Rojas E, Stokes C L, Mears D, Atwater I
Laboratory of Cell Biology and Genetics, National Institutes of Health, NIDDK, Bethesda, Maryland 20893.
J Membr Biol. 1995 Jan;143(1):65-77. doi: 10.1007/BF00232524.
A conventional patch clamp amplifier was used to test the feasibility of measuring whole-cell ionic currents under voltage clamp conditions from beta-cells in intact mouse islets of Langerhans perifused with bicarbonate Krebs buffer at 37 degrees C. Cells impaled with a high resistance microelectrode (ca. 0.150 G omega) were identified as beta-cells by the characteristic burst pattern of electrical activity induced by 11 mM glucose. Voltage-dependent outward K+ currents were enhanced by glucose both in the presence and absence of physiological bicarbonate buffer and also by bicarbonate regardless of the presence or absence of glucose. For comparison with the usual patch clamp protocol, similar measurements were made from single rat beta-cells at room temperature; glucose did not enhance the outward currents in these cells. Voltage-dependent inward currents were recorded in the presence of tetraethylammonium (TEA), an effective blocker of the K+ channels known to be present in the beta-cell membrane. Inward currents exhibited a fast component with activation-inactivation kinetics and a delayed component with a rather slow inactivation; inward currents were dependent on Ca2+ in the extracellular solution. These results suggest the presence of either two types of voltage-gated Ca2+ channels or a single type with fast and slow inactivation. We conclude that it is feasible to use a single intracellular microelectrode to measure voltage-gated membrane currents in the beta-cell within the intact islet at 37 degrees C, under conditions that support normal glucose-induced insulin secretion and that glucose enhances an as yet unidentified voltage-dependent outward K+ current.
使用传统的膜片钳放大器，在37℃下用含碳酸氢盐的Krebs缓冲液灌流完整小鼠胰岛中的β细胞，测试在电压钳制条件下测量全细胞离子电流的可行性。用高电阻微电极（约0.150 GΩ）刺入的细胞，通过11 mM葡萄糖诱导的特征性电活动爆发模式被鉴定为β细胞。无论有无生理碳酸氢盐缓冲液，葡萄糖均能增强电压依赖性外向K⁺电流，且无论有无葡萄糖，碳酸氢盐也能增强该电流。为了与常规膜片钳实验方案进行比较，在室温下对单个大鼠β细胞进行了类似测量；葡萄糖并未增强这些细胞的外向电流。在存在四乙铵（TEA）的情况下记录到电压依赖性内向电流，TEA是已知存在于β细胞膜中的K⁺通道的有效阻滞剂。内向电流表现出具有激活 - 失活动力学的快速成分和失活相当缓慢的延迟成分；内向电流依赖于细胞外溶液中的Ca²⁺。这些结果表明存在两种类型的电压门控Ca²⁺通道或一种具有快速和缓慢失活的单一类型通道。我们得出结论，在支持正常葡萄糖诱导的胰岛素分泌的条件下，在37℃完整胰岛内使用单个细胞内微电极测量β细胞中的电压门控膜电流是可行的，并且葡萄糖增强了一种尚未确定的电压依赖性外向K⁺电流。
