma and trigger action potential output. Layer 5 extratelencephalic (ET) prefrontal cortex (PFC) neurons project to the brainstem and various thalamic nuclei and are therefore well positioned to integrate task-relevant sensory signals and guide motor actions. We used current clamp and outside-out patch clamp recording to investigate dendritic spike generation in ET neurons from male wild-type and Fmr1 knockout (FX) mice. The threshold for dendritic spikes was more depolarized in FX neurons compared to wild-type. Analysis of voltage responses to simulated in vivo 'noisy' current injections showed that a larger dendritic input stimulus was required to elicit dendritic spikes in FX ET dendrites compared to wild-type. Patch clamp recordings revealed that the dendritic Na+ conductance was significantly smaller in FX ET dendrites. Taken together, our results suggest that the generation of Na+ -dependent dendritic spikes is impaired in ET neurons of the PFC in FX mice. Considering our prior findings that somatic D-type K+ and dendritic hyperpolarization-activated cyclic nucleotide-gated-channel function is reduced in ET neurons, we suggest that dendritic integration by PFC circuits is fundamentally altered in Fragile X syndrome. KEY POINTS: Dendritic spike threshold is depolarized in layer 5 prefrontal cortex neurons in Fmr1 knockout (FX) mice. Simultaneous somatic and dendritic recording with white noise current injections revealed that larger dendritic stimuli were required to elicit dendritic spikes in FX extratelencephalic (ET) neurons. Outside-out patch clamp recording revealed that dendritic sodium conductance density was lower in FX ET neurons.

Inhibition of SlSKOR by SlCIPK23-SlCBL1/9 uncovers CIPK-CBL-target network rewiring in land plants.

Transport of K+ to the xylem is a key process in the mineral nutrition of the shoots. Although CIPK-CBL complexes have been widely shown to regulate K+ uptake transport systems, no information is available about the xylem ones. Here, we studied the physiological roles of the voltage-gated K+ channel SlSKOR and its regulation by the SlCIPK23-SlCBL1/9 complexes in tomato plants. We phenotyped gene-edited slskor and slcipk23 tomato knockout mutants and carried out two-electrode voltage-clamp (TEVC) and BiFC assays in Xenopus oocytes as key approaches. SlSKOR was preferentially expressed in the root stele and was important not only for K+ transport to shoots but also, indirectly, for that of Ca2+ , Mg2+ , Na+ , NO3 - , and Cl- . Surprisingly, the SlCIPK23-SlCBL1/9 complexes turned out to be negative regulators of SlSKOR. Inhibition of SlSKOR by SlCIPK23-SlCBL1/9 was observed in Xenopus oocytes and tomato plants. Regulation of SKOR-like channels by CIPK23-CBL1 complexes was also present in Medicago, grapevine, and lettuce but not in Arabidopsis and saltwater cress. Our results provide a molecular framework for coordinating root K+ uptake and its translocation to the shoot by SlCIPK23-SlCBL1/9 in tomato plants. Moreover, they evidenced that CIPK-CBL-target networks have evolved differently in land plants.

Dimer interaction in the Hv1 proton channel.

The voltage-gated proton channel Hv1 is a member of the voltage-gated ion channel superfamily, which stands out in design: It is a dimer of two voltage-sensing domains (VSDs), each containing a pore pathway, a voltage sensor (S4), and a gate (S1) and forming its own ion channel. Opening of the two channels in the dimer is cooperative. Part of the cooperativity is due to association between coiled-coil domains that extend intracellularly from the S4s. Interactions between the transmembrane portions of the subunits may also contribute, but the nature of transmembrane packing is unclear. Using functional analysis of a mutagenesis scan, biochemistry, and modeling, we find that the subunits form a dimer interface along the entire length of S1, and also have intersubunit contacts between S1 and S4. These interactions exert a strong effect on gating, in particular on the stability of the open state. Our results suggest that gating in Hv1 is tuned by extensive VSD-VSD interactions between the gates and voltage sensors of the dimeric channel.

Chloride channels with ClC-1-like properties differentially regulate the excitability of dopamine receptor D1- and D2-expressing striatal medium spiny neurons.

