Jingzhaotoxin-X, a gating modifier of Kv4.2 and Kv4. 3 potassium channels purified from the venom of the Chinese tarantula Chilobrachys jingzhao

The tarantula Chilobrachys jingzhao is one of the largest venomous spiders in China. In previous studies, we purified and characterized at least eight peptides from C. jingzhao venom. In this report, we describe the purification and characterization of Jingzhaotoxin-X (JZTX-X), which selectively blocks Kv4.2 and Kv4.3 potassium channels. Methods: JZTX-X was purified using a combination of cation-exchange HPLC and reverse-phase HPLC. The amino-acid sequence was determined by automated Edman degradation and confirmed by mass spectrometry (MS). Voltage-gated ion channel currents were recorded in HEK293t cells transiently transfected with a variety of ion channel constructs. In addition, the hyperalgesic activity of JZTX-X and the toxin´s effect on motor function were assessed in mice. Results: JZTX-X contained 31 amino acids, with six cysteine residues that formed three disulfide bonds within an inhibitory cysteine knot (ICK) topology. In whole-cell voltage-clamp experiments, JZTX-X inhibited Kv4.2 and Kv4.3 potassium channels in a concentration- and voltage-dependent manner, without affecting other ion channels (Kv1.1, 1.2, 1.3, 2.1, delayed rectifier potassium channels, high- and low-voltage-activated Ca2+ channels, and voltage-gated sodium channels Nav1.5 and 1.7). JZTX-X also shifted the voltage-dependent channel activation to more depolarized potentials, whereas extreme depolarization caused reversible toxin binding to Kv4.2 channels. JZTX-X shifted the Kv4.2 and Kv4.3 activities towards a resting state, since at the resting potential the toxin completely inhibited the channels, even in the absence of an applied physical stimulus. Intrathecal or intraplantar injection of JZTX-X caused a long-lasting decrease in the mechanical nociceptive threshold (hyperalgesia) but had no effect on motor function as assessed in the rotarod test. Conclusions: JZTX-X selectively suppresses Kv4.2 and Kv4.3 potassium channel activity in a concentration- and voltage-dependent manner and causes long-lasting mechanical hyperalgesia.(AU)

Expression of Potassium Channels in Uterine Smooth Muscle Cells from Patients with Adenomyosis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Adenomyosis (AM) has impaired contraction. This study aimed to explore the expression of potassium channels related to contraction in myometrial smooth muscle cells (MSMCs) of AM.</p><p><b>METHODS</b>Uterine tissue samples from 22 patients (cases) with histologically confirmed AM and 12 (controls) with cervical intraepithelial neoplasia were collected for both immunohistochemistry and real-time polymerase chain reaction to detect the expression of large conductance calcium- and voltage-sensitive K + channel (BKCa)-α/β subunits, voltage-gated potassium channel (Kv) 4.2, and Kv4.3. Student's t-test was used to compare the expression.</p><p><b>RESULTS</b>The BKCa-α/β subunits, Kv4.2, and Kv4.3 were located in smooth muscle cells, glandular epithelium, and stromal cells. However, BKCa-β subunit expression in endometrial glands of the controls was weak, and Kv4.3 was almost undetectable in the controls. The expression of BKCa-α messenger RNA (mRNA) (0.62 ± 0.19-fold decrease, P < 0.05) and Kv4.3 mRNA (0.67 ± 0.20-fold decrease, P < 0.05) decreased significantly in the MSMCs of the control group compared with the AM group. However, there were no significant differences in BKCa-β subunit mRNA or Kv4.2 mRNA.</p><p><b>CONCLUSIONS</b>The BKCa-α mRNA and the Kv4.3 mRNA are expressed significantly higher in AM than those in the control group, that might cause the abnormal uterus smooth muscle contractility, change the microcirculation of uterus to accumulate the inflammatory factors, impair the endometrium further, and aggravate the pain.</p>

Shal-type (Kv4.x) potassium channel pore blockers from scorpion venoms / 生理学报

Voltage-gated potassium channels (Kv4.1, Kv4.2 and Kv4.3) encoded by the members of the KCND/Kv4 (Shal) channel family mediate the native, fast inactivating (A-type) K(+) current (IA) described both in heart and neurons. This IA current is specifically blocked by short scorpion toxins that belong to the α-KTx15 subfamily and which act as pore blockers, a different mode of action by comparison to spider toxins known as gating modifiers. This review summarizes our present chemical and pharmacological knowledge on the α-KTx15 toxins.

