应用PCR技术对先天性长QT综合征KCNQ1 基因进行定点突变的研究

利用聚合酶链反应（PCR）技术对长QT综合征（LQTS）KCNQ1基因进行定点突变的研究。首先设计两对引物（包含预定的突变）,通过3轮PCR扩增,扩增出含有所需突变位点的片段,然后将片段克隆入T载体中,通过酶切连接的方法将突变点引入到pIRES2-EGFP-KCNQ1中,随后用Effectene转染试剂介导转染HEK293细胞。结果在真核表达载体pIRES2-EGFP-KCNQ1基础上获得了KCNQ1 cDNA C934T的突变体,测序表明在序列中发生了预期的突变。将含突变点的pIRES2-EGFP-KCNQ1转染HEK293细胞后,在荧光显微镜下观察到被转染的HEK293细胞发出绿色荧光,表明含突变点的pIRES2-EGFP-KCNQ1得到了表达。Abstract: To study PCR site-directed mutagenesis of long QT syndrome KCNQ1 gene in vitro. The site-directed mutagenesis of LQTS gene KCNQ1 was made by PCR. Two sets of primers were designed according to the sequence of KCNQ1 cDNA, and mismatch was introduced into primers. Mutagenesis was performed in a three-step PCR. The amplified fragments from the third PCR which contained the mutation site were subcloned into the T-vecor PCR2.1.Then the fragments containing the mutation site was obtained from PCR2.1 with restriction enzyme digestion and was inserted into the same restriction site of pIRES2-EGFP-KCNQ1. With Effectene Transfection Reagent, pIRES2-EGFP-KCNQ1 was transfected into HEK293 cell. The sequencing analysis showed that the mutation site was correct. Mutation from T to C in 934 site of KCNQ1 cDNA was found. Under the fluorescence microscope, the green fluorescence was spread in the transfected HEK293 cell, meaning the pIRES2-EGFP-KCNQ1 containing the mutation site was expressed correctly.     

Novel KV7 ion channel openers for the treatment of epilepsy and implications for detrusor tissue contraction

Neuronal voltage-gated potassium channels, K_V7s, are the molecular mediators of the M current and regulate membrane excitability in the central and peripheral neuronal systems. Herein, we report novel small molecule K_V7 openers that demonstrate anti-seizure activities in electroshock and pentylenetetrazol-induced seizure models without influencing Rotarod readouts in mice. The anti-seizure activity was determined to be proportional to the unbound concentration in the brain. K_V7 channels are also expressed in the bladder smooth muscle (detrusor) and activation of these channels may cause localized undesired effects. Therefore, the impact of individual K_V7 isoforms was investigated in human detrusor tissue using a panel of K_V7 openers with distinct activity profiles among K_V7 isoforms. KCNQ4 and KCNQ5 mRNA were highly expressed in detrusor tissue, yet a compound that has significantly reduced activity on homomeric K_V7.4 did not reduce detrusor contraction. This may suggest that the homomeric K_V7.4 channel plays a less significant role in bladder contraction and further investigation is needed.     

Novel gene hKCNE4 slows the activation of the KCNQ1 channel

The KCNE genes encode small, single transmembrane domain peptides that associate with pore-forming potassium channel subunits to form mixed complexes with unique characteristics. We have identified a novel member of the human KCNE gene family, hKCNE4. The hKCNE4 gene encodes 170 amino acid protein and is localized to chromosome 2q35-36. The protein sequence shows 90% homology to mouse KCNE4 and 38% identity to human KCNE1. Northern blot analysis revealed that hKCNE4 is expressed strongly in heart, skeletal muscle, and kidney, less in placenta, lung, and liver, and weakly in brain and blood cells. Electrophysiological study showed that hKCNE4 modulates the activation of the KCNQ1 channel.     