Dynamic chloride (Cl-) regulation is critical for synaptic inhibition. In mature neurons, Cl- influx and extrusion are primarily controlled by ligand-gated anion channels (GABAA and glycine receptors) and the potassium chloride cotransporter K+-Cl- cotransporter 2 (KCC2), respectively. Here, we report for the first time, to our knowledge, a presence of a new source of Cl- influx in striatal neurons with properties similar to chloride voltage-gated channel 1 (ClC-1). Using whole cell patch-clamp recordings, we detected an outwardly rectifying voltage-dependent current that was impermeable to the large anion methanesulfonate (MsO-). The anionic current was sensitive to the ClC-1 inhibitor 9-anthracenecarboxylic acid (9-AC) and the nonspecific blocker phloretin. The mean fractions of anionic current inhibition by MsO-, 9-AC, and phloretin were not significantly different, indicating that anionic current was caused by active ClC-1-like channels. In addition, we found that Cl- current was not sensitive to the transmembrane protein 16A (TMEM16A; Ano1) inhibitor Ani9 and that the outward Cl- rectification was preserved even at a very high intracellular Ca2+ concentration (2 mM), indicating that TMEM16B (Ano2) did not contribute to the total current. Western blotting and immunohistochemical analyses confirmed the presence of ClC-1 channels in the striatum mainly localized to the somata of striatal neurons. Finally, we found that 9-AC decreased action potential firing frequencies and increased excitability in medium spiny neurons (MSNs) expressing dopamine type 1 (D1) and type 2 (D2) receptors in the brain slices, respectively. We conclude that ClC-1-like channels are preferentially located at the somata of MSNs, are functional, and can modulate neuronal excitability.

Interplay between genetics and lifestyle on pain susceptibility in women with fibromyalgia: the al-Ándalus project.

OBJECTIVES: It is widely acknowledged that the experience of pain is promoted by both genetic susceptibility and environmental factors such as engaging in physical activity (PA), and that pain-related cognitions are also important. Thus, the purpose of the present study was to test the association of 64 polymorphisms (34 candidate genes) and the gene-gene, gene-PA and gene-sedentary behaviour interactions with pain and pain-related cognitions in women with FM. METHODS: Saliva samples from 274 women with FM [mean (s.d.) age 51.7 (7.7) years] were collected for extracting DNA. We measured PA and sedentary behaviour by accelerometers for a week, pain with algometry and questionnaires, and pain-related cognitions with questionnaires. To assess the robustness of the results, a meta-analysis was also performed. RESULTS: The rs6311 and rs6313 polymorphisms (5-hydroxytryptamine receptor 2A, HTR2A) were individually related to algometer scores. The interaction of rs4818 (catechol-O-methyltransferase, COMT) and rs1799971 (opioid receptor µ gene, OPRM1) was related to pain catastrophizing. Five gene-behaviour interactions were significant: the interactions of sedentary behaviour with rs1383914 (adrenoceptor alpha 1A, ADRA1A), rs6860 (charged multivesicular body protein 1A, CHMP1A), rs4680 (COMT), rs165599 (COMT) and rs12994338 (SCN9A) on bodily pain subscale of the Short Form 36. Furthermore, the meta-analysis showed an association between rs4680 (COMT) and severity of FM symptoms (codominant model, P-value 0.032). CONCLUSION: The HTR2A gene (individually), COMT and OPRM1 gene-gene interaction, and the interactions of sedentary behaviour with ADRA1A, CHMP1A, COMT and SCN9A genes were associated with pain-related outcomes. Collectively, findings from the present study indicate a modest contribution of genetics and gene-sedentary behaviour interaction to pain and pain catastrophizing in women with FM. Future research should examine whether reducing sedentary behaviour is particularly beneficial for reducing pain in women with genetic susceptibility to pain.

CACNA1B facilitates breast cancer cell growth and migration by regulating cyclin D1 and EMT: the implication of CACNA1B in breast cancer.