Inhibition of Jingzhaotoxin-V on Kv4.3 channel / 生理学报

Kv4.3 channel is present in many mammalian tissues, predominantly in the heart and central nervous system. Its currents are transient, characterized by rapid activation and inactivation. In the hearts of most mammals, it is responsible for repolarization of the action potential of ventricular myocytes and is important in the regulation of the heart rate. Because of its central role in this important physiological process, Kv4.3 channel is a promising target for anti-arrhythmic drug development. Jingzhaotoxin-V (JZTX-V) is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. Whole-cell patch clamp recording showed that it partly blocked the transient outward potassium channels in dorsal root ganglion neurons of adult rats with an IC(50) value of 52.3 nmol/L. To investigate the effect of JZTX-V on Kv4.3 channel, JZTX-V was synthesized using the solid-phase chemical synthesis and separated by reverse phase high performance liquid chromatography (HPLC). The purity was tested by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MOLDI-TOF mass spectrometry). Two-electrode voltage-clamp technique was used to characterize the action of JZTX-V on Kv4.3 channels expressed in Xenopus laevis oocytes. As a result, JZTX-V displayed fast kinetics of inhibition and recovery from inactivation. Furthermore, it could inhibit Kv4.3 channel current in a time- and concentration-dependent manner with an IC(50) value of 425.1 nmol/L. The application of JZTX-V affected the activation and inactivation characteristics of Kv4.3 channel and caused a shift of the current-voltage relationship curve and the steady-state inactivation curve to depolarizing direction by approximately 29 mV and 10 mV, respectively. So we deduced that JZTX-V is a gating modifier toxin of Kv4.3 channel. Present findings should be helpful to develop JZTX-V into a molecular probe and drug candidate targeting to Kv4.3 channel in the myocardium.

Atrial myocytes KChIP2 mRNA expression in rheumatic heart disease patients with atrial fibrillation / 中华心血管病杂志

<p><b>OBJECTIVE</b>To detect the KChIP2 mRNA level in rheumatic heart disease patients with or without atrial fibrillation (AF) by real-time PCR.</p><p><b>METHODS</b>Right atrial appendage samples from rheumatic heart disease patients with (n = 17) or without AF (n = 13) were obtained during cardiac surgery. Total RNA was extracted from the atrial tissues, and the KChIP2 and Kv4.3 mRNA were detected by SYBR Green I real-time PCR with the GAPDH as the house keeping gene.</p><p><b>RESULT</b>The ratio of KChIP2/GAPDH (0.1468 +/- 0.0452 vs. 0.2200 +/- 0.0388, P<0.01) and the ratio of Kv4.3/GAPDH (0.3946 +/- 0.1826 vs. 0.5257 +/- 0.1427, P<0.05) were significantly lower in AF patients compared to non-AF patients.</p><p><b>CONCLUSION</b>Down-regulated atrial KChIP2 and Kv4.3 mRNA expressions in rheumatic heart disease patients with chronic AF might be one of the molecular bases responsible for the down-regulation of the I(to) current density of AF.</p>

Characteristics of the action potentials and the underlying ionic mechanisms in the cardiomyocytes from rabbit pulmonary vein sleeves / 生理学报

