| Background and Objective:Ion channels of biomembrane play an important role in many life activities. Among of them, K+ channel is a big family which include delayed rectifying potassium channels, calcium activated potassium channels, inwardly rectifying potassium and so on. Small conductance calcium-activated potassium channel (SK), one of the calcium activated potassium channels (Kca), plays an important role in excitable cells. There are four different subtypes of SK, and four mammalian SK channels (SKI, SK2, SK3 and SK4) have been cloned. Among of these subtypes, the SK2 is most sensitive to apamin. It is also potassium sensitive, voltage independent and high sensitive to Ca2+. SK2 channels are widely expressed in different tissues, such as the central nervous system, smooth muscules, heart, and so on.As a Ca2+ activated potassium channel, the SK2 there is a high sensitivity to the change of Ca2+ concentration in cell plasma. Following Ca2+ concentration increase, the SK2 is activated and led to K+ influx, which contributed to the repolarization of action potential. There are mainly two pathways to cause Ca2+ concentration increase in the muscle cell. One is the Ca2+ influx through voltage dependent Ca2 +channels (VDCCs), and another is the release of Ca2+ from intracellular sarcoplasm icreticμlum (mainly through Ryanodine receptors, RyRs). RyRs is multigene families which mediate Ca2+release from sarcoplasm icreticμlum. Three different RyRs isoforms (RyR1, RyR2, RyR3) have been identified and cloned. The RyR2 mainly distribute to myocardial cells in mouse and human. The mechanism of RyR2 regulates Ca2+ concentration is the Ca2+induced Ca2+release. When cardiac myocyte in depolarization, the L-calcium channel (LCCs) open and lead to a few calcium influx. The increase of the intracellular calcium concentration leads to lots of calcium release from SR. This is called calcium induced calcium release, which provides most of cytoplasm calcium to excitation-contraction coupling in the skeletal and cardiac muscle. RyR2 activation can rapidly increase the intracellular Ca2+concentration to more than 10 folds.Evidences have showed that there is a functional coupling between L-Ca2+ channel and SK channel in the myocardium. RyRs, SK channels and L- Ca2+channel are all related to the increase of incelluar Ca2+. It is well known that following myocardial cells calcium influx via LCCs, the RyR2 is activated and lead to more calcium release from SR. An important phenomenon is that SK2 has also been activated following the increase of calcium concentration in the cell. But, whether there is a functional coupling between SK channels and RyR channels in myocardial muscle has no yet been reported up to date. So we suppose that might there is a functional interaction between SK2 and RyR2 channels in myocardial cells. Therefore, the present study is designed for the purpose to investigate the relationship between SK2 channels activation and the cytoplasma Ca2+ increase which released from RyR2 channels in myocardial cells. The results may be provided a new window to the therapy of atrial arrhythmias.Methods:1. Mature (16-22W), male and female C57B1/6J mice weighing between 20-25g were used in the study.2. Single mice cardiac myocyte was isolated from the mouse heart by Langendorff therapy device at the temperature of 37-38℃and the constant perfusion rate,first the model of the aortic root intubation of the mouse heart was prepared, and then the heart was retrogradely perfused with Ca+-released tyrode,enzyme and KB solutions. CollagenaseⅡProtease and bovine serum albumin were used when digestion. 3. The whole-cell patch clamp method was used to record the whole cell current of the small-conductance-calcium k+ channels.The current density elicited from a holding potential of -55 mV,and the voltage steps from -120 mV to 60 mV with the filter at 2 kHz and the sample filter at 10 kHz.4. Statistical data are presented as the mean±SEM. The data were acquired with pulse 8.67 software, analyzed by SPSS 13.0, and fitted curves and mapped by Origin 6.0 software. Statistical comparisons were made using one-way ANOVA. P values of less than 0.05 were taken as significant; ns=no statistically significant difference.Results:1 Single spindle cells were acquired by acute isolation.2 The apamin sensitive current showed an decreasing tendency when the Voltage increased from the current density-voltage relationship curve. The currents were inwardly currents, with reversal potential approximately -73 mV, which was corresponding with the Nernst Equation.3. The current could be blocked by apamin. Compared with control, the apamin-sensitive currents of the caffeine group showed a significant increase in the current density in atrial myocytes, but the ryanodine and thapsigargin group showed a significant decrease. When the Ca2+ released from the sarcoplasmic reticulum, the RyR2 channels were interfered, the apamin sensitive current was also changed. Taken together, the results showed that the state of SK2 channels (open or closed) could be changed by the condition of RyR2 channels.Conclusions:There may be functionally coupling between SK2 channels and RyR2 channels in myocardium cells. |
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