PURPOSE: This study mainly aimed to explore the influences of Calcium Voltage-Gated Channel Subunit Alpha1 B (CACNA1B) on the development of breast cancer and the related mechanism. MATERIALS AND METHODS: The information of patients with breast cancer from TCGA database was used for analyses of CACNA1B expression and its prognostic value. Loss- and gain- of functions of CACNA1B were conducted in MCF7 and Bcap-37 cells, respectively. CCK-8, colony formation and transwell assays were applied for evaluating the cell viability and motility. Western blot was used for protein expression detection. RESULTS: We revealed that highly expressed CACNA1B in breast cancer tissues was related to poor prognosis according to the data gained from TCGA database. The outcomes of functional assays showed that depletion of CACNA1B restrained MCF7 cell growth, invasion and migration and high-expression of CACNA1B fortified the growth, invasion and migration in Bcap-37 cells. Finally, we manifested that silencing CACNA1B obviously raised the protein expression level of E-cadherin and reduced the protein levels of Cyclin D1, N-cadherin and Snail in MCF7 cells, whilst, over-expression of CACNA1B reduced the level of E-cadherin and increased the expression of Cyclin D1, N-cadherin and Snail in Bcap-37 cells. CONCLUSIONS: These results identified CACNA1B as a forwarder of the growth, invasion and migration in breast cancer cells.

Interaction of KCNA5, CX43, and CX40 proteins in the atrial muscle of patients with atrial fibrillation.

The objective of the study was to investigate the expression levels of potassium voltage-gated channel subfamily A member 5 (KCNA5), connexin 43 (Cx43), and connexin 40 (Cx40) in the left atrial appendage of patients with atrial fibrillation (AF) and the interactions between them. We gathered tissue samples from patients with persistent AF and sinus rhythm and used fluorescence quantitative polymerase chain reaction to evaluate messenger RNA (mRNA) changes of KCNA5, Cx43, and Cx40. Then, we studied the protein levels of KCNA5, Cx43, and Cx40 by immunofluorescence and western blot analysis and the interactions between these proteins were identified by immunoprecipitation and immunofluorescence colocation, respectively. Compared with the control group, the mRNA and protein levels of KCNA5, Cx43, and Cx40 in the AF group were decreased and the positive expression of KCNA5, Cx43, and Cx40 protein was also decreased by immunofluorescence staining in the AF group. In addition, immunoprecipitation and immunofluorescence colocation revealed that KCNA5 was coexpressed with Cx43 and Cx40 proteins. The expressions of KCNA5, Cx43, and Cx40 were substantially downregulated in the myocardium of patients with AF and KCNA5 interacted with Cx43 and Cx40 proteins, respectively.

Pulp inflammation induces Kv1.1 K+ channel down-regulation in rat thalamus.

OBJECTIVES: Signals from inflamed tooth pulp activate thalamic neurons to evoke central sensitization. We aimed to gain insights into the mechanisms mediating the early phase of pulpal inflammation-induced thalamic neural and glial activation. MATERIALS AND METHODS: Pulpal inflammation was induced via the application of mustard oil (MO) to the upper first molar of Wistar rats with local anesthesia (LA) or saline injection. After 0.5, 1, 2, and 24 hr, contralateral thalami were subjected to microarrays, a real-time polymerase chain reaction and immunohistochemistry to identify differentially expressed genes and assess potassium voltage-gated channel subfamily A member 1 (Kv1.1)-expressing axons and glial fibrillary acidic protein (GFAP)-expressing astrocytes. RESULTS: The Kv1.1 gene (Kcna1) was down-regulated and the density of Kv1.1-expressing axons decreased in non-anesthetized rats, but not in anesthetized rats 1 hr after the MO treatment. The density of GFAP-expressing astrocytes increased in both groups until 24 hr after the MO treatment, with a greater increase being observed in the saline-injection group than in the LA group. CONCLUSIONS: MO induced the transient down-regulation of Kcna1, transiently reduced the density of Kv1.1-expressing axons, and increased astrocytes in thalami within 1 hr of pulpal application. These results suggest central sensitization represented by neuronal hyperexcitability and astrocyte activation.

Absolute Quantification of Nav1.5 Expression by Targeted Mass Spectrometry.