To investigate the characteristics of action potentials and their ionic mechanism in cardiomyocytes from rabbit pulmonary vein sleeves (PVC), and to compare them with those in left atrial cardiomyocytes (LAC), the technique of whole-cell patch clamp was applied. We used current-clamp technique to record action potentials, and voltage-clamp technique to record ionic currents. PVC had longer action potential duration (APD) than LAC, and therefore a second plateau response could be induced easily, suggesting a strong tendency of early afterdepolarization (EAD) genesis in PVC. Non-selective cation current (I(NSCC)) was first recorded in both LAC and PVC. This I(NSCC)was permeable to K(+), Na(+) and Cs(+), sensitive to GdCl3 but not sensitive to 4-AP. The current densities of inward rectifier potassium current (I(K1)), transient outward potassium current (I(To)) and I(NSCC) were all significantly less in PVC than those in LAC. These differences in repolarizing ionic currents between PVC and LAC form a basis of the differences in their action potential configurations and might be an important ionic mechanism of the arrhythmogenic characteristics of pulmonary vein muscle sleeves.

Expressions of L-type calcium channel and potassium channel Kv4.3 in rapid paced primary cultured atrial myocytes / 中华心血管病杂志

<p><b>OBJECTIVE</b>To study the expressions of L-type calcium channel alpha1c and potassium channel Kv4.3 at early stages of atrial fibrillation in a rapid paced primary cultured atrial myocyte model.</p><p><b>METHODS</b>Primary rat atrial myocytes were cultured and a rapid paced cell model was established. The atrial cells were divided into five groups with pacing durations within 0 and 24 h. Reverse transcription-polymerase chain reaction and Western blot were applied to detect the messenger ribonucleic acid (mRNA) and proteins of L-type calcium channel alpha1c and potassium channel Kv4.3, respectively.</p><p><b>RESULTS</b>mRNA expression of L-type calcium channel alpha1c reduced after 6 h of rapid pacing and continued to decline as the pacing process. The decrease of L-type calcium channel alpha1c protein was paralleled with mRNA expression and reached the lowest levels at 24 h. Similarly, changes of potassium channel Kv4.3 protein and mRNA were paralleled. Kv4.3 mRNA was not altered within the first 6 h. It was reduced after 12 h. However, longer pacing periods did not further decrease mRNA and protein expression levels of potassium channel Kv4.3.</p><p><b>CONCLUSIONS</b>Expressions of L-type calcium channel alpha1c and potassium channel Kv4.3 were both reduced at different levels in early phase of rapid pacing atrial myocytes. It implicates the occurrence of ion channel remodeling of atrial myocytes, which may serve as molecular mechanism of electrical remodeling in the development of atrial fibrillation.</p>

KCNE2 modulates the function of Kv4.3 channel / 南方医科大学学报

<p><b>OBJECTIVE</b>To understand the role of KCNE2 in functional regulation of Kv4.3, the major alpha subunit of transient outward current (I(to)) in human heart.</p><p><b>METHODS</b>The cDNAs of Kv4.3 or Kv4.3 plus KCNE2 were transfected into COS-7 cells and 24-36 h after the transfection, the channel proteins were expressed in the surface membrane of the cells and the channel currents were recorded with patch-clamp technique in whole-cell mode.</p><p><b>RESULTS</b>KCNE2 played an important role in modulating the channel function. The recorded current density was decreased in cells co-expressing KCNE2 and Kv4.3 to 152.96-/+33.71 pA/pF (n=16) as compared with Kv4.3-expressing cells with a mean current density of 375.13-/+112.87 pA/pF (n=11). At the recording voltage of 60 mV, KCNE2 increased the time to peak (TTP) of the current. TTP in only Kv4.3-expressing cells was 4.82-/+0.32 ms (n=11), significantly shorter than the TTP of 20.41-/+2.13 ms (n=16) in cells co-expressing Kv4.3 and KCNE2 (P<0.05). In the presence of KCNE2, the voltage-dependent inactivation of Kv4.3 showed a positive shift. The voltage of half maximum inactivation (V(0.5)) was decreased significantly from -53.62-/+1.24 mV (n=8) in Kv4.3 group to -46.58-/+1.6 mV (n=10) in KCNE2 co-expression group (P<0.05). KCNE2 accelerated the recovery of the channel from inactivation, reducing the recovery time constant (tau) from 193.43-/+17.98 ms to 137.71-/+18.29 ms.</p><p><b>CONCLUSION</b>KCNE2 might serve as an important beta subunit and play a role in the regulation of I(to) function in human heart.</p>

