摘要：
AIM: To establish a perforated patch recording (PPR) mode with β-escin and compare L-type calcium current (Ica,L)recorded under PPR and normal whole-cell recording (WCR) condition in isolated guinea-pig ventricular myocytes. METHODS: Single myocytes were dissociated by enzymatic dissociation method. Β-escin was added to the pipette solution to perforate the cell membrane and obtain PPR mode. Ica,L was recorded using PPR and WCR techniques. RESULTS: β-Escin 20, 25, and 30 μmol/L could permeabilize the cell membrane and obtain PPR mode. With β-escin 25 μmol/L, the success rate was highest (16/17, 94 %) and the time required for permibilization was 2-15 (8+4) min. Run-down of Ica,L was considerably slower in PPR than in WCR condition. The amplitude of Ica,L was decreased by 36 % at 20 min after the formation of WCR, while it was slowly decreased by 8 % at 30 min after the formation of PPR. The current-voltage relation (I-V) curves, activation and inactivation curves of Ica,L were not significantly different between WCR and PPR. The inactivation rate of Ica,L was slower in PPR than in WCR, the faster inactivation time constant (τf) was longer in PPR than in WCR at membrane potentials of -20 mV - +10 mV (n=6, P＜0.05), and the slower time constant (τs) was also longer in PPR than in WCR at membrane potentials of-10 mV - +10 mV (n=6, P＜0.05). There was no significant difference between the activation rate in WCR and PPR. CONCLUSION: Using β-escin 25 μmol/L can easily obtain stable PPR in isolated guinea-pig ventricular myocytes, and this method is useful in dealing with channels, which show run-down under normal WCR such as L-type Ca channel.%目的:用β-e sci n在豚鼠心室肌细胞建立穿孔膜片箝技术(PPR),并记录L-型钙电流(ICa,L),与传统的全细胞记录(WCR)模式相比较.方法:用酶消化法分离单个心室肌细胞,将β-escin配在电极内液中穿孔心室肌细胞膜形成PPR模式.用WCR及PPR技术记录心室肌细胞ICa,L.结果:β-escin 20,25,30 μmol/L可在心室肌细胞膜穿孔形成PPR模式,用β-escin 25 μmol/L成功率最高(16/17,94%).用PPR模式记录的ICa,L其衰减明显比WCR方式记录的慢,ICa,L幅值在WCR形成后20 min减小36%,而在PPR形成后30 min仅缓慢减小8%.在两种模式下,ICa,L的I-V曲线,激活和失活曲线无显著差异.在PPR模式下,ICa,L的失活速率比在WCR模式下慢,在-20 mV-+10 mV电压下,其快失活相时间常数(τf)比WCR长(n=6,P＜0.05),在-10 mV-+10 mV电压下,其慢失活相时间常数(τs)也比WCR长(n=6,P＜0.05).在两种方式下,ICa,L的激活速率无显著差异.结论:用β-escin 25 μmol/L在豚鼠心室肌细胞能得到较稳定的PPR模式,此方法可以用于在普通的WCR模式下衰减较明显的电流如L-型钙电流的记录.