Nav1.5 is the pore forming α-subunit of the cardiac voltage-gated sodium channel that initiates cardiac action potential and regulates the human heartbeat. A normal level of Nav1.5 is crucial to cardiac function and health. Over- or under-expression of Nav1.5 can cause various cardiac diseases ranging from short PR intervals to Brugada syndromes. An assay that can directly quantify the protein amount in biological samples would be a priori to accurately diagnose and treat Nav1.5-associated cardiac diseases. Due to its large size (>200 KD), multipass transmembrane domains (24 transmembrane passes), and heavy modifications, Nav1.5 poses special quantitation challenges. To date, only the relative quantities of this protein have been measured in biological samples. Here, we describe the first targeted and mass spectrometry (MS)-based quantitative assay that can provide the copy numbers of Nav1.5 in cells with a well-defined lower limit of quantification (LLOQ) and precision. Applying the developed assay, we successfully quantified transiently expressed Nav1.5 in as few as 1.5 million Chinese hamster ovary (CHO) cells. The obtained quantity was 3 ± 2 fmol on the column and 3 ± 2 × 104 copies/cell. To our knowledge, this is the first absolute quantity of Nav1.5 measured in a biological sample.

In Vivo Attenuation of M-Current Suppression Impairs Consolidation of Object Recognition Memory.

The M-current is a low voltage-activated potassium current generated by neuronal Kv7 channels. A prominent role of the M-current is to a create transient increase of neuronal excitability in response to neurotransmitters through the suppression of this current. Accordingly, M-current suppression is assumed to be involved in higher brain functions including learning and memory. However, there is little evidence supporting such a role to date. To address this gap, we examined behavioral tasks to assess learning and memory in homozygous Kv7.2 knock-in mice, Kv7.2(S559A), which show reduced M-current suppression while maintaining a normal basal M-current activity in neurons. We found that Kv7.2(S559A) mice had normal object location memory and contextual fear memory, but impaired long-term object recognition memory. Furthermore, short-term memory for object recognition was intact in Kv7.2(S559A) mice. The deficit in long-term object recognition memory was restored by the administration of a selective Kv7 channel inhibitor, XE991, when delivered during the memory consolidation phase. Lastly, c-Fos induction 2 h after training in Kv7.2(S559A) mice was normal in the hippocampus, which corresponds to intact object location memory, but was reduced in the perirhinal cortex, which corresponds to impaired long-term object recognition memory. Together, these results support the overall conclusion that M-current suppression is important for memory consolidation of specific types of memories.SIGNIFICANCE STATEMENT Dynamic regulation of neuronal excitation is a fundamental mechanism for information processing in the brain, which is mediated by changes in synaptic transmissions or by changes in ion channel activity. Some neurotransmitters can facilitate action potential firing by suppression of a low voltage-activated potassium current, M-current. We demonstrate that M-current suppression is critical for establishment of long-term object recognition memory, but is not required for establishment of hippocampus-dependent location memory or contextual memory. This study suggests that M-current suppression is important for stable encoding of specific types of memories.

Genetic predictors of sick sinus syndrome.

Sick sinus syndrome (SSS) encompasses a group of conduction disorders characterized by the inability of sinoatrial node to perform its pacemaker function. Our aim was to identify genetic predictors of SSS in a prospective cohort of patients admitted to the clinic for pacemaker implantation using single-locus and multilocus approaches. We performed genotyping for polymorphic markers of CLCNKA (rs10927887), SCN10A (rs6795970), FNDC3B (rs9647379), MIR146A (rs2910164), SYT10 (rs7980799), MYH6 (rs365990), and KCNE1 (rs1805127) genes in the group of 284 patients with SSS and 243 healthy individuals. Associations between the studied loci and SSS were tested using logistic regression under recessive genetic model using sex and age as covariates. Multilocus analysis was performed using Markov chain Monte Carlo method implemented in the APSampler program. Correction for multiple testing was performed using Benjamini-Hochberg procedure. We detected an individual association between KCNE1 rs1805127*A allele and SSS in the total study group (OR 0.43, PFDR = 0.028) and in the subgroup of patients with 2nd or 3rd degree sinoatrial block (OR 0.17, PFDR = 0.033), and identified seven allelic patterns associated with the disease. SCN10A rs6795970*T and MIR146A rs2910164*C alleles were present in all seven combinations associated with SSS. The highest risk of SSS was conferred by the combination SCN10A rs6795970*T+FNDC3B rs9647379*C+MIR146A rs2910164*C+SYT10 rs7980799*C+KCNE1 rs1805127*G (OR 2.98, CI 1.77-5.00, P = 1.27 × 10-5, PFDR = 0.022). Our findings suggest that KCNE1 rs1805127 polymorphism may play a role in susceptibility to sinoatrial node dysfunction, particularly presenting as 2nd or 3rd degree sinoatrial block, and the risk-modifying effect of other studied loci is better detected using multilocus approach.