Design and evaluation of pyrazolopyrimidines as KCNQ channel modulators

Effective treatments of neuropathic pain have been a focus of many discovery programs. KCNQ (kv7) are voltage gated potassium channel openers that have the potential for the treatment of CNS disorders including neuropathic pain. Clinical studies have suggested agents such as Retigabine to be a modulator of pain-like effects such as hyperalgesia and allodynia. In this paper, we describe the discovery and evaluation of a series of novel pyrazolopyrimidines and their affinity for potassium channels KCNQ2/3. These pyrazolopyrimidines have also shown good efficacy in the capsaicin-induced acute and secondary mechanical allodynia model and excellent pharmacokinetic properties, which may be superior to Retigabine.     

Expression profile and characterisation of a truncated KCNQ5 splice variant

Ion channels encoded by KCNQ genes (1-5) are key regulators of membrane properties in many cell types. The KCNQ5 gene was the last to be identified and has three splice variants that are expressed in human brain and skeletal muscle. The KCNQ5 encoded channel possesses M-current properties and so far no channelopathy has been associated with any of the three variants. We now show that only the shortest KCNQ5 variant, which has exon 9 deleted, was expressed in a variety of murine vascular smooth muscle. In Xenopus oocytes, this variant generated currents with amplitudes, activation kinetics and biophysical properties similar to the full-length variant normally expressed in neuronal tissue. Furthermore sensitivity to block by XE991 and activation by retigabine were also similar between both variants. These data represent an exhaustive characterisation of a truncated KCNQ5 splice variant that may contribute to the native XE991-sensitive channel in murine vasculature.     

Studies of the effect of ionomycin on the KCNQ4 channel expressed in Xenopus oocytes

The effect of ionomycin on the human KCNQ4 channels expressed in Xenopus leavis oocytes was investigated. KCNQ4 channels expressed in Xenopus oocytes were measured using two-electrode voltage clamp. The activation of KCNQ4 current had slow activation kinetics and low threshold (approximately -50 mV). The expressed current of KCNQ4 showed the half-maximal activation (V(1/2)) was -17.8 mV and blocked almost completely by KCNQ4 channel blockers, linopirdine (300 microM) or bepridil (200 microM). The significant increase of KCNQ4 outward current induced by ionomycin (calcium salt) is about 1.7-fold of control current amplitude at +60 mV and shifted V(1/2) by approximately -8 mV (from -17.8 to -26.0 mV). This effect of ionomycin could be reversed by the further addition of BAPTA-AM (0.3 mM), a membrane-permeable calcium chelator. Furthermore, the increased effect of ionomycin on KCNQ4 current is abolished by pretreatment of linopirdine or bepridil. In contrast, direct cytoplasmic injection of calcium medium (up to 1 mM calcium, 50 nl) did not mimic the effect of ionomycin. In conclusion, the effect of ionomycin on enhancement of KCNQ4 current is independent of intracellular calcium mobilization and possibly acts on intramembrane hydrophobic site of KCNQ4 protein expressed in Xenopus oocytes.     

Voltage-gated K channel KCNQ1 regulates insulin secretion in MIN6 β-cell line

KCNQ1, located on 11p15.5, encodes a voltage-gated K⁺ channel with six transmembrane regions, and loss-of-function mutations in the KCNQ1 gene cause hereditary long QT syndrome. Recent genetic studies have identified that single nucleotide polymorphisms located in intron 15 of the KCNQ1 gene are strongly associated with type 2 diabetes and impaired insulin secretion. In order to understand the role of KCNQ1 in insulin secretion, we introduced KCNQ1 into the MIN6 mouse β-cell line using a retrovirus-mediated gene transfer system. In KCNQ1 transferred MIN6 cells, both the density of the KCNQ1 current and the density of the total K⁺ current were significantly increased. In addition, insulin secretion by glucose, pyruvate, or tolbutamide was significantly impaired by KCNQ1-overexpressing MIN6 cells. These results suggest that increased KCNQ1 protein expression limits insulin secretion from pancreatic β-cells by regulating the potassium channel current.     

