Objective This study aims to achieve high-yield functional expression of the human voltage-gated potassium channel K_V3.1 using an Escherichia coli cell-free protein synthesis system, thereby providing a novel synthetic approach for drug screening, structural analysis and functional characterization of K_V3.1. Methods K_V3.1 was expressed in an Escherichia coli cell-free protein synthesis system for 10 hours in the presence of peptide surfactant A₆K. The secondary structure of K_V3.1 was analyzed by circular dichroism spectroscopy. The potassium channel activity of the recombinant protein liposome K_V3.1-A₆K was investigated using fluorescent dyes Oxonol VI as indicators, which are capable of reflecting alterations in membrane potential. Results Soluble K_V3.1 protein was successfully synthesized, achieving a purified yield of up to 1.2 mg/mL via an Escherichia coli cell-free protein synthesis system. Circular dichroism spectroscopy revealed that K_V3.1 exhibited characteristic α-helical secondary structures. Membrane potential fluorescence assays demonstrated that the K_V3.1-A₆K proteoliposomes, which were reconstructed with surfactant peptide A₆K, exhibited remarkable potassium ion permeability. Conclusion This study successfully achieved high-yield expression of human K_V3.1 with activity using an Escherichia coli-based cell-free protein synthesis system. This innovative method not only significantly enhances the expression yield of K_V3.1, but also maintains its functional activity, thereby establishing a novel and efficient synthetic platform for drug screening and advancing our understanding of structure-function relationships in K_V3.1 research.
Humans ; Escherichia coli/metabolism* ; Shaw Potassium Channels/biosynthesis* ; Cell-Free System ; Circular Dichroism ; Protein Biosynthesis ; Recombinant Proteins/metabolism* ; Membrane Potentials ; Shab Potassium Channels

In patients with epilepsy, depression is a common comorbidity but difficult to be treated because many antidepressants cause pro-convulsive effects. Thus, it is important to identify the risk of seizures associated with antidepressants. To determine whether paroxetine, a very potent selective serotonin reuptake inhibitor (SSRI), interacts with ion channels that modulate neuronal excitability, we examined the effects of paroxetine on Kv3.1 potassium channels, which contribute to highfrequency firing of interneurons, using the whole-cell patch-clamp technique. Kv3.1 channels were cloned from rat neurons and expressed in Chinese hamster ovary cells. Paroxetine reversibly reduced the amplitude of Kv3.1 current, with an IC₅₀ value of 9.43 µM and a Hill coefficient of 1.43, and also accelerated the decay of Kv3.1 current. The paroxetine-induced inhibition of Kv3.1 channels was voltage-dependent even when the channels were fully open. The binding (k₊₁) and unbinding (k₋₁) rate constants for the paroxetine effect were 4.5 µM⁻¹s⁻¹ and 35.8 s⁻¹, respectively, yielding a calculated K(D) value of 7.9 µM. The analyses of Kv3.1 tail current indicated that paroxetine did not affect ion selectivity and slowed its deactivation time course, resulting in a tail crossover phenomenon. Paroxetine inhibited Kv3.1 channels in a usedependent manner. Taken together, these results suggest that paroxetine blocks the open state of Kv3.1 channels. Given the role of Kv3.1 in fast spiking of interneurons, our data imply that the blockade of Kv3.1 by paroxetine might elevate epileptic activity of neural networks by interfering with repetitive firing of inhibitory neurons.
Animals ; Antidepressive Agents ; Clone Cells ; Comorbidity ; Cricetinae ; Cricetulus ; Depression ; Epilepsy ; Female ; Fires ; Humans ; Interneurons ; Ion Channels ; Neurons ; Ovary ; Paroxetine* ; Patch-Clamp Techniques ; Rats ; Seizures ; Serotonin ; Shaw Potassium Channels* ; Tail

We have reported that hypoxia increases the activation of 15-lipoxygenase (15-LO), which converts arachidonic acid (AA) into 15-hydroxyeicosatetraenoic acid (15-HETE) in small pulmonary arteries (PAs). Through inhibition of Kv channels, 15-HETE causes more robust concentration-dependent contraction of PA rings from the hypoxic compared to the normoxic controls. However, the subtypes of Kv channels inhibited by 15-HETE are incompletely understood. The aim of the present study was to identify the contribution of Kv3.4 channel in the process of pulmonary vasoconstriction induced by 15-HETE using the tension studies of PA rings from rat with Kv3.4 channel blocker in tissue bath; to explore the role of vascular endothelium in15-HETE-induced pulmonary vasoconstriction through denuded endothelia of PA rings; and to define the downregulation of 15-HETE on the expression of Kv3.4 channel in cultured pulmonary artery smooth muscle cells (PASMCs) with RT-PCR and Western blot. In the present study, healthy Wistar rats were divided randomly into two groups: Group A with normal oxygen supply and group B with hypoxia. Six days later, the rats were killed. Pulmonary artery rings were prepared for organ bath experiments. Firstly, different concentrations of 15-HETE (10~1 000 nmol/L) were added to the Krebs solution. The isometric tension was recorded using a four-channel force-displacement transducer. Then Kv3.4 channel blocker, 100 nmol/L BDS-I, was added, followed by adding 1 mumol/L 15-HETE, and the isometric tension was recorded. Furthermore, RT-PCR and Western blot were employed to identify the influence of 15-HETE on the expression of Kv3.4 channel in cultured rat PASMCs.The results showed the PA tension was significantly increased both in groups A and B by 15-HETE in a concentration-dependent manner (P<0.05), especially in group B (P<0.05 compared to control); denuded endothelia enhanced 15-HETE concentration-related constrictions in rat PA rings; Kv3.4 channel blocker, BDS-I, significantly decreased the PA ring constriction induced by 15-HETE (P<0.05); the expressions of Kv3.4 mRNA and protein in rat PASMCs were significantly downregulated by 15-HETE (P<0.05). Based on all the information above, we conclude that Kv3.4 channel is involved in vasoconstriction induced by 15-HETE in rat PAs.
Animals ; Cells, Cultured ; Female ; Hydroxyeicosatetraenoic Acids ; pharmacology ; Hypertension, Pulmonary ; physiopathology ; Hypoxia ; physiopathology ; Male ; Muscle, Smooth, Vascular ; cytology ; pathology ; Pulmonary Artery ; cytology ; physiopathology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Shaw Potassium Channels ; antagonists & inhibitors ; genetics ; metabolism ; Vasoconstriction ; drug effects