On the control of the proton current in the voltage-gated proton channel Hv1.

The nature of the action of voltage-activated proton transport proteins is a conundrum of great current interest. Here we approach this issue by exploring the action of Hv1, a voltage-gated proton channel found in different cells in humans and other organisms. Our study focuses on evaluating the free energy of transporting a proton through the channel, as well as the effect of the proton transfer through D112, in both the closed and open channel conformations. It is found that D112 allows a transported proton to bypass the electrostatic barrier of the open channel, while not being able to help in passing the barrier in the closed form. This reflects the change in position of the gating arginine residues relative to D112, upon voltage activation. Significantly, the effect of D112 accounts for the observed trend in selectivity by overcoming the electrostatic barrier at its highest point. Thus, the calculations provide a structure/function correlation for the Hv1 system. The present work also clarifies that the action of Hv1 is not controlled by a Grotthuss mechanism but, as is always the case, by the protein electrostatic potential at the rate-limiting barriers.

Activation of KCNQ4 as a Therapeutic Strategy to Treat Hearing Loss.

Potassium voltage-gated channel subfamily q member 4 (KCNQ4) is a voltage-gated potassium channel that plays essential roles in maintaining ion homeostasis and regulating hair cell membrane potential. Reduction of the activity of the KCNQ4 channel owing to genetic mutations is responsible for nonsyndromic hearing loss, a typically late-onset, initially high-frequency loss progressing over time. In addition, variants of KCNQ4 have also been associated with noise-induced hearing loss and age-related hearing loss. Therefore, the discovery of small compounds activating or potentiating KCNQ4 is an important strategy for the curative treatment of hearing loss. In this review, we updated the current concept of the physiological role of KCNQ4 in the inner ear and the pathologic mechanism underlying the role of KCNQ4 variants with regard to hearing loss. Finally, we focused on currently developed KCNQ4 activators and their pros and cons, paving the way for the future development of specific KCNQ4 activators as a remedy for hearing loss.

A polybasic motif in alternatively spliced KChIP2 isoforms prevents Ca2+ regulation of Kv4 channels.

The Kv4 family of A-type voltage-gated K+ channels regulates the excitability in hippocampal pyramidal neuron dendrites and are key determinants of dendritic integration, spike timing-dependent plasticity, long-term potentiation, and learning. Kv4.2 channel expression is down-regulated following hippocampal seizures and in epilepsy, suggesting A-type currents as therapeutic targets. In addition to pore-forming Kv4 subunits, modulatory auxiliary subunits called K+ channel-interacting proteins (KChIPs) modulate Kv4 expression and activity and are required to recapitulate native hippocampal A-type currents in heterologous expression systems. KChIP mRNAs contain multiple start sites and alternative exons that generate considerable N-terminal variation and functional diversity in shaping Kv4 currents. As members of the EF-hand domain-containing neuronal Ca2+ sensor protein family, KChIP auxiliary proteins may convey Ca2+ sensitivity upon Kv4 channels; however, to what degree intracellular Ca2+ regulates KChIP-Kv4.2 complexes is unclear. To answer this question, we expressed KChIP2 with Kv4.2 in HEK293T cells, and, with whole-cell patch-clamp electrophysiology, measured an ∼1.5-fold increase in Kv4.2 current density in the presence of elevated intracellular Ca2+ Intriguingly, the Ca2+ regulation of Kv4 current was specific to KChIP2b and KChIP2c splice isoforms that lack a putative polybasic domain that is present in longer KChIP2a1 and KChIP2a isoforms. Site-directed acidification of the basic residues within the polybasic motif of KChIP2a1 rescued Ca2+-mediated regulation of Kv4 current density. These results support divergent Ca2+ regulation of Kv4 channels mediated by alternative splicing of KChIP2 isoforms. They suggest that distinct KChIP-Kv4 interactions may differentially control excitability and function of hippocampal dendrites.

A mutually induced conformational fit underlies Ca2+-directed interactions between calmodulin and the proximal C terminus of KCNQ4 K+ channels.