mRNA expression of voltage-dependent potassium channels in the brain of rats after middle cerebral artery occlusion / 药学学报

<p><b>AIM</b>To study the mRNA expression changes in the brain of rats after middle cerebral artery occlusion.</p><p><b>METHODS</b>Middle cerebral artery occlusion was used to induce ischemia in rat brain. The mRNA expression of voltage-dependent potassium channel subtypes, including Kv1.4, Kv1.5, Kv2.1 and Kv4.2, were detected in rat hippocampus and cortex by RT-PCR.</p><p><b>RESULTS</b>Middle cerebral artery occlusion induced a significant neurological injury in rats. After ischemia 2 h, the mRNA of Kv1.4, Kv2.1 and Kv4.2 in hippocampus increased by 50%, 67% and 90% , respectively. And the mRNA of Kv1.4 and Kv4.2 maintained at a high level in hippocampus after ischemia 24 h. In cortex, the mRNA level of all the four subtypes were not changed significantly after ischemia 2 h, but the mRNA of Kv2.1 and Kv4.2 increased by 70% and 62% after ischemia 24 h, respectively.</p><p><b>CONCLUSION</b>The mRNA expression levels of voltage-dependent potassium channels were up-regulated in rat hippocampus and cortex after middle cerebral artery occlusion.</p>

Electrophysiological correspondence between Kv4.2 current and transient outward potassium current in the cultured rat hippocampal neuron / 生理学报

The present study was carried out to determine the functional properties of Kv4.2 expressed in mammalian cells in comparison with native transient potassium outward current (I(A)) in the hippocampal neurons. Transient transfection, cell culture and whole cell voltage clamp techniques were used. The results showed that I(A) in cultured rat hippocampal neurons and Kv4.2 expressed in HEK293 cells both displayed "A"-type current properties. The activation curves of I(A) and Kv4.2 were better fitted by simple Boltzmann function with V(1/2) 10.0+/-3.3 mV, k 13.9+/-2.6 mV for I(A) and V1/2 -9.7+/-4.1 mV, k 15.8+/-5.7 mV for Kv4.2, respectively. The steady-state inactivation curves of I(A) had a midpoint of -93.0+/-11.4 mV and a slope of 9.0+/-1.5 mV. The voltage-dependence of inactivation for Kv4.2 exhibited midpoint and slope values of -59.4+/-12.2 mV and 8.0+/-3.1 mV, respectively. The time constants (tau) of recovery from inactivation of I(A) and Kv4.2 were 27.9+/-14.1 ms and 172.8+/-10.0 ms, respectively. These results suggest that Kv4.2 is probably a major isoform contributing to I(A) in hippocampus neurons.

Effects of AlCl3 on transient outward K+ current and delayed rectifier K(+) current in acutely isolated rat hippocampal CA1 neurons / 生理学报

The effects of aluminum chloride (AlCl3) on the transient outward potassium and delayed rectifier K(+) current in hippocampal CA1 neurons of rats were studied by the whole-cell patch clamp technique. It was found that AlCl3 reduced the transient outward potassium current and delayed rectifier K(+) current in a dose-dependent manner. 1000 micromol/L AlCl3 resulted in change in voltage and slope of the half-activation and the half-inactivation of I(A) and I(K). These results imply that AlCl3 may damage potassium channel of the hippocampal CA1 neurons from rats and this may be related to the mechanism of the damage to the central nervous system by aluminum.