Functional Significance of K+ Channel β-Subunit KCNE3 in Auditory Neurons

The KCNE3 β-subunit interacts with and regulates the voltage-dependent gating, kinetics, and pharmacology of a variety of Kv channels in neurons. Because a single neuron may express multiple KCNE3 partners, it is impossible to predict the overall functional relevance of the single transmembrane domain peptide on the pore-forming K⁺ channel subunits with which it associates. In the inner ear, the role of KCNE3 is undefined, despite its association with Meniere disease and tinnitus. To gain insights on the functional significance of KCNE3 in auditory neurons, we examined the properties of spiral ganglion neurons (SGNs) in Kcne3 null mutant neurons relative to their age-matched controls. We demonstrate that null deletion of Kcne3 abolishes characteristic wide variations in the resting membrane potentials of SGNs and yields age-dependent alterations in action potential and firing properties of neurons along the contour of the cochlear axis, in comparison with age-matched wild-type neurons. The properties of basal SGNs were markedly altered in Kcne3^−/− mice compared with the wild-type controls; these include reduced action potential latency, amplitude, and increased firing frequency. Analyses of the underlying conductance demonstrate that null mutation of Kcne3 results in enhanced outward K⁺ currents, which is sufficient to explain the ensuing membrane potential changes. Additionally, we have demonstrated that KCNE3 may regulate the activity of Kv4.2 channels in SGNs. Finally, there were developmentally mediated compensatory changes that occurred such that, by 8 weeks after birth, the electrical properties of the null mutant neurons were virtually indistinguishable from the wild-type neurons, suggesting that ion channel remodeling in auditory neurons progresses beyond hearing onset.     

化合物QO-58对KCNQ1/KCNE1/IKs通道电流调节作用的研究

慢激活延迟整流钾电流(slowly activated delayed rectifier potassium current,IKs)由KCNQ1通道与KCNE1通道共同编码。KCNQl或KCNEl通道电流功能上调能够引发短QT综合征。全新化学结构化合物QO-58对KCNQ1-5通道具有开放作用。该文采用电生理膜片钳技术探讨QO-58对KCNQl/KCNEl/IKs通道电流作用,观察QO-58的心脏电生理毒性。结果表明,化合物QO-58能够浓度依赖性地增大KCNQ1/KCNE1通道电流,并且引起KCNQ1/KCNE1通道电流电压关系曲线向超极化方向移动。QO-58能够轻微增大豚鼠心室肌IKs通道电流,但对豚鼠乳头肌动作电位时程无显著影响。结果提示,QO-58心脏电生理毒性较低,具有进一步研发成为治疗兴奋性增强等相关疾病的新型药物的潜力。     

Identification of a protein–protein interaction between KCNE1 and the activation gate machinery of KCNQ1

KCNQ1 channels assemble with KCNE1 transmembrane (TM) peptides to form voltage-gated K⁺ channel complexes with slow activation gate opening. The cytoplasmic C-terminal domain that abuts the KCNE1 TM segment has been implicated in regulating KCNQ1 gating, yet its interaction with KCNQ1 has not been described. Here, we identified a protein–protein interaction between the KCNE1 C-terminal domain and the KCNQ1 S6 activation gate and S4–S5 linker. Using cysteine cross-linking, we biochemically screened over 300 cysteine pairs in the KCNQ1–KCNE1 complex and identified three residues in KCNQ1 (H363C, P369C, and I257C) that formed disulfide bonds with cysteine residues in the KCNE1 C-terminal domain. Statistical analysis of cross-link efficiency showed that H363C preferentially reacted with KCNE1 residues H73C, S74C, and D76C, whereas P369C showed preference for only D76C. Electrophysiological investigation of the mutant K⁺ channel complexes revealed that the KCNQ1 residue, H363C, formed cross-links not only with KCNE1 subunits, but also with neighboring KCNQ1 subunits in the complex. Cross-link formation involving the H363C residue was state dependent, primarily occurring when the KCNQ1–KCNE1 complex was closed. Based on these biochemical and electrophysiological data, we generated a closed-state model of the KCNQ1–KCNE1 cytoplasmic region where these protein–protein interactions are poised to slow activation gate opening.     

