Protein Kinase C Controls the Excitability of Cortical Pyramidal Neurons by Regulating Kv2.2 Channel Activity / 神经科学通报·英文版

The family of voltage-gated potassium Kv2 channels consists of the Kv2.1 and Kv2.2 subtypes. Kv2.1 is constitutively highly phosphorylated in neurons and its function relies on its phosphorylation state. Whether the function of Kv2.2 is also dependent on its phosphorylation state remains unknown. Here, we investigated whether Kv2.2 channels can be phosphorylated by protein kinase C (PKC) and examined the effects of PKC-induced phosphorylation on their activity and function. Activation of PKC inhibited Kv2.2 currents and altered their steady-state activation in HEK293 cells. Point mutations and specific antibodies against phosphorylated S481 or S488 demonstrated the importance of these residues for the PKC-dependent modulation of Kv2.2. In layer II pyramidal neurons in cortical slices, activation of PKC similarly regulated native Kv2.2 channels and simultaneously reduced the frequency of action potentials. In conclusion, this study provides the first evidence to our knowledge that PKC-induced phosphorylation of the Kv2.2 channel controls the excitability of cortical pyramidal neurons.

Electrophysiology mechanisms of 4-butyl-alpha-agarofuran: a new anxiolytic and antidepressant drug / 药学学报

To investigate the electrophysiology mechanisms of new anxiolytic and antidepressant drug: 4-butyl-alpha-agarofuran (AF-5), patch clamp-recording was used to test the effects of AF-5 on voltage-dependent sodium currents, voltage-dependent potassium currents, L-type voltage-dependent calcium currents and GABA dependent Cl(-) currents in primary cultured rat cortical neurons. Effects of AF-5 on Kv2.1 currents, expressed stably in HEK293 cells, were also tested. Our results showed that, delayed rectifier potassium currents (I(K(DR, L-type voltage-dependent calcium currents (I(LC-ca)) in primary cultured rat cortical neurons and Kv2.1 currents in HEK293 cells were significantly inhibited by AF-5, with IC50 as 6.17, 4.4 and 5.29 micromol x L(-1) respectively. However, voltage-dependent sodium currents (I(Na)), GABA dependent Cl(-) currents and transient outward potassium currents (I(K(A)) in primary cultured rat cortical neurons were not significantly blocked by AF-5. Our results concluded that, blocked I(K(DR)) and I(L-Ca) currents may be one of the mechanisms of anxiolytic and antidepression actions of AF-5.

Synthesis, refolding and identification of pharmacological activities of neurotoxin JZTX-XI and R3A-JZTX-XI / 生物工程学报

Kv2.1 channel currents in pancreatic beta-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion. Because of its central role in this important physiological process, Kv2.1 channel is a promising target for the treatment of type 2 diabetes. Jingzhaotoxin-XI (JZTX-XI) is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. Two-microelectrode voltage clamp experiments had showed that the toxin inhibited Kv2.1 potassium currents expressed in Xenopus Laevis oocytes. In order to investigate the structure-function relationship of JZTX-XI, the natural toxin and a mutant of JZTX-XI in which Arg3 was replaced by Ala, were synthesized by solid-phase chemistry method with Fmoc-protected amino acids on the PS3 automated peptide synthesizer. Reverse-phase high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) were used to monitor the oxidative refolding process of synthetic linear peptides to find the optimal renaturation conditions of these toxins. The experiments also proved that the relative molecular masses of refolded peptides were in accordance with their theoretical molecular masses. RP-HPLC chromatogram of co-injected native and refolded JZTX-XI was a single peak. Under the whole-cell patch-clamp mode, JZTX-XI could completely inhibit hKv2.1 and hNav1.5 channels currents expressed in HEK293T cells with IC50 values of 95.8 nmol/L and 437.1 nmol/L respectively. The mutant R3A-JZTX-XI could also inhibit hKv2.1 and hNav1.5 channel currents expressed in HEK293T cells with IC50 values of 1.22 micromol/L and 1.96 micromol/L respectively. However, the prohibitive levels of R3A-JZTX-XI on hKv2.1 and hNav1.5 channels were reduced by about 12.7 times and 4.5 times respectively, indicating that Arg3 was a key amino acid residue relative to the hKv2.1 channel activity of JZTX-XI, but it is also an amino acid residue correlated with the binding activity of JZTX-XI to hNav1.5 channel. Our findings should be helpful to develop JZTX-XI into a molecular probe and drug candidate targeting to Kv2.1 potassium channel in the pancreas.