Calmodulin (CaM) conveys intracellular Ca2+ signals to KCNQ (Kv7, "M-type") K+ channels and many other ion channels. Whether this "calmodulation" involves a dramatic structural rearrangement or only slight perturbations of the CaM/KCNQ complex is as yet unclear. A consensus structural model of conformational shifts occurring between low nanomolar and physiologically high intracellular [Ca2+] is still under debate. Here, we used various techniques of biophysical chemical analyses to investigate the interactions between CaM and synthetic peptides corresponding to the A and B domains of the KCNQ4 subtype. We found that in the absence of CaM, the peptides are disordered, whereas Ca2+/CaM imposed helical structure on both KCNQ A and B domains. Isothermal titration calorimetry revealed that Ca2+/CaM has higher affinity for the B domain than for the A domain of KCNQ2-4 and much higher affinity for the B domain when prebound with the A domain. X-ray crystallography confirmed that these discrete peptides spontaneously form a complex with Ca2+/CaM, similar to previous reports of CaM binding KCNQ-AB domains that are linked together. Microscale thermophoresis and heteronuclear single-quantum coherence NMR spectroscopy indicated the C-lobe of Ca2+-free CaM to interact with the KCNQ4 B domain (Kd ∼10-20 µm), with increasing Ca2+ molar ratios shifting the CaM-B domain interactions via only the CaM C-lobe to also include the N-lobe. Our findings suggest that in response to increased Ca2+, CaM undergoes lobe switching that imposes a dramatic mutually induced conformational fit to both the proximal C terminus of KCNQ4 channels and CaM, likely underlying Ca2+-dependent regulation of KCNQ gating.

Analysis of SCN9A Gene Variants for Acute and Chronic Postoperative Pain and Morphine Consumption After Total Hysterectomy.

BACKGROUND: Single nucleotide polymorphisms (SNPs) of the voltage-gated sodium channel alpha subunit gene (SCN9A) have been associated with pain in various settings. The aim of this study was to investigate the association of the SNPs to evaluate the influence of common gene variants on chronic postoperative pain (CPSP) and other related pain variables in a cohort of patients who underwent a scheduled hysterectomy. METHODS: DNA samples from a cohort of 1,075 patients who underwent a scheduled total hysterectomy in our hospital were genotyped for three common SCN9A SNPs using TaqMan assays. Multivariate logistic regression models were used to quantify the association between independent covariates such as pain threshold, pain endurance, pain scores, morphine use, and the presence of chronic pain. RESULTS: Frequencies of the minor alleles were different between the different ethnic groups. There was a statistically significant association of rs16851799 with morphine consumption and self-reported postoperative pain for the 1,038 subjects genotyped, with the TT genotype reporting higher pain and using more morphine. For the subpopulation of 446 subjects with chronic pain data, there was a similar association with self-reported postoperative pain and tolerance of pressure pain. Univariate analysis also showed a statistically significant association of rs16851799 with CPSP, whereas multivariable analysis revealed a similar association of rs4387806 with this outcome. There were three haplotypes with different relative frequencies for the CPSP and non-CPSP groups. CONCLUSIONS: Our results showed that SCN9A polymorphisms could play a role in acute pain perception and the susceptibility to chronic pain.

Long Non-Coding KCNQ1OT1 Promotes Oxygen-Glucose-Deprivation/Reoxygenation-Induced Neurons Injury Through Regulating MIR-153-3p/FOXO3 Axis.