KCNE2的两种突变体I57T和V65M对Kv4.3通道功能的调节

Mink相关蛋白1（MiRP1）是由KCNE基因家族成员KCNE2编码的具有一个跨膜结构的小分子蛋白质,发生在KCNE2上的相关突变能够引起遗传性长QT间期综合症（long QT syn＆ome,LQT6）,但其机制不明．以往的工作表明,MiRP1调节瞬间外向钾电流（transient outward current,Ito）的功能,对维持心电稳定性具有重要的调节作用．在哺乳细胞系COS-7表达系统利用膜片钳全细胞记录方式,研究了两种LQT6相关的突变体157T和V65M对Kv4.3通道功能的影响,从MiRP1对Ito功能调控的改变探讨LQT6引起心律失常的电生理机制．结果表明,KCNE2与Kv4.3共表达后对通道功能具有明显的调控作用,使通道的激活和失活明显减慢,电压依赖性失活发生正向移位,同时加快Kv4.3通道从失活中的恢复．157T与Kv4．3共表达的通道,门控动力学以及通道的恢复特性更接近Kv4.3单独表达的通道,表现为丧失KCNE2的功能—“loss of function”,而V65M的作用则与之刚好相反,对Kv4.3门控动力学和恢复特性的调节较KCNE2更强,同时,使通道电流密度明显降低,表现为增强KCNE2的功能——“gain of function”．由此推论,KCNE2对k功能有重要的调节作用,发生在KCNE2基因上的突变,无论是增强（V65M）还是减弱（157T）KCNE2的功能都可能通过改变Ito在心脏电稳定性中的贡献,从而使心脏在某些条件下发生心律失常．     

KCNE2的两种突变体I57T和V65M对Kv4.3通道功能的调节

Mink相关蛋白1(MiRP1)是由KCNE基因家族成员KCNE2编码的具有一个跨膜结构的小分子蛋白质,发生在KCNE2上的相关突变能够引起遗传性长QT间期综合症(long QT syndrome,LQT6),但其机制不明.以往的工作表明,MiRP1调节瞬间外向钾电流(transient outwatd current,Ito)的功能,对维持心电稳定性具有重要的调节作用.在哺乳细胞系COS-7表达系统利用膜片钳全细胞记录方式,研究了两种LQT6相关的突变体157T和V65M对Kv4.3通道功能的影响,从MiRP1对Ito功能调控的改变探讨LQT6引起心律失常的电生理机制.结果表明,KCNE2与Kv4.3共表达后对通道功能具有明显的调控作用,使通道的激活和失活明显减慢,电压依赖性失活发生正向移位,同时加快Kv4.3通道从失活中的恢复.157T与Kv4.3共表达的通道,门控动力学以及通道的恢复特性更接近Kv4.3单独表达的通道,表现为丧失KCNE2的功能--"loss of function",而V65M的作用则与之刚好相反,对Kv4.3门控动力学和恢复特性的调节较KCNE2更强,同时,使通道电流密度明显降低,表现为增强KCNE2的功能--"gain of function".由此推论,KCNE2对Ito功能有重要的调节作用,发生在KCNE2基因上的突变,无论是增强(V65M)还是减弱(I57T)KCNE2的功能都可能通过改变Ito在心脏电稳定性中的贡献,从而使心脏在某些条件下发生心律失常.     