Expressions of voltage-gated K+ channel 2.1 and 2.2 in rat bladder with detrusor hyperreflexia / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Voltage-gated K+ channel (Kv) plays a critical role in the modulation of detrusor contraction. This study was conducted to investigate the expressions of Kv2.1 and Kv2.2 in rat bladder with detrusor hyperreflexia (DH).</p><p><b>METHODS</b>Thirty adult female Sprague-Dawley rats (200-220 g) were randomly divided into the control group and the experimental group. The experimental group was subjected to spinal cord injury (SCI). In the controls, the surgical procedure was identical with the exception that dura and spinal cord were transected. Four weeks after SCI, in vivo cystometry and mechanical pulling tests of isolated detrusor strips were performed. mRNA was extracted from the detrusors of normal and DH rats for the detection of expression of Kv2.1 and Kv2.2 by RT-PCR. Differences in expression between normal and overactive detrusors were identified by gel imaging.</p><p><b>RESULTS</b>Fourteen rats in the experimental group exhibited uninhibited bladder contraction (>8 cmH2O) before voiding after SCI. One rat died from infection. The frequency of DH in the experimental group was significantly different from that in the control group with or without treatment with 4-aminopyridine (4-AP) (P < 0.05), while the amplitude of DH did not change markedly. The rates of variation of the automatic contractile frequency and amplitude were (66.8 +/- 12.4)% and (42.6 +/- 12.6)% respectively in the control group, and (38.4 +/- 9.8)% and (28.0 +/- 4.6)% respectively in the DH group. 4-AP increased the automatic contractile frequency apart from the automatic contractile amplitude in both the control and DH groups (P < 0.05). 4-AP increased the rate of variation of the automatic contractile frequency more markedly in the control group than in the DH group (P < 0.05). Significant expression of Kv2.2 was not detected in bladders in the control group. Compared to the mRNA levels of beta-actin, the mRNA level of Kv2.1 was 1.26 +/- 0.12 in the control group and 0.66 +/- 0.08 in the DH group. SCI significantly reduced the mRNA level of Kv2.1 in rat bladders with DH (P < 0.05).</p><p><b>CONCLUSIONS</b>Our study showed that the mRNA level of Kv2.1 decreased significantly in rat bladder with DH, which was one of the important pathogenetic mechanisms for DH, and suggested that Kv2.1 might be one of the therapeutic targets for bladder overactivity.</p>

The role of subtypes of voltage-gated K+ channels in pulmonary vasoconstriction induced by 15-hydroeicosatetraenoic acid / 药学学报

<p><b>AIM</b>To observe the effect of subtypes of Kv channels in rat pulmonary artery smooth muscle cells (PASMCs) on the process of pulmonary vasoconstriction induced by 15-HETE.</p><p><b>METHODS</b>In the present study, ring of rabbit PA with specific Kv channel blockers were employed to functionally identify certain channel subtypes that took part in the process of 15-HETE induced pulmonary vasoconstriction; RT-PCR and Western blotting analysis were also used to measure the expression of subtypes of Kv in PASMCs exposed to 15-HETE,chronic hypoxia.</p><p><b>RESULTS</b>Blocking of Kv1. 1, Kv1. 2, Kv1. 3 and Kv1. 6 channels did not affect 15-HETE induced vasoconstriction in normoxic rats; 15-HETE did not affect expression of Kv1. 1 and Kv1. 2 channels; 15-HETE significantly downregulated the expression of mRNA and protein of Kv1. 5 and Kv2. 1 in rat PASMCs.</p><p><b>CONCLUSION</b>The results suggested that hypoxia may block Kv1. 5 and Kv2. 1 channels via 15-HETE mediated mechanism, leading to decrease numbers of functional Kv1. 5 and Kv2. 1 channels in PASMCs, leading to PA vasoconstriction.</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>

Effect of 17beta-estradiol on Kv2.1 current and delayed rectifier potassium current in cultured rat hippocampal neurons / 药学学报

<p><b>AIM</b>To study the effects of 17beta-estradiol on Kv2.1 potassium channel current and delayed rectifier potassium current (IK) in cultured rat hippocampal neurons.</p><p><b>METHODS</b>The effects of 17beta-estradiol on Kv2.1 channel current and IK in cultured rat hippocampal neurons were observed using the whole cell patch clamp techniques.</p><p><b>RESULTS</b>17beta-Estradiol was shown to reduce the amplitude of Kv2.1 current and IK in concentration-dependent manners. The IC50s of 17beta-estradiol blocking Kv2.1 and IK were 2.4 and 4.0 micromol x L(-1), respectively. 17beta-Estradiol (3 micromol x l(-1)) significantly shifted the steady-state activation and inactivation curves of Kv2.1 current to negative potentials. However, it only produced the shift of the steady-state activation curve of IK to the negative potential without effect on the steady-state inactivation of IK.</p><p><b>CONCLUSION</b>17beta-Estradiol inhibits Kv2.1 and IK of hippocampus at similar level. The inhibition of 17beta-estradiol on IK current may be partially via blocking Kv2.1 current.</p>

Effects of chronic exposure to beta-amyloid-peptide25-35 on the mRNA expressions of voltage-gated outward potassium channel subunits in cultured rat hippocampal neurons / 药学学报

<p><b>AIM</b>To investigate mRNA expression changes of voltage-gated outward potassium channel subtypes in cultured rat hippocampal neurons after chronic exposure to beta-amyloid-petitde25-35 (beta-AP25-35).</p><p><b>METHODS</b>mRNA expression was detected by RT-PCR, comparative expression levels were determined by imaging densitometer.</p><p><b>RESULTS</b>Delayed rectifying (Kv2.1, Kv1.5), transient outward (Kv1.4, Kv4.2) and large conductance calcium-activated (rSlo) potassium channel mRNA were expressed in cultured rat hippocampal. In the presence of beta-AP25-35 3 mumol.L-1 for 24 h, the relative expression level of Kv2.1 was significantly increased (n = 3, P < 0.05); the other subtypes were not changed obviously (n = 3, P > 0.05). The increase of Kv2.1 mRNA mainly happened between 24 and 36 h after exposure to beta-AP25-35. After exposure to beta-AP25-35 for 60 h, Kv2.1 mRNA decreased significantly (n = 3, P < 0.01).</p><p><b>CONCLUSION</b>The upregulation of Kv2.1 on transcription levels may be involved in the enhancement of delayed rectifying outward potassium (Ik) current induced by beta-AP25-35.</p>