BACKGROUND: Long non-coding RNAs (LncRNAs) have been reported to play important roles in the pathogenesis and development of many diseases, including cerebral ischemia and reperfusion (I/R) injury. In this study, we aimed to investigate the role of LncRNA-Potassium Voltage-Gated Channel Subfamily Q Member 1 opposite strand/antisense transcript 1 (KCNQ1OT1) in cerebral I/R induced neuronal injury, and its underlying mechanisms. METHODS: Primary mouse cerebral cortical neurons treated with oxygen-glucose deprivation and reoxygenation (OGD/R) in vitro and mice subjected to middle cerebral artery occlusion (MCAO) and reperfusion were used to mimic cerebral I/R injury. Small inference RNA (siRNA) was used to knockdown KCNQ1OT1 or microRNA-153-3p (miR-153-3p). Dual-luciferase assay was performed to detect the interaction between KCNQ1OT1 and miR-153-3p and interaction between miR-153-3p and Fork head box O3a (Foxo3). Flow cytometry analysis was performed to detect neuronal apoptosis. qRT-PCR and Western blotting were performed to detect RNA and protein expressions. RESULTS: KCNQ1OT1 and Foxo3 expressions were significantly increased in neurons subjected to I/R injury in vitro and in vivo, and miR-153-3p expression were significantly decreased. Knockdown of KCNQ1OT1 or overexpression of miR-153-3p weakened OGD/R-induced neuronal injury and regulated Foxo3 expressions. Dual-luciferase analysis showed that KCNQ1OT1 directly interacted with miR-153-3p and Foxo3 is a direct target of miR-153-3p. CONCLUSIONS: Our results indicate that LncRNA-KCNQ1OT1 promotes OGD/R-induced neuronal injury at least partially through acting as a competing endogenous RNA (ceRNA) for miR-153-3p to regulate Foxo3a expression, suggesting LncRNA-KCNQ1OT1 as a potential therapeutic target for cerebral I/R injury.

A recurrent mutation in KCNQ4 in Korean families with nonsyndromic hearing loss and rescue of the channel activity by KCNQ activators.

Mutations in potassium voltage-gated channel subfamily Q member 4 (KCNQ4) are etiologically linked to nonsyndromic hearing loss (NSHL), deafness nonsyndromic autosomal dominant 2 (DFNA2). To identify causative mutations of hearing loss in 98 Korean families, we performed whole exome sequencing. In four independent families with NSHL, we identified a cosegregating heterozygous missense mutation, c.140T>C (p.Leu47Pro), in KCNQ4. Individuals with the c.140T>C KCNQ4 mutation shared a haplotype flanking the mutated nucleotide, suggesting that this mutation may have arisen from a common ancestor in Korea. The mutant KCNQ4 protein could reach the plasma membrane and interact with wild-type (WT) KCNQ4, excluding a trafficking defect; however, it exhibited significantly decreased voltage-gated potassium channel activity and fast deactivation kinetics compared with WT KCNQ4. In addition, when co-expressed with WT KCNQ4, mutant KCNQ4 protein exerted a dominant-negative effect. Interestingly, the channel activity of the p.Leu47Pro KCNQ4 protein was rescued by the KCNQ activators MaxiPost and zinc pyrithione. The c.140T>C (p.Leu47Pro) mutation in KCNQ4 causes progressive NSHL; however, the defective channel activity of the mutant protein can be rescued using channel activators. Hence, in individuals with the c.140T>C mutation, NSHL is potentially treatable, or its progression may be delayed by KCNQ activators.

Role of a conserved glutamine in the function of voltage-gated Ca2+ channels revealed by a mutation in human CACNA1D.

Voltage-gated Cav Ca2+ channels play crucial roles in regulating gene transcription, neuronal excitability, and synaptic transmission. Natural or pathological variations in Cav channels have yielded rich insights into the molecular determinants controlling channel function. Here, we report the consequences of a natural, putatively disease-associated mutation in the CACNA1D gene encoding the pore-forming Cav1.3 α1 subunit. The mutation causes a substitution of a glutamine residue that is highly conserved in the extracellular S1-S2 loop of domain II in all Cav channels with a histidine and was identified by whole-exome sequencing of an individual with moderate hearing impairment, developmental delay, and epilepsy. When introduced into the rat Cav1.3 cDNA, Q558H significantly decreased the density of Ca2+ currents in transfected HEK293T cells. Gating current analyses and cell-surface biotinylation experiments suggested that the smaller current amplitudes caused by Q558H were because of decreased numbers of functional Cav1.3 channels at the cell surface. The substitution also produced more sustained Ca2+ currents by weakening voltage-dependent inactivation. When inserted into the corresponding locus of Cav2.1, the substitution had similar effects as in Cav1.3. However, the substitution introduced in Cav3.1 reduced current density, but had no effects on voltage-dependent inactivation. Our results reveal a critical extracellular determinant of current density for all Cav family members and of voltage-dependent inactivation of Cav1.3 and Cav2.1 channels.