Disruption of the K+ Channel β-Subunit KCNE3 Reveals an Important Role in Intestinal and Tracheal Cl? Transport

The KCNE3 β-subunit constitutively opens outwardly rectifying KCNQ1 (Kv7.1) K⁺ channels by abolishing their voltage-dependent gating. The resulting KCNQ1/KCNE3 heteromers display enhanced sensitivity to K⁺ channel inhibitors like chromanol 293B. KCNE3 was also suggested to modify biophysical properties of several other K⁺ channels, and a mutation in KCNE3 was proposed to underlie forms of human periodic paralysis. To investigate physiological roles of KCNE3, we now disrupted its gene in mice. kcne3^−/− mice were viable and fertile and displayed neither periodic paralysis nor other obvious skeletal muscle abnormalities. KCNQ1/KCNE3 heteromers are present in basolateral membranes of intestinal and tracheal epithelial cells where they might facilitate transepithelial Cl⁻ secretion through basolateral recycling of K⁺ ions and by increasing the electrochemical driving force for apical Cl⁻ exit. Indeed, cAMP-stimulated electrogenic Cl⁻ secretion across tracheal and intestinal epithelia was drastically reduced in kcne3^−/− mice. Because the abundance and subcellular localization of KCNQ1 was unchanged in kcne3^−/− mice, the modification of biophysical properties of KCNQ1 by KCNE3 is essential for its role in intestinal and tracheal transport. Further, these results suggest KCNE3 as a potential modifier gene in cystic fibrosis.     

How Does KCNE1 Regulate the Kv7.1 Potassium Channel? Model-Structure,Mutations, and Dynamics of the Kv7.1-KCNE1 Complex

The voltage-gated potassium channel Kv7.1 and its auxiliary subunit KCNE1 are expressed in the heart and give rise to the major repolarization current. The interaction of Kv7.1 with the single transmembrane helix of KCNE1 considerably slows channel activation and deactivation, raises single-channel conductance, and prevents slow voltage-dependent inactivation. We built a Kv7.1-KCNE1 model-structure. The model-structure agrees with previous disulfide mapping studies and enables us to derive molecular interpretations of electrophysiological recordings that we obtained for two KCNE1 mutations. An elastic network analysis of Kv7.1 fluctuations in the presence and absence of KCNE1 suggests a mechanistic perspective on the known effects of KCNE1 on Kv7.1 function: slow deactivation is attributed to the low mobility of the voltage-sensor domains upon KCNE1 binding, abolishment of voltage-dependent inactivation could result from decreased fluctuations in the external vestibule, and amalgamation of the fluctuations in the pore region is associated with enhanced ion conductivity.     

Molecular Cloning and Functional Expression of the Equine K+ Channel KV11.1 (Ether à Go-Go-Related/KCNH2 Gene) and the Regulatory Subunit KCNE2 from Equine Myocardium

The KCNH2 and KCNE2 genes encode the cardiac voltage-gated K⁺ channel K_V11.1 and its auxiliary β subunit KCNE2. K_V11.1 is critical for repolarization of the cardiac action potential. In humans, mutations or drug therapy affecting the K_V11.1 channel are associated with prolongation of the QT intervals on the ECG and increased risk of ventricular tachyarrhythmia and sudden cardiac death—conditions known as congenital or acquired Long QT syndrome (LQTS), respectively. In horses, sudden, unexplained deaths are a well-known problem. We sequenced the cDNA of the KCNH2 and KCNE2 genes using RACE and conventional PCR on mRNA purified from equine myocardial tissue. Equine K_V11.1 and KCNE2 cDNA had a high homology to human genes (93 and 88%, respectively). Equine and human K_V11.1 and K_V11.1/KCNE2 were expressed in Xenopus laevis oocytes and investigated by two-electrode voltage-clamp. Equine K_V11.1 currents were larger compared to human K_V11.1, and the voltage dependence of activation was shifted to more negative values with V_1/2 = -14.2±1.1 mV and -17.3±0.7, respectively. The onset of inactivation was slower for equine K_V11.1 compared to the human homolog. These differences in kinetics may account for the larger amplitude of the equine current. Furthermore, the equine K_V11.1 channel was susceptible to pharmacological block with terfenadine. The physiological importance of K_V11.1 was investigated in equine right ventricular wedge preparations. Terfenadine prolonged action potential duration and the effect was most pronounced at slow pacing. In conclusion, these findings indicate that horses could be disposed to both congenital and acquired LQTS.