Inactivation gating of Kv7.1 channels does not involve concerted cooperative subunit interactions

Inactivation is an intrinsic property of numerous voltage-gated K⁺ (Kv) channels and can occur by N-type or/and C-type mechanisms. N-type inactivation is a fast, voltage independent process, coupled to activation, with each inactivation particle of a tetrameric channel acting independently. In N-type inactivation, a single inactivation particle is necessary and sufficient to occlude the pore. C-type inactivation is a slower process, involving the outermost region of the pore and is mediated by a concerted, highly cooperative interaction between all four subunits. Inactivation of Kv7.1 channels does not exhibit the hallmarks of N- and C-type inactivation. Inactivation of WT Kv7.1 channels can be revealed by hooked tail currents that reflects the recovery from a fast and voltage-independent inactivation process. However, several Kv7.1 mutants such as the pore mutant L273F generate an additional voltage-dependent slow inactivation. The subunit interactions during this slow inactivation gating remain unexplored. The goal of the present study was to study the nature of subunit interactions along Kv7.1 inactivation gating, using concatenated tetrameric Kv7.1 channel and introducing sequentially into each of the four subunits the slow inactivating pore mutation L273F. Incorporating an incremental number of inactivating mutant subunits did not affect the inactivation kinetics but slowed down the recovery kinetics from inactivation. Results indicate that Kv7.1 inactivation gating is not compatible with a concerted cooperative process. Instead, adding an inactivating subunit L273F into the Kv7.1 tetramer incrementally stabilizes the inactivated state, which suggests that like for activation gating, Kv7.1 slow inactivation gating is not a concerted process.     

Chemical control of metabolically-engineered voltage-gated K+ channels

Metabolic oligosaccharide engineering is a powerful approach for installing unnatural glycans with unique functional groups into the glycocalyx of living cells and animals. Using this approach, we showed that K⁺ channel complexes decorated with thiol-containing sialic acids were irreversibly inhibited with scorpion toxins bearing a pendant maleimide group. Irreversible inhibition required a glycosylated K⁺ channel subunit and was completely reversible with mild reductant when the tether connecting the toxin to the maleimide contained a disulfide bond. Cleavage of the disulfide bond not only restored function, but delivered a biotin moiety to the modified K⁺ channel subunit, providing a novel approach for preferentially labeling wild type K⁺ channel complexes functioning in cells.     

Congenital long QT syndrome caused by the F275S KCNQ1 mutation: Mechanism of impaired channel function

Congenital long QT syndrome is characterized by a prolongation of ventricular repolarization and recurrent episodes of life-threatening ventricular tachyarrhythmias, often leading to sudden death. We previously identified a missense mutation F275S located within the S5 transmembrane domain of the KCNQ1 ion channel in a Chinese family with long QT syndrome. We used oocyte expression of the KCNQ1 polypeptide to study the effects of the F275S mutation on channel properties. Expression of the F275 mutant, or co-expression with the wild-type S275 polypeptide, significantly decreased channel current amplitudes. Moreover, the F275S substitution decreased the rates of channel activation and deactivation. In transfected HEK293 cells fluorescence microscopy revealed that the F275S mutation perturbed the subcelluar localization of the ion channel. These results indicate that the F275S KCNQ1 mutation leads to impaired polypeptide trafficking that in turn leads to reduction of channel ion currents and altered gating kinetics.     

Ketamine exerts its sustained antidepressant effects via cell-type-specific regulation of Kcnq2

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The contribution of Kv7 channels to pregnant mouse and human myometrial contractility

Premature birth accounts for approximately 75% of neonatal mortality and morbidity in the developed world. Despite this, methods for identifying and treating women at risk of preterm labour are limited and many women still present in preterm labour requiring tocolytic therapy to suppress uterine contractility. The aim of this study was to assess the utility of Kv7 channel activators as potential uterine smooth muscle (myometrium) relaxants in tissues from pregnant mice and women. Myometrium was obtained from early and late pregnant mice and from lipopolysaccharide (LPS)‐injected mice (day 15 of gestation; model of infection in pregnancy). Human myometrium was obtained at the time of Caesarean section from women at term (38–41 weeks). RT‐PCR/qRT‐PCR detected KCNQ and KCNE expression in mouse and human myometrium. In mice, there was a global suppression of all KCNQ isoforms, except KCNQ3, in early pregnancy (n= 6, P < 0.001 versus late pregnant); expression subsequently increased in late pregnancy (n= 6). KCNE isoforms were also gestationally regulated (P < 0.05). KCNQ and KCNE isoform expression was slightly down‐regulated in myometrium from LPS‐treated‐mice versus controls (P < 0.05, n= 3–4). XE991 (10 μM, Kv7 inhibitor) significantly increased spontaneous myometrial contractions in vitro in both human and mouse myometrial tissues (P < 0.05) and retigabine/flupirtine (20 μM, Kv7 channel activators) caused profound myometrial relaxation (P < 0.05). In summary, Kv7 activators suppressed myometrial contraction and KCNQ gene expression was sustained throughout gestation, particularly at term. Consequently, activation of the encoded channels represents a novel mechanism for treatment of preterm labour.     

