Reconstitution of large conductance calcium-activated potassium channels into artificial planar lipid bilayers / 生理学报

This study was aimed to establish a method to create a stable planar lipid bilayer membranes (PLBMs), in which large conductance calcium-activated potassium channels (BK) were reconstituted. Using spreading method, PLBMs were prepared by decane lipid fluid consisting of N-weathered mixture of phosphatidylcholine and cholesterol at 3:1 ratio. After successful incorporation of BKchannel into PLBMs, single channel characteristics of BKwere studied by patch clamp method. The results showed that i) the single channel conductance of BKwas (206.8 ± 16.9) pS; ii) the activities of BKchannel were voltage dependent; iii) in the bath solution without Ca, there was almost no BKchannel activities regardless of under hyperpolarization or repolarization conditions; iv) under the condition of +40 mV membrane potential, BKchannels were activated in a Caconcentration dependent manner; v) when [Ca] was increased from 1 μmol/L to 100 μmol/L, both the channel open probability and the average open time were increased, and the average close time was decreased from (32.2 ± 2.8) ms to (2.1 ± 1.8) ms; vi) the reverse potential of the reconstituted BKwas -30 mV when [K] was at 40/140 mmol/L (Cis/Trans). These results suggest that the spreading method could serve as a new method for preparing PLBMs and the reconstituted BKinto PLBMs showed similar electrophysiological characteristics to natural BKchannels, so the PLBMs with incorporated BKcan be used in the studies of pharmacology and dynamics of BKchannel.

β-estradiol activates BK(Ca) in mesenteric artery smooth muscle cells of post-menopause women / 生理学报

The aim of the present study was to study the effect of β-estradiol (β-E(2)) on the large-conductance Ca(2+)-activated potassium (BK(Ca)) channel in mesenteric artery smooth muscle cells (SMCs). The mesenteric arteries were obtained from post-menopause female patients with abdominal surgery, and the SMCs were isolated from the arteries using an enzymatic disassociation. According to the sources, the SMCs were divided into non-hypertension (NH) and essential hypertension (EH) groups. Single channel patch clamp technique was used to investigate the effect of β-E(2) and ICI 182780 (a specific blocker of estrogen receptor) on BK(Ca) in the SMCs. The results showed the opening of BK(Ca) in the SMCs was voltage and calcium dependent, and could be blocked by IbTX. β-E(2) (100 μmol/L) significantly increased open probability (Po) of BK(Ca) in both NH and EH groups. After β-E(2) treatment, NH group showed higher Po of BK(Ca) compared with EH group. ICI 182780 could inhibit the activating effect of β-E(2) on BK(Ca) in no matter NH or EH groups. These results suggest β-E(2) activates BK(Ca) in mesenteric artery SMCs from post-menopause women via estrogen receptor, but hypertension may decline the activating effect of β-E(2) on BK(Ca).

Construction and identification of the expression plasmid of SK2 (KCNN2) gene from human atrial myocytes with overlapping PCR / 中国应用生理学杂志

<p><b>OBJECTIVE</b>Small conductance calcium activated potassium channels type 2 (SK2) play a crucial role in atrial repolarization. It is difficult to acquire the full-length of its coded gene KCNN2 by RT-PCR with one step. We aim to get the full-length of KCNN2 gene and construct the plasmid by Overlapping PCR, and further more discuss the application of Overlapping PCR.</p><p><b>METHODS</b>Total RNA was extracted from human right atrial tissue and cDNA was acquired with reverse transcription. Overlapping PCR was conducted with three pairs of primers which were designed according to the sequence of KCNN2 (AY258141) gene. The expression plasmid of pIRES-hrGFP-SK2 was constructed by directed cloning with restriction enzyme site and identified by enzyme cutting and sequencing.</p><p><b>RESULTS</b>Three parts of PCR amplification were consistent with predicted size. The sequence of the plasmid was consistent with the gene-bank data except two sites, however, which were the same as gene in different tissues.</p><p><b>CONCLUSION</b>The expression plasmid pIRES-hrGFP-SK2 was constructed successfully. Overlapping PCR is a good choice for amplifying these genes with long size or low expression.</p>

Increased small conductance calcium-activated potassium channel (SK2 channel) current in atrial myocytes of patients with persistent atrial fibrillation / 中华心血管病杂志

<p><b>OBJECTIVE</b>To compare the amplitude of the SK2 current (small conductance calcium-activated potassium channel) in human atrial myocytes with or without persistent atrial fibrillation (AF).</p><p><b>METHODS</b>Right atrial appendage was obtained from 15 patients with sinus rate (SR) and 7 patients with AF underwent surgical valve replacement. Single myocyte was isolated by enzymatic dissociation method and the SK2 channel current density was recorded using whole-cell patch clamp techniques to detect the changes. Immunofluorescence was used to observe SK2 channel protein distribution on right atrial appendage.</p><p><b>RESULTS</b>Using the whole cell patch-clamp recording techniques, an inward rectifier K(+) mix currents could be obtained from both SR (n = 15) and AF (n = 7) samples, I(K1) mix currents density in single myocyte of AF group was significantly increased than in SR group [(-16.42 ± 5.32) pA/pF vs (-6.59 ± 2.24) pA/pF, P < 0.01], which could be partially inhibited by apamin (100 nmol/L). The apamin-sensitive current was obtained by subtraction of the currents before and after treatment with apamin. SK2 current density was significantly increased in AF group than that of SR group [(-9.81 ± 2.54) pA/pF vs (-3.67 ± 0.37) pA/pF, P < 0.01]. SK2 channel protein was evidenced with immunofluorescence method in right atrial appendage from AF group and SR group.</p><p><b>CONCLUSION</b>SK2 channel protein and current were present in atrial myocytes. The SK2 current density was significantly increased in AF group than in SR group suggesting that the increase of SK2 current might contribute to the electrical remodeling in AF patients.</p>

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>