Skeletal muscle Kv7 (KCNQ) channels in myoblast differentiation and proliferation

Voltage-dependent K⁺ channels (Kv) are involved in myocyte proliferation and differentiation by triggering changes in membrane potential and regulating cell volume. Since Kv7 channels may participate in these events, the purpose of this study was to investigate whether skeletal muscle Kv7.1 and Kv7.5 were involved during proliferation and myogenesis. Here we report that, while myotube formation did not regulate Kv7 channels, Kv7.5 was up-regulated during cell cycle progression. Although, Kv7.1 mRNA also increased during the G₁-phase, pharmacological evidence mainly involves Kv7.5 in myoblast growth. Our results indicate that the cell cycle-dependent expression of Kv7.5 is involved in skeletal muscle cell proliferation.     

Single channel kinetic analysis of the cAMP effect on IKs mutants,S209F and S27D/S92D

The I_Ks current is important in the heart’s response to sympathetic stimulation. β-adrenergic stimulation increases the amount of I_Ks and creates a repolarization reserve that shortens the cardiac action potential duration. We have recently shown that 8-CPT-cAMP, a membrane-permeable cAMP analog, changes the channel kinetics and causes it to open more quickly and more often, as well as to higher subconductance levels, which produces an increase in the I_Ks current. The mechanism proposed to underlie these kinetic changes is increased activation of the voltage sensors. The present study extends our previous work and shows detailed subconductance analysis of the effects of 8-CPT-cAMP on an enhanced gating mutant (S209F) and on a double pseudo-phosphorylated I_Ks channel (S27D/S92D). 8-CPT-cAMP still produced kinetic changes in S209F + KCNE1, further enhancing gating, while S27D/S92D + KCNE1 showed no significant response to 8-CPT-cAMP, suggesting that these last two mutations fully recapitulate the effect of channel phosphorylation by cAMP.     

Some Pharmacological Properties Of Neural KCNQ Channels

The lecture summarizes data on the structural and pharmacological properties of KCNQ channels, in particular KCNQ2-5, associated primarily with the nervous system. The KCNQ channels are known to play a crucial role in the control of neuronal excitability. Two classes of drugs altering KCNQ channel activity and therefore producing considerable changes in neuronal excitability are described. These are KCNQ/M channel blocking agents (linopirdine, TEA) and KCNQ channel enhancers (retigabine).     

A truncated splice variant of KCNQ1 cloned from rat heart

KCNQ1 encodes a pore-forming subunit of potassium channels. Mutations in this gene cause inherited diseases, i.e., Romano-Ward syndrome and Jervell and Lange-Nielsen syndrome. A truncated isoform of KCNQ1 was reported to be expressed physiologically and to suppress a delayed rectifier potassium current dominant-negatively in human heart. However, it is not known whether this way of modulation occurs in other species. We cloned another truncated splice variant of KCNQ1 (tr-rKCNQ1) from rat heart. Judging from the deleted sequence of the tr-rKCNQ1, the genomic structure of rat in this portion might be different from those of human and mouse. Otherwise, an unknown exon might exist. RT-PCR analysis demonstrated that the tr-rKCNQ1 was expressed in fetal and neonatal hearts. When this gene was expressed along with a full-length KCNQ1, it suppressed potassium currents, whether a regulatory subunit minK was co-expressed or not.